Publications

Publications in peer reviewed journals

100 Publications found
  • Verrucomicrobia use hundreds of enzymes to digest the algal polysaccharide fucoidan.

    Sichert A, Corzett CH, Schechter MS, Unfried F, Markert S, Becher D, Fernandez-Guerra A, Liebeke M, Schweder T, Polz MF, Hehemann JH
    2020 - Nat Microbiol, 8: 1026-1039

    Abstract: 

    Brown algae are important players in the global carbon cycle by fixing carbon dioxide into 1 Gt of biomass annually, yet the fate of fucoidan-their major cell wall polysaccharide-remains poorly understood. Microbial degradation of fucoidans is slower than that of other polysaccharides, suggesting that fucoidans are more recalcitrant and may sequester carbon in the ocean. This may be due to the complex, branched and highly sulfated structure of fucoidans, which also varies among species of brown algae. Here, we show that 'Lentimonas' sp. CC4, belonging to the Verrucomicrobia, acquired a remarkably complex machinery for the degradation of six different fucoidans. The strain accumulated 284 putative fucoidanases, including glycoside hydrolases, sulfatases and carbohydrate esterases, which are primarily located on a 0.89-megabase pair plasmid. Proteomics reveals that these enzymes assemble into substrate-specific pathways requiring about 100 enzymes per fucoidan from different species of brown algae. These enzymes depolymerize fucoidan into fucose, which is metabolized in a proteome-costly bacterial microcompartment that spatially constrains the metabolism of the toxic intermediate lactaldehyde. Marine metagenomes and microbial genomes show that Verrucomicrobia including 'Lentimonas' are abundant and highly specialized degraders of fucoidans and other complex polysaccharides. Overall, the complexity of the pathways underscores why fucoidans are probably recalcitrant and more slowly degraded, since only highly specialized organisms can effectively degrade them in the ocean.

  • Diarrhoeal events can trigger long-term Clostridium difficile colonization with recurrent blooms.

    VanInsberghe D, Elsherbini JA, Varian B, Poutahidis T, Erdman S, Polz MF
    2020 - Nat Microbiol, 4: 642-650

    Abstract: 

    Although Clostridium difficile is widely considered an antibiotic- and hospital-associated pathogen, recent evidence indicates that this is an insufficient depiction of the risks and reservoirs. A common thread that links all major risk factors of infection is their association with gastrointestinal disturbances, but this relationship to C. difficile colonization has never been tested directly. Here, we show that disturbances caused by diarrhoeal events trigger susceptibility to C. difficile colonization. Using survey data of the human gut microbiome, we detected C. difficile colonization and blooms in people recovering from food poisoning and Vibrio cholerae infections. Carriers remained colonized for year-long time scales and experienced highly variable patterns of C. difficile abundance, where increased shedding over short periods of 1-2 d interrupted week-long periods in which C. difficile was undetectable. Given that short shedding events were often linked to gastrointestinal disturbances, our results help explain why C. difficile is frequently detected as a co-infecting pathogen in patients with diarrhoea. To directly test the impact of diarrhoea on susceptibility to colonization, we developed a mouse model of variable disturbance intensity, which allowed us to monitor colonization in the absence of disease. As mice exposed to avirulent C. difficile spores ingested increasing quantities of laxatives, more individuals experienced C. difficile blooms. Our results indicate that the likelihood of colonization is highest in the days immediately following acute disturbances, suggesting that this could be an important window during which transmission could be interrupted and the incidence of infection lowered.

  • Culture-independent tracking of Vibrio cholerae lineages reveals complex spatiotemporal dynamics in a natural population.

    Kirchberger PC, Orata FD, Nasreen T, Kauffman KM, Tarr CL, Case RJ, Polz MF, Boucher YF
    2020 - Environ. Microbiol., in press

    Abstract: 

    Populations of the bacterium Vibrio cholerae consist of dozens of distinct lineages, with primarily (but not exclusively) members of the pandemic generating lineage capable of causing the diarrhoeal disease cholera. Assessing the composition and temporal dynamics of such populations requires extensive isolation efforts and thus only rarely covers large geographic areas or timeframes exhaustively. We developed a culture-independent amplicon sequencing strategy based on the protein-coding gene viuB (vibriobactin utilization) to study the structure of a V. cholerae population over the course of a summer. We show that the 26 co-occurring V. cholerae lineages continuously compete for limited space on nutrient-rich particles where only a few of them can grow to large numbers. Differential abundance of lineages between locations and size-fractions associated with a particle-attached or free-swimming lifestyle could reflect adaptation to various environmental niches. In particular, a major V. cholerae lineage occasionally grows to large numbers on particles but remain undetectable using isolation-based methods, indicating selective culturability for some members of the species. We thus demonstrate that isolation-based studies may not accurately reflect the structure and complex dynamics of V. cholerae populations and provide a scalable high-throughput method for both epidemiological and ecological approaches to studying this species.

  • Summer phyto- and bacterioplankton communities during low and high productivity scenarios in the Western Antarctic Peninsula

    Fuentes S, Arroyo JI, Rodriguez-Marconi S, Masotti I, Alarcon-Schumacher T, Polz MF, Trefault N, de la Iglesia R, Díez B
    2019 - Polar Biology, 42: 159-169

    Abstract: 

    Phytoplankton blooms taking place during the warm season drive high productivity in Antarctic coastal seawaters. Important temporal and spatial variations exist in productivity patterns, indicating local constraints influencing the phototrophic community. Surface water in Chile Bay (Greenwich Island, South Shetlands) is influenced by freshwater from the melting of sea ice and surrounding glaciers; however, it is not a widely studied system. The phyto- and bacterioplankton communities in Chile Bay were studied over two consecutive summers; during a low productivity period (chlorophyll a < 0.05 mg m−3) and an ascendant phototrophic bloom (chlorophyll a up to 2.38 mg m−3). Microbial communities were analyzed by 16S rRNA—including plastidial—gene sequencing. Diatoms (mainly Thalassiosirales) were the most abundant phytoplankton, particularly during the ascendant bloom. Bacterioplankton in the low productivity period was less diverse and dominated by a few operational taxonomic units (OTUs), related to Colwellia and Pseudoalteromonas. Alpha diversity was higher during the bloom, where several Bacteroidetes taxa absent in the low productivity period were present. Network analysis indicated that phytoplankton relative abundance was correlated with bacterioplankton phylogenetic diversity and the abundance of several bacterial taxa. Hubs—the most connected OTUs in the network—were not the most abundant OTUs and included some poorly described taxa in Antarctica, such as Neptunomonas and Ekhidna. In summary, the results of this study indicate that in Antarctic Peninsula coastal waters, such as Chile Bay, higher bacterioplankton community diversity occurs during a phototrophic bloom. This is likely a result of primary production, providing a source of fresh organic matter to bacterioplankton.

  • Maintenance of Sympatric and Allopatric Populations in Free-Living Terrestrial Bacteria.

    Chase AB, Arevalo P, Brodie EL, Polz MF, Karaoz U, Martiny JBH
    2019 - mBio, 5: e02361-19

    Abstract: 

    For free-living bacteria and archaea, the equivalent of the biological species concept does not exist, creating several obstacles to the study of the processes contributing to microbial diversification. These obstacles are particularly high in soil, where high bacterial diversity inhibits the study of closely related genotypes and therefore the factors structuring microbial populations. Here, we isolated strains within a single ecotype from surface soil (leaf litter) across a regional climate gradient and investigated the phylogenetic structure, recombination, and flexible gene content of this genomic diversity to infer patterns of gene flow. Our results indicate that microbial populations are delineated by gene flow discontinuities, with distinct populations cooccurring at multiple sites. Bacterial population structure was further delineated by genomic features allowing for the identification of candidate genes possibly contributing to local adaptation. These results suggest that the genetic structure within this bacterium is maintained both by ecological specialization in localized microenvironments (isolation by environment) and by dispersal limitation between geographic locations (isolation by distance). Due to the promiscuous exchange of genetic material and asexual reproduction, delineating microbial species (and, by extension, populations) remains challenging. Because of this, the vast majority of microbial studies assessing population structure often compare divergent strains from disparate environments under varied selective pressures. Here, we investigated the population structure within a single bacterial ecotype, a unit equivalent to a eukaryotic species, defined as highly clustered genotypic and phenotypic strains with the same ecological niche. Using a combination of genomic and computational analyses, we assessed the phylogenetic structure, extent of recombination, and flexible gene content of this genomic diversity to infer patterns of gene flow. To our knowledge, this study is the first to do so for a dominant soil bacterium. Our results indicate that bacterial soil populations, similarly to those in other environments, are structured by gene flow discontinuities and exhibit distributional patterns consistent with both isolation by distance and isolation by environment. Thus, both dispersal limitation and local environments contribute to the divergence among closely related soil bacteria as observed in macroorganisms.

  • A Reverse Ecology Approach Based on a Biological Definition of Microbial Populations.

    Arevalo P, VanInsberghe D, Elsherbini J, Gore J, Polz MF
    2019 - Cell, 4: 820-834.e14

    Abstract: 

    Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology.

  • Genomic Features for Desiccation Tolerance and Sugar Biosynthesis in the Extremophile sp. UTEX B3054.

    Urrejola C, Alcorta J, Salas L, Vásquez M, Polz MF, Vicuña R, Díez B
    2019 - Front Microbiol, 950

    Abstract: 

    For tolerating extreme desiccation, cyanobacteria are known to produce both compatible solutes at intracellular level and a copious amount of exopolysaccharides as a protective coat. However, these molecules make cyanobacterial cells refractory to a broad spectrum of cell disruption methods, hindering genome sequencing, and molecular studies. In fact, few genomes are already available from cyanobacteria from extremely desiccated environments such as deserts. In this work, we report the 5.4 Mbp draft genome (with 100% of completeness in 105 contigs) of sp. UTEX B3054 (subsection I; Order Chroococcales), a cultivable sugar-rich and hardly breakable hypolithic cyanobacterium from the Atacama Desert. Our analyses focused on genomic features related to sugar-biosynthesis and adaptation to dryness. Among other findings, screening of genome revealed a unique genetic potential related to the biosynthesis and regulation of compatible solutes and polysaccharides. For instance, our findings showed for the first time a novel genomic arrangement exclusive of Chroococcaceae cyanobacteria associated with the recycling of trehalose, a compatible solute involved in desiccation tolerance. Additionally, we performed a comparative genome survey and analyses to entirely predict the highly diverse pool of glycosyltransferases enzymes, key players in polysaccharide biosynthesis and the formation of a protective coat to dryness. We expect that this work will set the fundamental genomic framework for further research on microbial tolerance to desiccation and to a wide range of other extreme environmental conditions. The study of microorganisms like sp. UTEX B3054 will contribute to expand our limited understanding regarding water optimization and molecular mechanisms allowing extremophiles to thrive in xeric environments such as the Atacama Desert.

  • Polyclonal symbiont populations in hydrothermal vent tubeworms and the environment.

    Polzin J, Arevalo P, Nussbaumer T, Polz MF, Bright M
    2019 - Proc. Biol. Sci., 1896: 20181281

    Abstract: 

    Horizontally transmitted symbioses usually house multiple and variable symbiont genotypes that are acquired from a much more diverse environmental pool via partner choice mechanisms. However, in the deep-sea hydrothermal vent tubeworm Riftia pachyptila (Vestimentifera, Siboglinidae), it has been suggested that the Candidatus Endoriftia persephone symbiont is monoclonal. Here, we show with high-coverage metagenomics that adult R. pachyptila house a polyclonal symbiont population consisting of one dominant and several low-frequency variants. This dominance of one genotype is confirmed by multilocus gene sequencing of amplified housekeeping genes in a broad range of host individuals where three out of four loci ( atpA, uvrD and recA) revealed no genomic differences, while one locus ( gyrB) was more diverse in adults than in juveniles. We also analysed a metagenome of free-living Endoriftia and found that the free-living population showed greater sequence variability than the host-associated population. Most juveniles and adults shared a specific dominant genotype, while other genotypes can dominate in few individuals. We suggest that although generally permissive, partner choice is selective enough to restrict uptake of some genotypes present in the environment.

  • Interactions in self-assembled microbial communities saturate with diversity.

    Yu X, Polz MF, Alm EJ
    2019 - ISME J, 6: 1602-1617

    Abstract: 

    How the diversity of organisms competing for or sharing resources influences community function is an important question in ecology but has rarely been explored in natural microbial communities. These generally contain large numbers of species making it difficult to disentangle how the effects of different interactions scale with diversity. Here, we show that changing diversity affects measures of community function in relatively simple communities but that increasing richness beyond a threshold has little detectable effect. We generated self-assembled communities with a wide range of diversity by growth of cells from serially diluted seawater on brown algal leachate. We subsequently isolated the most abundant taxa from these communities via dilution-to-extinction in order to compare productivity functions of the entire community to those of individual taxa. To parse the effect of different types of organismal interactions, we defined relative total function (RTF) as an index for positive or negative effects of diversity on community function. Our analysis identified three overall regimes with increasing diversity. At low richness (<12 taxa), positive and negative effects of interactions were both weak, while at moderate richness (12-26 taxa), community resource uptake increased but the carbon use efficiency decreased. Finally, beyond 26 taxa, the effect of interactions on community function saturated and further diversity increases did not affect community function. Although more diverse communities had overall greater access to resources, on average individual taxa within these communities had lower resource availability and reduced carbon use efficiency. Our results thus suggest competition and complementation simultaneously increase with diversity but both saturate at a threshold.

  • Bacterial community structure in a sympagic habitat expanding with global warming: brackish ice brine at 85-90 °N.

    Fernández-Gómez B, Díez B, Polz MF, Arroyo JI, Alfaro FD, Marchandon G, Sanhueza C, Farías L, Trefault N, Marquet PA, Molina-Montenegro MA, Sylvander P, Snoeijs-Leijonmalm P
    2019 - ISME J, 2: 316-333

    Abstract: 

    Larger volumes of sea ice have been thawing in the Central Arctic Ocean (CAO) during the last decades than during the past 800,000 years. Brackish brine (fed by meltwater inside the ice) is an expanding sympagic habitat in summer all over the CAO. We report for the first time the structure of bacterial communities in this brine. They are composed of psychrophilic extremophiles, many of them related to phylotypes known from Arctic and Antarctic regions. Community structure displayed strong habitat segregation between brackish ice brine (IB; salinity 2.4-9.6) and immediate sub-ice seawater (SW; salinity 33.3-34.9), expressed at all taxonomic levels (class to genus), by dominant phylotypes as well as by the rare biosphere, and with specialists dominating IB and generalists SW. The dominant phylotypes in IB were related to Candidatus Aquiluna and Flavobacterium, those in SW to Balneatrix and ZD0405, and those shared between the habitats to Halomonas, Polaribacter and Shewanella. A meta-analysis for the oligotrophic CAO showed a pattern with Flavobacteriia dominating in melt ponds, Flavobacteriia and Gammaproteobacteria in solid ice cores, Flavobacteriia, Gamma- and Betaproteobacteria, and Actinobacteria in brine, and Alphaproteobacteria in SW. Based on our results, we expect that the roles of Actinobacteria and Betaproteobacteria in the CAO will increase with global warming owing to the increased production of meltwater in summer. IB contained three times more phylotypes than SW and may act as an insurance reservoir for bacterial diversity that can act as a recruitment base when environmental conditions change.

  • Streamlining standard bacteriophage methods for higher throughput.

    Kauffman KM, Polz MF
    2018 - MethodsX, 159-172

    Abstract: 

    A universal tool in the culture-based study of bacterial viruses (bacteriophages, or phages) is the agar overlay, which is used in the isolation of new viruses, and in their quantification and purification. Here, simple optimizations that increase efficiency and throughput in agar overlay based isolation and cultivation of virus-host systems are presented. The agar overlay is streamlined to minimize steps and materials. Serial purification of viruses from viral colonies (plaques) is optimized to eliminate steps by combining purification by serial re-streaking with the optimized agar overlay approach. Finally, recommendations are made for efficient archival and storage of virus plaques. In sum, this work presents: •Tube-free Agar Overlays: rapid plaque assays with fewer steps and materials•Molten Streaking for Singles: rapid tube-free serial purification of viruses•Archiving Plaques: saving virus purification for later.

  • Ancestral gene acquisition as the key to virulence potential in environmental Vibrio populations.

    Bruto M, Labreuche Y, James A, Piel D, Chenivesse S, Petton B, Polz MF, Le Roux F
    2018 - ISME J, 12: 2954-2966

    Abstract: 

    Diseases of marine animals caused by bacteria of the genus Vibrio are on the rise worldwide. Understanding the eco-evolutionary dynamics of these infectious agents is important for predicting and managing these diseases. Yet, compared to Vibrio infecting humans, knowledge of their role as animal pathogens is scarce. Here we ask how widespread is virulence among ecologically differentiated Vibrio populations, and what is the nature and frequency of virulence genes within these populations? We use a combination of population genomics and molecular genetics to assay hundreds of Vibrio strains for their virulence in the oyster Crassostrea gigas, a unique animal model that allows high-throughput infection assays. We show that within the diverse Splendidus clade, virulence represents an ancestral trait but has been lost from several populations. Two loci are necessary for virulence, the first being widely distributed across the Splendidus clade and consisting of an exported conserved protein (R5.7). The second is a MARTX toxin cluster, which only occurs within V. splendidus and is for the first time associated with virulence in marine invertebrates. Varying frequencies of both loci among populations indicate different selective pressures and alternative ecological roles, based on which we suggest strategies for epidemiological surveys.

  • Viruses of the Nahant Collection, characterization of 251 marine Vibrionaceae viruses.

    Kauffman KM, Brown JM, Sharma RS, VanInsberghe D, Elsherbini J, Polz MF, Kelly L
    2018 - Sci Data, 180114

    Abstract: 

    Viruses are highly discriminating in their interactions with host cells and are thought to play a major role in maintaining diversity of environmental microbes. However, large-scale ecological and genomic studies of co-occurring virus-host pairs, required to characterize the mechanistic and genomic foundations of virus-host interactions, are lacking. Here, we present the largest dataset of cultivated and sequenced co-occurring virus-host pairs that captures ecologically representative fine-scale diversity. Using the ubiquitous and ecologically diverse marine Vibrionaceae as a host platform, we isolate and sequence 251 dsDNA viruses and their hosts from three time points within a 93-day time-series study. The virus collection includes representatives of the three Caudovirales tailed virus morphotypes, a novel family of nontailed viruses, and the smallest (10,046 bp) and largest (348,911 bp) Vibrio virus genomes described. We provide general characterization and annotation of the viruses and describe read-mapping protocols to standardize genome presentation. The rich ecological and genomic contextualization of hosts and viruses make the Nahant Collection a unique platform for high-resolution studies of environmental virus-host infection networks.

  • Widespread distribution of prophage-encoded virulence factors in marine Vibrio communities.

    Castillo D, Kauffman K, Hussain F, Kalatzis P, Rørbo N, Polz MF, Middelboe M
    2018 - Sci Rep, 1: 9973

    Abstract: 

    Prophages are known to encode important virulence factors in the human pathogen Vibrio cholerae. However, little is known about the occurrence and composition of prophage-encoded traits in environmental vibrios. A database of 5,674 prophage-like elements constructed from 1,874 Vibrio genome sequences, covering sixty-four species, revealed that prophage-like elements encoding possible properties such as virulence and antibiotic resistance are widely distributed among environmental vibrios, including strains classified as non-pathogenic. Moreover, we found that 45% of Vibrio species harbored a complete prophage-like element belonging to the Inoviridae family, which encode the zonula occludens toxin (Zot) previously described in the V. cholerae. Interestingly, these zot-encoding prophages were found in a variety of Vibrio strains covering both clinical and marine isolates, including strains from deep sea hydrothermal vents and deep subseafloor sediments. In addition, the observation that a spacer from the CRISPR locus in the marine fish pathogen V. anguillarum strain PF7 had 95% sequence identity with a zot gene from the Inoviridae prophage found in V. anguillarum strain PF4, suggests acquired resistance to inoviruses in this species. Altogether, our results contribute to the understanding of the role of prophages as drivers of evolution and virulence in the marine Vibrio bacteria.

  • Inter-individual variability in copepod microbiomes reveals bacterial networks linked to host physiology.

    Datta MS, Almada AA, Baumgartner MF, Mincer TJ, Tarrant AM, Polz MF
    2018 - ISME J, 9: 2103-2113

    Abstract: 

    Copepods harbor diverse bacterial communities, which collectively carry out key biogeochemical transformations in the ocean. However, bulk copepod sampling averages over the variability in their associated bacterial communities, thereby limiting our understanding of the nature and specificity of copepod-bacteria associations. Here, we characterize the bacterial communities associated with nearly 200 individual Calanus finmarchicus copepods transitioning from active growth to diapause. We find that all individual copepods sampled share a small set of "core" operational taxonomic units (OTUs), a subset of which have also been found associated with other marine copepod species in different geographic locations. However, most OTUs are patchily distributed across individual copepods, thereby driving community differences across individuals. Among patchily distributed OTUs, we identified groups of OTUs correlated with common ecological drivers. For instance, a group of OTUs positively correlated with recent copepod feeding served to differentiate largely active growing copepods from those entering diapause. Together, our results underscore the power of individual-level sampling for understanding host-microbiome relationships.

  • Function and functional redundancy in microbial systems.

    Louca S, Polz MF, Mazel F, Albright MBN, Huber JA, O'Connor MI, Ackermann M, Hahn AS, Srivastava DS, Crowe SA, Doebeli M, Parfrey LW
    2018 - Nat Ecol Evol, 6: 936-943

    Abstract: 

    Microbial communities often exhibit incredible taxonomic diversity, raising questions regarding the mechanisms enabling species coexistence and the role of this diversity in community functioning. On the one hand, many coexisting but taxonomically distinct microorganisms can encode the same energy-yielding metabolic functions, and this functional redundancy contrasts with the expectation that species should occupy distinct metabolic niches. On the other hand, the identity of taxa encoding each function can vary substantially across space or time with little effect on the function, and this taxonomic variability is frequently thought to result from ecological drift between equivalent organisms. Here, we synthesize the powerful paradigm emerging from these two patterns, connecting the roles of function, functional redundancy and taxonomy in microbial systems. We conclude that both patterns are unlikely to be the result of ecological drift, but are inevitable emergent properties of open microbial systems resulting mainly from biotic interactions and environmental and spatial processes.

  • Evolution of a Vegetarian Vibrio: Metabolic Specialization of Vibrio breoganii to Macroalgal Substrates.

    Corzett CH, Elsherbini J, Chien DM, Hehemann JH, Henschel A, Preheim SP, Yu X, Alm EJ, Polz MF
    2018 - J. Bacteriol., 15: in press

    Abstract: 

    While most are considered generalists that thrive on diverse substrates, including animal-derived material, we show that has specialized for the consumption of marine macroalga-derived substrates. Genomic and physiological comparisons of with other isolates revealed the ability to degrade alginate, laminarin, and additional glycans present in algal cell walls. Moreover, the widely conserved ability to hydrolyze animal-derived polymers, including chitin and glycogen, was lost, along with the ability to efficiently grow on a variety of amino acids. Ecological data showing associations with particulate algal material but not zooplankton further support this shift in niche preference, and the loss of motility appears to reflect a sessile macroalga-associated lifestyle. Together, these findings indicate that algal polysaccharides have become a major source of carbon and energy in , and these ecophysiological adaptations may facilitate transient commensal associations with marine invertebrates that feed on algae. Vibrios are often considered animal specialists or generalists. Here, we show that has undergone massive genomic changes to become specialized on algal carbohydrates. Accompanying genomic changes include massive gene import and loss. These vibrios may help us better understand how algal biomass is degraded in the environment and may serve as a blueprint on how to optimize the conversion of algae to biofuels.

  • A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria.

    Kauffman KM, Hussain FA, Yang J, Arevalo P, Brown JM, Chang WK, VanInsberghe D, Elsherbini J, Sharma RS, Cutler MB, Kelly L, Polz MF
    2018 - Nature, 7690: 118-122

    Abstract: 

    The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.

  • High resolution time series reveals cohesive but short-lived communities in coastal plankton.

    Martin-Platero AM, Cleary B, Kauffman K, Preheim SP, McGillicuddy DJ, Alm EJ, Polz MF
    2018 - Nat Commun, 1: 266

    Abstract: 

    Because microbial plankton in the ocean comprise diverse bacteria, algae, and protists that are subject to environmental forcing on multiple spatial and temporal scales, a fundamental open question is to what extent these organisms form ecologically cohesive communities. Here we show that although all taxa undergo large, near daily fluctuations in abundance, microbial plankton are organized into clearly defined communities whose turnover is rapid and sharp. We analyze a time series of 93 consecutive days of coastal plankton using a technique that allows inference of communities as modular units of interacting taxa by determining positive and negative correlations at different temporal frequencies. This approach shows both coordinated population expansions that demarcate community boundaries and high frequency of positive and negative associations among populations within communities. Our analysis thus highlights that the environmental variability of the coastal ocean is mirrored in sharp transitions of defined but ephemeral communities of organisms.

  • Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

    Badur AH, Plutz MJ, Yalamanchili G, Jagtap SS, Schweder T, Unfried F, Markert S, Polz MF, Hehemann JH, Rao CV
    2017 - J. Biol. Chem., 31: 13056-13067

    Abstract: 

    Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium One strain, 13B01, exhibited high extracellular alginate lyase activity compared with other strains. To identify the enzymes responsible for this high extracellular activity, we compared 13B01 with the previously characterized 12B01, which has low extracellular activity and lacks two alginate lyase genes present in 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions.

  • Natural resource landscapes of a marine bacterium reveal distinct fitness-determining genes across the genome.

    Takemura AF, Corzett CH, Hussain F, Arevalo P, Datta M, Yu X, Le Roux F, Polz MF
    2017 - Environ. Microbiol., 6: 2422-2433

    Abstract: 

    Heterotrophic bacteria exploit diverse microhabitats in the ocean, from particles to transient gradients. Yet the degree to which genes and pathways can contribute to an organism's fitness on such complex and variable natural resource landscapes remains poorly understood. Here, we determine the gene-by-gene fitness of a generalist saprophytic marine bacterium (Vibrio sp. F13 9CS106) on complex resources derived from its natural habitats - copepods (Apocyclops royi) and brown algae (Fucus vesiculosus) - and as reference substrates, glucose and the polysaccharide alginate, derived from brown algal cell walls. We find that resource complexity strongly buffers fitness costs of mutations, and that anabolic rather than catabolic pathways are more stringently required, likely due to functional redundancy in the latter. Moreover, while carbohydrate-rich algae requires several synthesis pathways, protein-rich Apocyclops does not, suggesting this ancestral habitat for Vibrios is a replete medium with metabolically redundant substrates. We also identify a candidate fitness trade-off for algal colonization: deletion of mshA increases mutant fitness. Our results demonstrate that gene fitness depends on habitat composition, and suggest that this generalist uses distinct resources in different natural habitats. The results further indicate that substrate replete conditions may lead to relatively relaxed selection on catabolic genes.

  • Microbial community assembly and evolution in subseafloor sediment.

    Starnawski P, Bataillon T, Ettema TJ, Jochum LM, Schreiber L, Chen X, Lever MA, Polz MF, Jørgensen BB, Schramm A, Kjeldsen KU
    2017 - Proc. Natl. Acad. Sci. U.S.A., 11: 2940-2945

    Abstract: 

    Bacterial and archaeal communities inhabiting the subsurface seabed live under strong energy limitation and have growth rates that are orders of magnitude slower than laboratory-grown cultures. It is not understood how subsurface microbial communities are assembled and whether populations undergo adaptive evolution or accumulate mutations as a result of impaired DNA repair under such energy-limited conditions. Here we use amplicon sequencing to explore changes of microbial communities during burial and isolation from the surface to the >5,000-y-old subsurface of marine sediment and identify a small core set of mostly uncultured bacteria and archaea that is present throughout the sediment column. These persisting populations constitute a small fraction of the entire community at the surface but become predominant in the subsurface. We followed patterns of genome diversity with depth in four dominant lineages of the persisting populations by mapping metagenomic sequence reads onto single-cell genomes. Nucleotide sequence diversity was uniformly low and did not change with age and depth of the sediment. Likewise, there was no detectable change in mutation rates and efficacy of selection. Our results indicate that subsurface microbial communities predominantly assemble by selective survival of taxa able to persist under extreme energy limitation.

  • Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes.

    Becker S, Scheffel A, Polz MF, Hehemann JH
    2017 - Appl. Environ. Microbiol., 9: in press

    Abstract: 

    Marine algae produce a variety of glycans, which fulfill diverse biological functions and fuel the carbon and energy demands of heterotrophic microbes. A common approach to analysis of marine organic matter uses acid to hydrolyze the glycans into measurable monosaccharides. The monosaccharides may be derived from different glycans that are built with the same monosaccharides, however, and this approach does not distinguish between glycans in natural samples. Here we use enzymes to digest selectively and thereby quantify laminarin in particulate organic matter. Environmental metaproteome data revealed carbohydrate-active enzymes from marine flavobacteria as tools for selective hydrolysis of the algal β-glucan laminarin. The enzymes digested laminarin into glucose and oligosaccharides, which we measured with standard methods to establish the amounts of laminarin in the samples. We cloned, expressed, purified, and characterized three new glycoside hydrolases (GHs) of bacteria: two are endo-β-1,3-glucanases, of the GH16 and GH17 families, and the other is a GH30 exo-β-1,6-glucanase. sp. nov strain Hel1_33_131 GH30 (FbGH30) removed the β-1,6-glucose side chains, and GH17A (FaGH17A) and FaGH16A hydrolyzed the β-1,3-glucose backbone of laminarin. Specificity profiling with a library of glucan oligosaccharides and polysaccharides revealed that FaGH17A and FbGH30 were highly specific enzymes, while FaGH16A also hydrolyzed mixed-linked glucans with β-1,4-glucose. Therefore, we chose the more specific FaGH17A and FbGH30 to quantify laminarin in two cultured diatoms, namely, and , and in seawater samples from the North Sea and the Arctic Ocean. Combined, these results demonstrate the potential of enzymes for faster, stereospecific, and sequence-specific analysis of select glycans in marine organic matter. Marine algae synthesize substantial amounts of the glucose polymer laminarin for energy and carbon storage. Its concentrations, rates of production by autotrophic organisms, and rates of digestion by heterotrophic organisms remain unknown. Here we present a method based on enzymes that hydrolyze laminarin and enable its quantification even in crude substrate mixtures, without purification. Compared to the commonly used acid hydrolysis, the enzymatic method presented here is faster and stereospecific and selectively cleaves laminarin in mixtures of glycans, releasing only glucose and oligosaccharides, which can be easily quantified with reducing sugar assays.

  • A Mobile Element in mutS Drives Hypermutation in a Marine Vibrio.

    Chu ND, Clarke SA, Timberlake S, Polz MF, Grossman AD, Alm EJ
    2017 - mBio, 1: in press

    Abstract: 

    Bacteria face a trade-off between genetic fidelity, which reduces deleterious mistakes in the genome, and genetic innovation, which allows organisms to adapt. Evidence suggests that many bacteria balance this trade-off by modulating their mutation rates, but few mechanisms have been described for such modulation. Following experimental evolution and whole-genome resequencing of the marine bacterium Vibrio splendidus 12B01, we discovered one such mechanism, which allows this bacterium to switch to an elevated mutation rate. This switch is driven by the excision of a mobile element residing in mutS, which encodes a DNA mismatch repair protein. When integrated within the bacterial genome, the mobile element provides independent promoter and translation start sequences for mutS-different from the bacterium's original mutS promoter region-which allow the bacterium to make a functional mutS gene product. Excision of this mobile element rejoins the mutS gene with host promoter and translation start sequences but leaves a 2-bp deletion in the mutS sequence, resulting in a frameshift and a hypermutator phenotype. We further identified hundreds of clinical and environmental bacteria across Betaproteobacteria and Gammaproteobacteria that possess putative mobile elements within the same amino acid motif in mutS In a subset of these bacteria, we detected excision of the element but not a frameshift mutation; the mobile elements leave an intact mutS coding sequence after excision. Our findings reveal a novel mechanism by which one bacterium alters its mutation rate and hint at a possible evolutionary role for mobile elements within mutS in other bacteria.
    DNA mutations are a double-edged sword. Most mutations are harmful; they can scramble precise genetic sequences honed over thousands of generations. However, in rare cases, mutations also produce beneficial new traits that allow populations to adapt to changing environments. Recent evidence suggests that some bacteria balance this trade-off by altering their mutation rates to suit their environment. To date, however, we know of few mechanisms that allow bacteria to change their mutation rates. We describe one such mechanism, driven by the action of a mobile element, in the marine bacterium Vibrio splendidus 12B01. We also found similar mobile genetic sequences in the mutS genes of many different bacteria, including clinical and agricultural pathogens. These mobile elements might play an as yet unknown role in the evolution of these important bacteria.

  • Physiological and gene expression responses to nitrogen regimes and temperatures in Mastigocladus sp. strain CHP1, a predominant thermotolerant cyanobacterium of hot springs.

    Alcamán ME, Alcorta J, Bergman B, Vásquez M, Polz MF, Díez B
    2017 - Syst. Appl. Microbiol., 2: 102-113

    Abstract: 

    Cyanobacteria are widely distributed primary producers with significant implications for the global biogeochemical cycles of carbon and nitrogen. Diazotrophic cyanobacteria of subsection V (Order Stigonematales) are particularly ubiquitous in photoautotrophic microbial mats of hot springs. The Stigonematal cyanobacterium strain CHP1 isolated from the Porcelana hot spring (Chile) was one of the major contributors of the new nitrogen through nitrogen fixation. Further morphological and genetic characterization verified that the strain CHP1 belongs to Stigonematales, and it formed a separate clade together with other thermophiles of the genera Fischerella and Mastigocladus. Strain CHP1 fixed maximum N in the light, independent of the temperature range. At 50°C nifH gene transcripts showed high expression during the light period, whereas the nifH gene expression at 45°C was arrhythmic. The strain displayed a high affinity for nitrate and a low tolerance for high ammonium concentrations, whereas the narB and glnA genes showed higher expression in light and at the beginning of the dark phase. It is proposed that Mastigocladus sp. strain CHP1 would represent a good model for the study of subsection V thermophilic cyanobacteria, and for understanding the adaptations of these photoautotrophic organisms inhabiting microbial mats in hot springs globally.

  • Endemicity of the cosmopolitan mesophilic chemolithoautotroph Sulfurimonas at deep-sea hydrothermal vents.

    Mino S, Nakagawa S, Makita H, Toki T, Miyazaki J, Sievert SM, Polz MF, Inagaki F, Godfroy A, Kato S, Watanabe H, Nunoura T, Nakamura K, Imachi H, Watsuji TO, Kojima S, Takai K, Sawabe T
    2017 - ISME J, 4: 909-919

    Abstract: 

    Rich animal and microbial communities have been found at deep-sea hydrothermal vents. Although the biogeography of vent macrofauna is well understood, the corresponding knowledge about vent microbial biogeography is lacking. Here, we apply the multilocus sequence analysis (MLSA) to assess the genetic variation of 109 Sulfurimonas strains with ⩾98% 16S rRNA gene sequence similarity, which were isolated from four different geographical regions (Okinawa Trough (OT), Mariana Volcanic Arc and Trough (MVAT), Central Indian Ridge (CIR) and Mid-Atlantic Ridge (MAR)). Sequence typing based on 11 protein-coding genes revealed high genetic variation, including some allele types that are widespread within regions, resulting in 102 nucleotide sequence types (STs). This genetic variation was predominantly due to mutation rather than recombination. Phylogenetic analysis of the 11 concatenated genes showed a clear geographical isolation corresponding to the hydrothermal regions they originated from, suggesting limited dispersal. Genetic differentiation among Sulfurimonas populations was primarily influenced by geographical distance rather than gas composition of vent fluid or habitat, although in situ environmental conditions of each microhabitat could not be examined. Nevertheless, Sulfurimonas may possess a higher dispersal capability compared with deep-sea hydrothermal vent thermophiles. This is the first report on MLSA of deep-sea hydrothermal vent Epsilonproteobacteria, which is indicative of allopatric speciation.

  • Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition.

    Bruto M, James A, Petton B, Labreuche Y, Chenivesse S, Alunno-Bruscia M, Polz MF, Le Roux F
    2017 - ISME J, 4: 1043-1052

    Abstract: 

    Vibrios are frequently associated with oyster mortality; however whether they are the primary causative agent or secondary opportunistic colonizers is not well understood. Here we combine analysis of natural infection dynamics, population genomics and molecular genetics to ask (i) to what extent oysters are passively colonized by Vibrio population present in the surrounding water, (ii) how populations turn over during pathogenicity events and (iii) what genetic factors are responsible for pathogenicity. We identified several populations of Vibrio preferentially associated with oyster tissues. Among these, Vibrio crassostreae is particularly abundant in diseased animals while nearly absent in the surrounding water, and its pathogenicity is correlated with the presence of a large mobilizable plasmid. We further demonstrate that the plasmid is essential for killing but not necessary for survival in tissues of oysters. Our results suggest that V. crassostreae first differentiated into a benign oyster colonizer that was secondarily turned into a pathogen by introgression of a virulence plasmid into the population, possibly facilitated by elevated host density in farming areas.

  • Direct single-cell biomass estimates for marine bacteria via Archimedes' principle.

    Cermak N, Becker JW, Knudsen SM, Chisholm SW, Manalis SR, Polz MF
    2017 - ISME J, 3: 825-828

    Abstract: 

    Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell's biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean.

  • Evidence for Ecological Flexibility in the Cosmopolitan Genus Curtobacterium.

    Chase AB, Arevalo P, Polz MF, Berlemont R, Martiny JB
    2016 - Front Microbiol, 1874

    Abstract: 

    Assigning ecological roles to bacterial taxa remains imperative to understanding how microbial communities will respond to changing environmental conditions. Here we analyze the genus , as it was found to be the most abundant taxon in a leaf litter community in southern California. Traditional characterization of this taxon predominantly associates it as the causal pathogen in the agricultural crops of dry beans. Therefore, we sought to investigate whether the abundance of this genus was because of its role as a plant pathogen or another ecological role. By collating >24,000 16S rRNA sequences with 120 genomes across the Microbacteriaceae family, we show that has a global distribution with a predominant presence in soil ecosystems. Moreover, this genus harbors a high diversity of genomic potential for the degradation of carbohydrates, specifically with regards to structural polysaccharides. We conclude that may be responsible for the degradation of organic matter within litter communities.

  • Bacterial evolution: Genomics of metabolic trade-offs.

    Polz MF, Cordero OX
    2016 - Nat Microbiol, 11: 16181

    Abstract: 

    The number of ribosomal operons in bacterial genomes correlates with both growth rate and carbon use efficiency, likely via proteome allocation trade-offs, providing clues into how microbial communities are structured to make best use of available nutrients.

  • Adaptive radiation by waves of gene transfer leads to fine-scale resource partitioning in marine microbes.

    Hehemann JH, Arevalo P, Datta MS, Yu X, Corzett CH, Henschel A, Preheim SP, Timberlake S, Alm EJ, Polz MF
    2016 - Nat Commun, 12860

    Abstract: 

    Adaptive radiations are important drivers of niche filling, since they rapidly adapt a single clade of organisms to ecological opportunities. Although thought to be common for animals and plants, adaptive radiations have remained difficult to document for microbes in the wild. Here we describe a recent adaptive radiation leading to fine-scale ecophysiological differentiation in the degradation of an algal glycan in a clade of closely related marine bacteria. Horizontal gene transfer is the primary driver in the diversification of the pathway leading to several ecophysiologically differentiated Vibrionaceae populations adapted to different physical forms of alginate. Pathway architecture is predictive of function and ecology, underscoring that horizontal gene transfer without extensive regulatory changes can rapidly assemble fully functional pathways in microbes.

  • A Small Number of Phylogenetically Distinct Clonal Complexes Dominate a Coastal Vibrio cholerae Population.

    Kirchberger PC, Orata FD, Barlow EJ, Kauffman KM, Case RJ, Polz MF, Boucher Y
    2016 - Appl. Environ. Microbiol., 18: 5576-86

    Abstract: 

    Vibrio cholerae is a ubiquitous aquatic microbe in temperate and tropical coastal areas. It is a diverse species, with many isolates that are harmless to humans, while others are highly pathogenic. Most notable among them are strains belonging to the pandemic O1/O139 serogroup lineage, which contains the causative agents of cholera. The environmental selective regimes that led to this diversity are key to understanding how pathogens evolve in environmental reservoirs. A local population of V. cholerae and its close relative Vibrio metoecus from a coastal pond and lagoon system was extensively sampled during two consecutive months across four size fractions (480 isolates). In stark contrast to previous studies, the observed population was highly clonal, with 60% of V. cholerae isolates falling into one of five clonal complexes, which varied in abundance in the short temporal scale sampled. V. cholerae clonal complexes had significantly different distributions across size fractions and the two environments sampled, the pond and the lagoon. Sequencing the genomes of 20 isolates representing these five V. cholerae clonal complexes revealed different evolutionary trajectories, with considerable variations in gene content with potential ecological significance. Showing genotypic differentiation and differential spatial distribution, the dominant clonal complexes are likely ecologically divergent. Temporal variation in the relative abundance of these complexes suggests that transient blooms of specific clones could dominate local diversity.
    Vibrio cholerae is commonly found in coastal areas worldwide, with only a single group of this bacterium capable of causing severe cholera outbreaks. However, the potential to evolve the ability to cause disease exists in many strains of this species in its aquatic reservoir. Understanding how pathogenic bacteria evolve requires the study of their natural environments. By extensive sampling in a geographically restricted location in the United States, we found that most cells of a V. cholerae population belong to only a small number of strains. Analysis of their genome composition and spatial distribution indicates differential environmental adaptations between these strains. Other strains exist in smaller numbers, and the population was found to be temporally varied. This suggests frequent bloom and collapse cycles on a time scale of weeks. These population dynamics make it possible that more virulent strains could stochastically rise to large numbers, allowing for infection to occur.

  • Population genomics of the symbiotic plasmids of sympatric nitrogen-fixing Rhizobium species associated with Phaseolus vulgaris.

    Pérez Carrascal OM, VanInsberghe D, Juárez S, Polz MF, Vinuesa P, González V
    2016 - Environ. Microbiol., 8: 2660-76

    Abstract: 

    Cultivated common beans are the primary protein source for millions of people around the world who subsist on low-input agriculture, enabled by the symbiotic N2 -fixation these legumes perform in association with rhizobia. Within a single agricultural plot, multiple Rhizobium species can nodulate bean roots, but it is unclear how genetically isolated these species remain in sympatry. To better understand this issue, we sequenced and compared the genomes of 33 strains isolated from the rhizosphere and root nodules of a particular bean variety grown in the same agricultural plot. We found that the Rhizobium species we observed coexist with low genetic recombination across their core genomes. Accessory plasmids thought to be necessary for the saprophytic lifestyle in soil show similar levels of genetic isolation, but with higher rates of recombination than the chromosomes. However, the symbiotic plasmids are extremely similar, with high rates of recombination and do not appear to have co-evolved with the chromosome or accessory plasmids. Therefore, while Rhizobium species are genetically isolated units within the microbial community, a common symbiotic plasmid allows all Rhizobium species to engage in symbiosis with the same host in a single agricultural plot.

  • Microbial interactions lead to rapid micro-scale successions on model marine particles.

    Datta MS, Sliwerska E, Gore J, Polz MF, Cordero OX
    2016 - Nat Commun, 11965

    Abstract: 

    In the ocean, organic particles harbour diverse bacterial communities, which collectively digest and recycle essential nutrients. Traits like motility and exo-enzyme production allow individual taxa to colonize and exploit particle resources, but it remains unclear how community dynamics emerge from these individual traits. Here we track the taxon and trait dynamics of bacteria attached to model marine particles and demonstrate that particle-attached communities undergo rapid, reproducible successions driven by ecological interactions. Motile, particle-degrading taxa are selected for during early successional stages. However, this selective pressure is later relaxed when secondary consumers invade, which are unable to use the particle resource but, instead, rely on carbon from primary degraders. This creates a trophic chain that shifts community metabolism away from the particle substrate. These results suggest that primary successions may shape particle-attached bacterial communities in the ocean and that rapid community-wide metabolic shifts could limit rates of marine particle degradation.

  • A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior.

    Bongrand C, Koch EJ, Moriano-Gutierrez S, Cordero OX, McFall-Ngai M, Polz MF, Ruby EG
    2016 - ISME J, 12: 2907-2917

    Abstract: 

    Newly hatched Euprymna scolopes squid obtain their specific light-organ symbionts from an array of Vibrio (Allivibrio) fischeri strains present in their environment. Two genetically distinct populations of this squid species have been identified, one in Kaneohe Bay (KB), and another in Maunaloa Bay (MB), Oahu. We asked whether symbionts isolated from squid in each of these populations outcompete isolates from the other population in mixed-infection experiments. No relationship was found between a strain's host source (KB or MB) and its ability to competitively colonize KB or MB juveniles in a mixed inoculum. Instead, two colonization behaviors were identified among the 11 KB and MB strains tested: a 'dominant' outcome, in which one strain outcompetes the other for colonization, and a 'sharing' outcome, in which two strains co-colonize the squid. A genome-level comparison of these and other V. fischeri strains suggested that the core genomic structure of this species is both syntenous and highly conserved over time and geographical distance. We also identified ~250 Kb of sequence, encoding 194 dispersed orfs, that was specific to those strains that expressed the dominant colonization behavior. Taken together, the results indicate a link between the genome content of V. fischeri strains and their colonization behavior when initiating a light-organ symbiosis.

  • Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals.

    Le Roux F, Wegner KM, Polz MF
    2016 - Trends Microbiol., 7: 568-580

    Abstract: 

    Disease dynamics in the wild are influenced by a number of ecological and evolutionary factors not addressed by traditional laboratory-based characterization of pathogens. Here we propose the oyster, Crassostrea gigas, as a model for studying the interaction of the environment, bacterial pathogens, and the host in disease dynamics. We show that an important first step is to ask whether the functional unit of pathogenesis is a bacterial clone, a population, or a consortium in order to assess triggers of disease outbreaks and devise appropriate monitoring tools. Moreover, the development of specific-pathogen-free (SPF) oysters has enabled assessment of the infection process under natural conditions. Finally, recent results show the importance of microbial interactions and host genetics in determining oyster health and disease.

  • Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population.

    Klose J, Polz MF, Wagner M, Schimak MP, Gollner S, Bright M
    2015 - Proc. Natl. Acad. Sci. U.S.A., 36: 11300-11305

    Abstract: 

    Theory predicts that horizontal acquisition of symbionts by plants and animals must be coupled to release and limited dispersal of symbionts for intergenerational persistence of mutualisms. For deep-sea hydrothermal vent tubeworms (Vestimentifera, Siboglinidae), it has been demonstrated that a few symbiotic bacteria infect aposymbiotic host larvae and grow in a newly formed organ, the trophosome. However, whether viable symbionts can be released to augment environmental populations has been doubtful, because (i) the adult worms lack obvious openings and (ii) the vast majority of symbionts has been regarded as terminally differentiated. Here we show experimentally that symbionts rapidly escape their hosts upon death and recruit to surfaces where they proliferate. Estimating symbiont release from our experiments taken together with well-known tubeworm density ranges, we suggest a few million to 1.5 billion symbionts seeding the environment upon death of a tubeworm clump. In situ observations show that such clumps have rapid turnover, suggesting that release of large numbers of symbionts may ensure effective dispersal to new sites followed by active larval colonization. Moreover, release of symbionts might enable adaptations that evolve within host individuals to spread within host populations and possibly to new environments.

  • Eco-Evolutionary Dynamics of Episomes among Ecologically Cohesive Bacterial Populations.

    Xue H, Cordero OX, Camas FM, Trimble W, Meyer F, Guglielmini J, Rocha EP, Polz MF
    2015 - mBio, 3: e00552-15

    Abstract: 

    Although plasmids and other episomes are recognized as key players in horizontal gene transfer among microbes, their diversity and dynamics among ecologically structured host populations in the wild remain poorly understood. Here, we show that natural populations of marine Vibrionaceae bacteria host large numbers of families of episomes, consisting of plasmids and a surprisingly high fraction of plasmid-like temperate phages. Episomes are unevenly distributed among host populations, and contrary to the notion that high-density communities in biofilms act as hot spots of gene transfer, we identified a strong bias for episomes to occur in free-living as opposed to particle-attached cells. Mapping of episomal families onto host phylogeny shows that, with the exception of all phage and a few plasmid families, most are of recent evolutionary origin and appear to have spread rapidly by horizontal transfer. Such high eco-evolutionary turnover is particularly surprising for plasmids that are, based on previously suggested categorization, putatively nontransmissible, indicating that this type of plasmid is indeed frequently transferred by currently unknown mechanisms. Finally, analysis of recent gene transfer among plasmids reveals a network of extensive exchange connecting nearly all episomes. Genes functioning in plasmid transfer and maintenance are frequently exchanged, suggesting that plasmids can be rapidly transformed from one category to another. The broad distribution of episomes among distantly related hosts and the observed promiscuous recombination patterns show how episomes can offer their hosts rapid assembly and dissemination of novel functions.
    Plasmids and other episomes are an integral part of bacterial biology in all environments, yet their study is heavily biased toward their role as vectors for antibiotic resistance genes. This study presents a comprehensive analysis of all episomes within several coexisting bacterial populations of Vibrionaceae from the coastal ocean and represents the largest-yet genomic survey of episomes from a single bacterial family. The host population framework allows analysis of the eco-evolutionary dynamics at unprecedented resolution, yielding several unexpected results. These include (i) discovery of novel, nonintegrative temperate phages, (ii) revision of a class of episomes, previously termed "nontransmissible," as highly transmissible, and (iii) surprisingly high evolutionary turnover of episomes, manifest as frequent birth, spread, and loss.

  • Microbial taxonomy in the post-genomic era: rebuilding from scratch?

    Thompson CC, Amaral GR, Campeão M, Edwards RA, Polz MF, Dutilh BE, Ussery DW, Sawabe T, Swings J, Thompson FL
    2015 - Arch. Microbiol., 3: 359-70

    Abstract: 

    Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. Its cornerstone, the prokaryote species has been re-evaluated twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. Ultimately, we will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.

  • Vibrio metoecus sp. nov., a close relative of Vibrio cholerae isolated from coastal brackish ponds and clinical specimens.

    Kirchberger PC, Turnsek M, Hunt DE, Haley BJ, Colwell RR, Polz MF, Tarr CL, Boucher Y
    2014 - Int. J. Syst. Evol. Microbiol., 9: 3208-14

    Abstract: 

    A Gram-staining-negative, curved-rod-shaped bacterium with close resemblance to Vibrio cholerae, the aetiological agent of cholera, was isolated over the course of several years from coastal brackish water (17 strains) and from clinical cases (two strains) in the United States. 16S rRNA gene identity with V. cholerae exceeded 98 % yet an average nucleotide identity based on genome data of around 86 % and multi locus sequence analysis of six housekeeping genes (mdh, adk, gyrB, recA, pgi and rpoB) clearly delineated these isolates as a distinct genotypic cluster within the V. cholerae-V. mimicus clade. Most standard identification techniques do not differentiate this cluster of isolates from V. cholerae. Only amplification of the ompW gene using V. cholerae-specific primers and a negative Voges-Proskauer test showed a difference between the two clusters. Additionally, all isolated strains differed phenotypically from V. cholerae in their ability to utilize N-acetyl-d-galactosamine and d-glucuronic acid as sole carbon sources. Furthermore, they were generally unable to infect the slime mould Dictyostelium discoideum, a widespread ability in V. cholerae. Based on these clear phenotypic differences that are not necessarily apparent in standard tests as well as average nucleotide identity and phylogeny of protein-coding genes, we propose the existence of a novel species, Vibrio metoecus sp. nov. with the type strain OP3H(T) ( = LMG 27764(T) = CIP 110643(T)). Due to its close resemblance to V. cholerae and the increasing number of strains isolated over the past several years, we suggest that V. metoecus sp. nov. is a relatively common species of the genus Vibrio, isolates of which have been identified as atypical isolates of V. cholerae in the past. Its isolation from clinical samples also indicates that strains of this species, like V. cholerae, are opportunistic pathogens.

  • Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope, temperature, and pH adaptations.

    Jagtap SS, Hehemann JH, Polz MF, Lee JK, Zhao H
    2014 - Appl. Environ. Microbiol., 14: 4207-14

    Abstract: 

    Marine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) from Vibrio splendidus 12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-β-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showed kcat/Km from 2,130 mg(-1) ml s(-1) for the trisaccharide to 224 mg(-1) ml s(-1) for larger oligomers of ∼50 residues, and 50.5 mg(-1) ml s(-1) for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.

  • Competition-dispersal tradeoff ecologically differentiates recently speciated marine bacterioplankton populations.

    Yawata Y, Cordero OX, Menolascina F, Hehemann JH, Polz MF, Stocker R
    2014 - Proc. Natl. Acad. Sci. U.S.A., 15: 5622-7

    Abstract: 

    Although competition-dispersal tradeoffs are commonly invoked to explain species coexistence for animals and plants in spatially structured environments, such mechanisms for coexistence remain unknown for microorganisms. Here we show that two recently speciated marine bacterioplankton populations pursue different behavioral strategies to exploit nutrient particles in adaptation to the landscape of ephemeral nutrient patches characteristic of ocean water. These differences are mediated primarily by differential colonization of and dispersal among particles. Whereas one population is specialized to colonize particles by attaching and growing biofilms, the other is specialized to disperse among particles by rapidly detecting and swimming toward new particles, implying that it can better exploit short-lived patches. Because the two populations are very similar in their genomic composition, metabolic abilities, chemotactic sensitivity, and swimming speed, this fine-scale behavioral adaptation may have been responsible for the onset of the ecological differentiation between them. These results demonstrate that the principles of spatial ecology, traditionally applied at macroscales, can be extended to the ocean's microscale to understand how the rich spatiotemporal structure of the resource landscape contributes to the fine-scale ecological differentiation and species coexistence among marine bacteria.

  • Ordering microbial diversity into ecologically and genetically cohesive units.

    Shapiro BJ, Polz MF
    2014 - Trends Microbiol., 5: 235-47

    Abstract: 

    We propose that microbial diversity must be viewed in light of gene flow and selection, which define units of genetic similarity, and of phenotype and ecological function, respectively. We discuss to what extent ecological and genetic units overlap to form cohesive populations in the wild, based on recent evolutionary modeling and on evidence from some of the first microbial populations studied with genomics. These show that if recombination is frequent and selection moderate, ecologically adaptive mutations or genes can spread within populations independently of their original genomic background (gene-specific sweeps). Alternatively, if the effect of recombination is smaller than selection, genome-wide selective sweeps should occur. In both cases, however, distinct units of overlapping ecological and genotypic similarity will form if microgeographic separation, likely involving ecological tradeoffs, induces barriers to gene flow. These predictions are supported by (meta)genomic data, which suggest that a 'reverse ecology' approach, in which genomic and gene flow information is used to make predictions about the nature of ecological units, is a powerful approach to ordering microbial diversity.

  • Explaining microbial genomic diversity in light of evolutionary ecology.

    Cordero OX, Polz MF
    2014 - Nat. Rev. Microbiol., 4: 263-73

    Abstract: 

    Comparisons of closely related microorganisms have shown that individual genomes can be highly diverse in terms of gene content. In this Review, we discuss several studies showing that much of this variation is associated with social and ecological interactions, which have an important role in the population biology of wild populations of bacteria and archaea. These interactions create frequency-dependent selective pressures that can either stabilize gene frequencies at intermediate levels in populations or promote fast gene turnover, which presents as low gene frequencies in genome surveys. Thus, interpretation of gene-content diversity requires the delineation of populations according to cohesive gene flow and ecology, as micro-evolutionary changes arise in response to local selection pressures and population dynamics.

  • Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level.

    Takemura AF, Chien DM, Polz MF
    2014 - Front Microbiol, 38

    Abstract: 

    The Vibrionaceae, which encompasses several potential pathogens, including V. cholerae, the causative agent of cholera, and V. vulnificus, the deadliest seafood-borne pathogen, are a well-studied family of marine bacteria that thrive in a diverse habitats. To elucidate the environmental conditions under which vibrios proliferate, numerous studies have examined correlations with bulk environmental variables-e.g., temperature, salinity, nitrogen, and phosphate-and association with potential host organisms. However, how meaningful these environmental associations are remains unclear because data are fragmented across studies with variable sampling and analysis methods. Here, we synthesize findings about Vibrio correlations and physical associations using a framework of increasingly fine environmental and taxonomic scales, to better understand their dynamics in the wild. We first conduct a meta-analysis to determine trends with respect to bulk water environmental variables, and find that while temperature and salinity are generally strongly predictive correlates, other parameters are inconsistent and overall patterns depend on taxonomic resolution. Based on the hypothesis that dynamics may better correlate with more narrowly defined niches, we review evidence for specific association with plants, algae, zooplankton, and animals. We find that Vibrio are attached to many organisms, though evidence for enrichment compared to the water column is often lacking. Additionally, contrary to the notion that they flourish predominantly while attached, Vibrio can have, at least temporarily, a free-living lifestyle and even engage in massive blooms. Fine-scale sampling from the water column has enabled identification of such lifestyle preferences for ecologically cohesive populations, and future efforts will benefit from similar analysis at fine genetic and environmental sampling scales to describe the conditions, habitats, and resources shaping Vibrio dynamics.

  • Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits.

    Goudenège D, Labreuche Y, Krin E, Ansquer D, Mangenot S, Calteau A, Médigue C, Mazel D, Polz MF, Le Roux F
    2013 - ISME J, 10: 1985-96

    Abstract: 

    Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed 'nigritoxin', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.

  • Horizontal gene transfer and the evolution of bacterial and archaeal population structure.

    Polz MF, Alm EJ, Hanage WP
    2013 - Trends Genet., 3: 170-5

    Abstract: 

    Many bacterial and archaeal lineages have a history of extensive and ongoing horizontal gene transfer and loss, as evidenced by the large differences in genome content even among otherwise closely related isolates. How ecologically cohesive populations might evolve and be maintained under such conditions of rapid gene turnover has remained controversial. Here we synthesize recent literature demonstrating the importance of habitat and niche in structuring horizontal gene transfer. This leads to a model of ecological speciation via gradual genetic isolation triggered by differential habitat-association of nascent populations. Further, we hypothesize that subpopulations can evolve through local gene-exchange networks by tapping into a gene pool that is adaptive towards local, continuously changing organismic interactions and is, to a large degree, responsible for the observed rapid gene turnover. Overall, these insights help to explain how bacteria and archaea form populations that display both ecological cohesion and high genomic diversity.

  • Reproducibility of Vibrionaceae population structure in coastal bacterioplankton.

    Szabó G, Preheim SP, Kauffman KM, David LA, Shapiro J, Alm EJ, Polz MF
    2013 - ISME J, 3: 509-19

    Abstract: 

    How reproducibly microbial populations assemble in the wild remains poorly understood. Here, we assess evidence for ecological specialization and predictability of fine-scale population structure and habitat association in coastal ocean Vibrionaceae across years. We compare Vibrionaceae lifestyles in the bacterioplankton (combinations of free-living, particle, or zooplankton associations) measured using the same sampling scheme in 2006 and 2009 to assess whether the same groups show the same environmental association year after year. This reveals complex dynamics with populations falling primarily into two categories: (i) nearly equally represented in each of the two samplings and (ii) highly skewed, often to an extent that they appear exclusive to one or the other sampling times. Importantly, populations recovered at the same abundance in both samplings occupied highly similar habitats suggesting predictable and robust environmental association while skewed abundances of some populations may be triggered by shifts in ecological conditions. The latter is supported by difference in the composition of large eukaryotic plankton between years, with samples in 2006 being dominated by copepods, and those in 2009 by diatoms. Overall, the comparison supports highly predictable population-habitat linkage but highlights the fact that complex, and often unmeasured, environmental dynamics in habitat occurrence may have strong effects on population dynamics.

  • Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations.

    Cordero OX, Ventouras LA, DeLong EF, Polz MF
    2012 - Proc. Natl. Acad. Sci. U.S.A., 49: 20059-64

    Abstract: 

    A common strategy among microbes living in iron-limited environments is the secretion of siderophores, which can bind poorly soluble iron and make it available to cells via active transport mechanisms. Such siderophore-iron complexes can be thought of as public goods that can be exploited by local communities and drive diversification, for example by the evolution of "cheating." However, it is unclear whether bacterial populations in the environment form stable enough communities such that social interactions significantly impact evolutionary dynamics. Here we show that public good games drive the evolution of iron acquisition strategies in wild populations of marine bacteria. We found that within nonclonal but ecologically cohesive genotypic clusters of closely related Vibrionaceae, only an intermediate percentage of genotypes are able to produce siderophores. Nonproducers within these clusters exhibited selective loss of siderophore biosynthetic pathways, whereas siderophore transport mechanisms were retained, suggesting that these nonproducers can act as cheaters that benefit from siderophore producers in their local environment. In support of this hypothesis, these nonproducers in iron-limited media suffer a significant decrease in growth, which can be alleviated by siderophores, presumably owing to the retention of transport mechanisms. Moreover, using ecological data of resource partitioning, we found that cheating coevolves with the ecological specialization toward association with larger particles in the water column, suggesting that these can harbor stable enough communities for dependencies among organisms to evolve.

  • Ecological populations of bacteria act as socially cohesive units of antibiotic production and resistance.

    Cordero OX, Wildschutte H, Kirkup B, Proehl S, Ngo L, Hussain F, Le Roux F, Mincer T, Polz MF
    2012 - Science, 6099: 1228-31

    Abstract: 

    In animals and plants, social structure can reduce conflict within populations and bias aggression toward competing populations; however, for bacteria in the wild it remains unknown whether such population-level organization exists. Here, we show that environmental bacteria are organized into socially cohesive units in which antagonism occurs between rather than within ecologically defined populations. By screening approximately 35,000 possible mutual interactions among Vibrionaceae isolates from the ocean, we show that genotypic clusters known to have cohesive habitat association also act as units in terms of antibiotic production and resistance. Genetic analyses show that within populations, broad-range antibiotics are produced by few genotypes, whereas all others are resistant, suggesting cooperation between conspecifics. Natural antibiotics may thus mediate competition between populations rather than solely increase the success of individuals.

  • Shape and evolution of the fundamental niche in marine Vibrio.

    Materna AC, Friedman J, Bauer C, David C, Chen S, Huang IB, Gillens A, Clarke SA, Polz MF, Alm EJ
    2012 - ISME J, 12: 2168-77

    Abstract: 

    Hutchinson's fundamental niche, defined by the physical and biological environments in which an organism can thrive in the absence of inter-species interactions, is an important theoretical concept in ecology. However, little is known about the overlap between the fundamental niche and the set of conditions species inhabit in nature, and about natural variation in fundamental niche shape and its change as species adapt to their environment. Here, we develop a custom-made dual gradient apparatus to map a cross-section of the fundamental niche for several marine bacterial species within the genus Vibrio based on their temperature and salinity tolerance, and compare tolerance limits to the environment where these species commonly occur. We interpret these niche shapes in light of a conceptual model comprising five basic niche shapes. We find that the fundamental niche encompasses a much wider set of conditions than those strains typically inhabit, especially for salinity. Moreover, though the conditions that strains typically inhabit agree well with the strains' temperature tolerance, they are negatively correlated with the strains' salinity tolerance. Such relationships can arise when the physiological response to different stressors is coupled, and we present evidence for such a coupling between temperature and salinity tolerance. Finally, comparison with well-documented ecological range in V. vulnificus suggests that biotic interactions limit the occurrence of this species at low-temperature-high-salinity conditions. Our findings highlight the complex interplay between the ecological, physiological and evolutionary determinants of niche morphology, and caution against making inferences based on a single ecological factor.

  • Population genomics of early events in the ecological differentiation of bacteria.

    Shapiro BJ, Friedman J, Cordero OX, Preheim SP, Timberlake SC, Szabó G, Polz MF, Alm EJ
    2012 - Science, 6077: 48-51

    Abstract: 

    Genetic exchange is common among bacteria, but its effect on population diversity during ecological differentiation remains controversial. A fundamental question is whether advantageous mutations lead to selection of clonal genomes or, as in sexual eukaryotes, sweep through populations on their own. Here, we show that in two recently diverged populations of ocean bacteria, ecological differentiation has occurred akin to a sexual mechanism: A few genome regions have swept through subpopulations in a habitat-specific manner, accompanied by gradual separation of gene pools as evidenced by increased habitat specificity of the most recent recombinations. These findings reconcile previous, seemingly contradictory empirical observations of the genetic structure of bacterial populations and point to a more unified process of differentiation in bacteria and sexual eukaryotes than previously thought.

  • High frequency of a novel filamentous phage, VCY φ, within an environmental Vibrio cholerae population.

    Xue H, Xu Y, Boucher Y, Polz MF
    2012 - Appl. Environ. Microbiol., 1: 28-33

    Abstract: 

    Environmental Vibrio cholerae strains isolated from a coastal brackish pond (Oyster Pond, Woods Hole, MA) carried a novel filamentous phage, VCY, which can exist as a host genome integrative form (IF) and a plasmid-like replicative form (RF). Outside the cell, the phage displays a morphology typical of Inovirus, with filamentous particles ∼1.8 μm in length and 7 nm in width. Four independent RF isolates had identical genomes, except for 8 single nucleotide polymorphisms clustered in two regions. The overall genome size is 7,103 bp with 11 putative open reading frames organized into three functional modules (replication, structure and assembly, and regulation). VCY shares sequence similarity with other filamentous phages (including cholera disease-associated CTX) in a highly mosaic manner, indicating evolution by horizontal gene transfer and recombination. VCY integrates in the vicinity of the putative translation initiation factor Sui1 in chromosome II of V. cholerae. A screen of 531 closely related host isolates showed that ∼40% harbored phages, with 27% and 13% carrying the IF and RF, respectively. The relative frequencies of the RF and IF differed among strains isolated from the pond or lagoon of Oyster Pond, suggesting that the host habitat influences intracellular phage biology. The overall high prevalence within the host population shows that filamentous phages can be an important component of the environmental biology of V. cholerae.

  • Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion.

    Wollenberg MS, Preheim SP, Polz MF, Ruby EG
    2012 - Environ. Microbiol., 3: 655-68

    Abstract: 

    This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains of Vibrio fischeri. These 'dark' V. fischeri strains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light production in dim strains of luminous bacteria. Surprisingly, the entire lux locus (eight genes) was absent in over 97% of these dark V. fischeri strains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within the V. fischeri clade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the model V. fischeri host, the Hawaiian bobtail squid Euprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species.

  • Crystal ball: We need more model systems.

    Polz MF
    2011 - Environ. Microbiol. Reports, 3: 18-19
  • Merging taxonomy with ecological population prediction in a case study of Vibrionaceae.

    Preheim SP, Timberlake S, Polz MF
    2011 - Appl. Environ. Microbiol., 20: 7195-206

    Abstract: 

    We synthesized population structure data from three studies that assessed the fine-scale distribution of Vibrionaceae among temporally and spatially distinct environmental categories in coastal seawater and animals. All studies used a dynamic model (AdaptML) to identify phylogenetically cohesive and ecologically distinct bacterial populations and their predicted habitats without relying on a predefined genetic cutoff or relationships to previously named species. Across the three studies, populations were highly overlapping, displaying similar phylogenetic characteristics (identity and diversity), and were predominantly congruent with taxonomic Vibrio species previously characterized as genotypic clusters by multilocus sequence analysis (MLSA). The environmental fidelity of these populations appears high, with 9 out of 12 reproducibly associating with the same predicted (micro)habitats when similar environmental categories were sampled. Overall, this meta-analysis provides information on the habitat predictability and structure of previously described species, demonstrating that MLSA-based taxonomy can, at least in some cases, serve to approximate ecologically cohesive populations.

  • A simple and efficient method for concentration of ocean viruses by chemical flocculation.

    John SG, Mendez CB, Deng L, Poulos B, Kauffman AK, Kern S, Brum J, Polz MF, Boyle EA, Sullivan MB
    2011 - Environ Microbiol Rep, 2: 195-202

    Abstract: 

    Ocean viruses alter ecosystems through host mortality, horizontal gene transfer and by facilitating remineralization of limiting nutrients. However, the study of wild viral populations is limited by inefficient and unreliable concentration techniques. Here, we develop a new technique to recover viruses from natural waters using iron-based flocculation and large-pore-size filtration, followed by resuspension of virus-containing precipitates in a pH 6 buffer. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (> 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research.

  • Local mobile gene pools rapidly cross species boundaries to create endemicity within global Vibrio cholerae populations.

    Boucher Y, Cordero OX, Takemura A, Hunt DE, Schliep K, Bapteste E, Lopez P, Tarr CL, Polz MF
    2011 - mBio, 2: in press

    Abstract: 

    Vibrio cholerae represents both an environmental pathogen and a widely distributed microbial species comprised of closely related strains occurring in the tropical to temperate coastal ocean across the globe (Colwell RR, Science 274:2025-2031, 1996; Griffith DC, Kelly-Hope LA, Miller MA, Am. J. Trop. Med. Hyg. 75:973-977, 2006; Reidl J, Klose KE, FEMS Microbiol. Rev. 26:125-139, 2002). However, although this implies dispersal and growth across diverse environmental conditions, how locally successful populations assemble from a possibly global gene pool, relatively unhindered by geographic boundaries, remains poorly understood. Here, we show that environmental Vibrio cholerae possesses two, largely distinct gene pools: one is vertically inherited and globally well mixed, and the other is local and rapidly transferred across species boundaries to generate an endemic population structure. While phylogeographic analysis of isolates collected from Bangladesh and the U.S. east coast suggested strong panmixis for protein-coding genes, there was geographic structure in integrons, which are the only genomic islands present in all strains of V. cholerae (Chun J, et al., Proc. Natl. Acad. Sci. U. S. A. 106:15442-15447, 2009) and are capable of acquiring and expressing mobile gene cassettes. Geographic differentiation in integrons arises from high gene turnover, with acquisition from a locally co-occurring sister species being up to twice as likely as exchange with conspecific but geographically distant V. cholerae populations. IMPORTANCE Functional predictions of integron genes show the predominance of secondary metabolism and cell surface modification, which is consistent with a role in competition and predation defense. We suggest that the integron gene pool's distinctness and tempo of sharing are adaptive in allowing rapid conversion of genomes to reflect local ecological constraints. Because the integron is frequently the main element differentiating clinical strains (Chun J, et al., Proc. Natl. Acad. Sci. U. S. A. 106:15442-15447, 2009) and its recombinogenic activity is directly stimulated by environmental stresses (Guerin E, et al., Science 324:1034, 2009), these observations are relevant for local emergence and subsequent dispersal.

  • DNA phosphorothioation is widespread and quantized in bacterial genomes.

    Wang L, Chen S, Vergin KL, Giovannoni SJ, Chan SW, DeMott MS, Taghizadeh K, Cordero OX, Cutler M, Timberlake S, Alm EJ, Polz MF, Pinhassi J, Deng Z, Dedon PC
    2011 - Proc. Natl. Acad. Sci. U.S.A., 7: 2963-8

    Abstract: 

    Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes.

  • Metapopulation structure of Vibrionaceae among coastal marine invertebrates.

    Preheim SP, Boucher Y, Wildschutte H, David LA, Veneziano D, Alm EJ, Polz MF
    2011 - Environ. Microbiol., 1: 265-275

    Abstract: 

    Although animal-associated microbial communities (microbiomes) are increasingly recognized to influence health, the extent to which animals represent highly selective habitats for microbes leading to predominance of high host specificity remains poorly understood. Here, we show that vibrios, which are well-known commensals and opportunistic pathogens of marine animals, overall display little host preference, likely because of efficient dispersal-colonization dynamics mediated by food items. We isolated 1753 strains from water and animal samples, which are linked in a food chain and display different degrees of similarity (respiratory and digestive tract of mussels and crabs, live and dead zooplankton, and whole water samples). Multilocus sequence data served as input for modelling and statistical analysis of spatiotemporal population structure. These data showed that the majority of populations occurred broadly within and among hosts, with the dominant population being a near perfect generalist with regard to seasons, host taxa and body regions. Zooplankton harboured the fewest and most specific populations, while crabs and mussels contained the highest diversity with little evidence for host preferences. Most mussel- and crab-associated populations were detected in water samples at similar frequencies, particularly in filter-feeding mussels where populations were also evenly distributed across host individuals. The higher variation among individuals observed in crabs and zooplankton is consistent with stochastic clonal expansions. These patterns suggest that evolution of a high degree of host specificity is surprisingly rare even though these animals represent long-lived habitats, and vibrios are consistent members of their microbiome. Instead, many of the populations show stronger association with planktonic (micro)habitats while the microbiome may be a fairly open system for vibrios in which high rates of immigration can outpace selection for specialization.

  • O-antigen diversity and lateral transfer of the wbe region among Vibrio splendidus isolates.

    Wildschutte H, Preheim SP, Hernandez Y, Polz MF
    2010 - Environ. Microbiol., 11: 2977-87

    Abstract: 

    The O-antigen is a highly diverse structure expressed on the outer surface of Gram-negative bacteria. The products responsible for O-antigen synthesis are encoded in the wbe region, which exhibits extensive genetic diversity. While heterogeneous O-antigens are observed within Vibrio species, characterization of these structures has been devoted almost exclusively to pathogens. Here, we investigate O-antigen diversity among coastal marine Vibrio splendidus-like isolates. The wbe region was first identified and characterized using the sequenced genomes of strains LGP32, 12B01 and Med222. These regions were genetically diverse, reflective of their expressed O-antigen. Additional isolates from physically distinct habitats in Plum Island Estuary (MA, USA), including within animal hosts and on suspended particles, were further characterized based on multilocus sequence analysis (MLSA) and O-antigen profiles. Results showed serotype diversity within an ecological setting. Among 48 isolates which were identical in three MLSA genes, 41 showed gpm genetic diversity, a gene closely linked to the wbe locus, and at least 12 expressed different O-antigen profiles further suggesting wbe genetic diversity. Our results demonstrate O-antigen hyper-variability among these environmental strains and suggest that frequent lateral gene transfer generates wbe extensive diversity among V. splendidus and its close relatives.

  • Vibrio chromosomes share common history.

    Kirkup BC, Chang L, Chang S, Gevers D, Polz MF
    2010 - BMC Microbiol., 137

    Abstract: 

    While most gamma proteobacteria have a single circular chromosome, Vibrionales have two circular chromosomes. Horizontal gene transfer is common among Vibrios, and in light of this genetic mobility, it is an open question to what extent the two chromosomes themselves share a common history since their formation.
    Single copy genes from each chromosome (142 genes from chromosome I and 42 genes from chromosome II) were identified from 19 sequenced Vibrionales genomes and their phylogenetic comparison suggests consistent phylogenies for each chromosome. Additionally, study of the gene organization and phylogeny of the respective origins of replication confirmed the shared history.
    Thus, while elements within the chromosomes may have experienced significant genetic mobility, the backbones share a common history. This allows conclusions based on multilocus sequence analysis (MLSA) for one chromosome to be applied equally to both chromosomes.

  • Diversity of active marine picoeukaryotes in the Eastern Mediterranean Sea unveiled using photosystem-II psbA transcripts.

    Man-Aharonovich D, Philosof A, Kirkup BC, Le Gall F, Yogev T, Berman-Frank I, Polz MF, Vaulot D, Beja O
    2010 - ISME J, 8: 1044-52

    Abstract: 

    In vast areas of the oceans, most of the primary production is performed by cells smaller than 2-3 mum in diameter (picophytoplankton). In recent years, several in situ molecular studies showed a broad genetic diversity of small eukaryotes by sequencing 18S rRNA genes. Compared with photosynthetic cyanobacteria that are dominated by two genera, Prochlorococcus and Synechococcus, marine photosynthetic picoeukaryotes (PPEs) are much more diverse, with virtually every algal class being represented. However, the genetic diversity and ecology of PPEs are still poorly described. Here, we show using in situ molecular analyses of psbA transcripts that PPEs in the Eastern Mediterranean Sea are highly diverse, probably very active, and dominated by groups belonging to the red algal lineages, Haptophyta, Heterokontophyta (also called Stramenopiles), and Cryptophyta.

  • A supervised learning approach for taxonomic classification of core-photosystem-II genes and transcripts in the marine environment.

    Tzahor S, Man-Aharonovich D, Kirkup BC, Yogev T, Berman-Frank I, Polz MF, Beja O, Mandel-Gutfreund Y
    2009 - BMC Genomics, 229

    Abstract: 

    Cyanobacteria of the genera Synechococcus and Prochlorococcus play a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Recently, genes encoding the photosystem II reaction center (psbA and psbD) were found in cyanophage genomes. This phenomenon suggested that the horizontal transfer of these genes may be involved in increasing phage fitness. To date, a very small percentage of marine bacteria and phages has been cultured. Thus, mapping genomic data extracted directly from the environment to its taxonomic origin is necessary for a better understanding of phage-host relationships and dynamics.
    To achieve an accurate and rapid taxonomic classification, we employed a computational approach combining a multi-class Support Vector Machine (SVM) with a codon usage position specific scoring matrix (cuPSSM). Our method has been applied successfully to classify core-photosystem-II gene fragments, including partial sequences coming directly from the ocean, to seven different taxonomic classes. Applying the method on a large set of DNA and RNA psbA clones from the Mediterranean Sea, we studied the distribution of cyanobacterial psbA genes and transcripts in their natural environment. Using our approach, we were able to simultaneously examine taxonomic and ecological distributions in the marine environment.
    The ability to accurately classify the origin of individual genes and transcripts coming directly from the environment is of great importance in studying marine ecology. The classification method presented in this paper could be applied further to classify other genes amplified from the environment, for which training data is available.

  • ACID: annotation of cassette and integron data.

    Joss MJ, Koenig JE, Labbate M, Polz MF, Gillings MR, Stokes HW, Doolittle WF, Boucher Y
    2009 - BMC Bioinformatics, 118

    Abstract: 

    Although integrons and their associated gene cassettes are present in ~10% of bacteria and can represent up to 3% of the genome in which they are found, very few have been properly identified and annotated in public databases. These genetic elements have been overlooked in comparison to other vectors that facilitate lateral gene transfer between microorganisms.
    By automating the identification of integron integrase genes and of the non-coding cassette-associated attC recombination sites, we were able to assemble a database containing all publicly available sequence information regarding these genetic elements. Specialists manually curated the database and this information was used to improve the automated detection and annotation of integrons and their encoded gene cassettes. ACID (annotation of cassette and integron data) can be searched using a range of queries and the data can be downloaded in a number of formats. Users can readily annotate their own data and integrate it into ACID using the tools provided.
    ACID is a community resource providing easy access to annotations of integrons and making tools available to detect them in novel sequence data. ACID also hosts a forum to prompt integron-related discussion, which can hopefully lead to a more universal definition of this genetic element.

  • Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity.

    Le Roux F, Zouine M, Chakroun N, Binesse J, Saulnier D, Bouchier C, Zidane N, Ma L, Rusniok C, Lajus A, Buchrieser C, Médigue C, Polz MF, Mazel D
    2009 - Environ. Microbiol., 8: 1959-70

    Abstract: 

    Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.

  • The bacterial species challenge: making sense of genetic and ecological diversity.

    Fraser C, Alm EJ, Polz MF, Spratt BG, Hanage WP
    2009 - Science, 5915: 741-6

    Abstract: 

    The Bacteria and Archaea are the most genetically diverse superkingdoms of life, and techniques for exploring that diversity are only just becoming widespread. Taxonomists classify these organisms into species in much the same way as they classify eukaryotes, but differences in their biology-including horizontal gene transfer between distantly related taxa and variable rates of homologous recombination-mean that we still do not understand what a bacterial species is. This is not merely a semantic question; evolutionary theory should be able to explain why species exist at all levels of the tree of life, and we need to be able to define species for practical applications in industry, agriculture, and medicine. Recent studies have emphasized the need to combine genetic diversity and distinct ecology in an attempt to define species in a coherent and convincing fashion. The resulting data may help to discriminate among the many theories of prokaryotic species that have been produced to date.

  • The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs.

    Stewart JR, Gast RJ, Fujioka RS, Solo-Gabriele HM, Meschke JS, Amaral-Zettler LA, Del Castillo E, Polz MF, Collier TK, Strom MS, Sinigalliano CD, Moeller PD, Holland AF
    2008 - Environ Health, S3

    Abstract: 

    Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

  • Resource partitioning and sympatric differentiation among closely related bacterioplankton.

    Hunt DE, David LA, Gevers D, Preheim SP, Alm EJ, Polz MF
    2008 - Science, 5879: 1081-5

    Abstract: 

    Identifying ecologically differentiated populations within complex microbial communities remains challenging, yet is critical for interpreting the evolution and ecology of microbes in the wild. Here we describe spatial and temporal resource partitioning among Vibrionaceae strains coexisting in coastal bacterioplankton. A quantitative model (AdaptML) establishes the evolutionary history of ecological differentiation, thus revealing populations specific for seasons and life-styles (combinations of free-living, particle, or zooplankton associations). These ecological population boundaries frequently occur at deep phylogenetic levels (consistent with named species); however, recent and perhaps ongoing adaptive radiation is evident in Vibrio splendidus, which comprises numerous ecologically distinct populations at different levels of phylogenetic differentiation. Thus, environmental specialization may be an important correlate or even trigger of speciation among sympatric microbes.

  • Rapid chemotactic response enables marine bacteria to exploit ephemeral microscale nutrient patches.

    Stocker R, Seymour JR, Samadani A, Hunt DE, Polz MF
    2008 - Proc. Natl. Acad. Sci. U.S.A., 11: 4209-14

    Abstract: 

    Because ocean water is typically resource-poor, bacteria may gain significant growth advantages if they can exploit the ephemeral nutrient patches originating from numerous, small sources. Although this interaction has been proposed to enhance biogeochemical transformation rates in the ocean, it remains questionable whether bacteria are able to efficiently use patches before physical mechanisms dissipate them. Here we show that the rapid chemotactic response of the marine bacterium Pseudoalteromonas haloplanktis substantially enhances its ability to exploit nutrient patches before they dissipate. We investigated two types of patches important in the ocean: nutrient pulses and nutrient plumes, generated for example from lysed algae and sinking organic particles, respectively. We used microfluidic devices to create patches with environmentally realistic dimensions and dynamics. The accumulation of P. haloplanktis in response to a nutrient pulse led to formation of bacterial hot spots within tens of seconds, resulting in a 10-fold higher nutrient exposure for the fastest 20% of the population compared with nonmotile cells. Moreover, the chemotactic response of P. haloplanktis was >10 times faster than the classic chemotaxis model Escherichia coli, leading to twice the nutrient exposure. We demonstrate that such rapid response allows P. haloplanktis to colonize nutrient plumes for realistic particle sinking speeds, with up to a 4-fold nutrient exposure compared with nonmotile cells. These results suggest that chemotactic swimming strategies of marine bacteria in patchy nutrient seascapes exert strong influence on carbon turnover rates by triggering the formation of microscale hot spots of bacterial productivity.

  • Conservation of the chitin utilization pathway in the Vibrionaceae.

    Hunt DE, Gevers D, Vahora NM, Polz MF
    2008 - Appl. Environ. Microbiol., 1: 44-51

    Abstract: 

    Vibrionaceae are regarded as important marine chitin degraders, and attachment to chitin regulates important biological functions; yet, the degree of chitin pathway conservation in Vibrionaceae is unknown. Here, a core chitin degradation pathway is proposed based on comparison of 19 Vibrio and Photobacterium genomes with a detailed metabolic map assembled for V. cholerae from published biochemical, genomic, and transcriptomic results. Further, to assess whether chitin degradation is a conserved property of Vibrionaceae, a set of 54 strains from 32 taxa were tested for the ability to grow on various forms of chitin. All strains grew on N-acetylglucosamine (GlcNAc), the monomer of chitin. The majority of isolates grew on alpha (crab shell) and beta (squid pen) chitin and contained chitinase A (chiA) genes. chiA sequencing and phylogenetic analysis suggest that this gene is a good indicator of chitin metabolism but appears subject to horizontal gene transfer and duplication. Overall, chitin metabolism appears to be a core function of Vibrionaceae, but individual pathway components exhibit dynamic evolutionary histories.

  • Adaptation and spectral tuning in divergent marine proteorhodopsins from the eastern Mediterranean and the Sargasso Seas.

    Sabehi G, Kirkup BC, Rozenberg M, Stambler N, Polz MF, Beja O
    2007 - ISME J, 1: 48-55

    Abstract: 

    Proteorhodopsins (PRs) phototrophy was recently discovered in oceanic surface waters. PRs have been observed in different marine environments and in diverse taxa, including the ubiquitous marine alphaproteobacterial SAR11 group and the uncultured gammaproteobacterial SAR86 group. Previously, two SAR86 PR subgroups, discovered in the Pacific Ocean, were shown to absorb light with different maxima, lambda max 527 nm (green) and lambda max 490 nm (blue) and their distribution was explained by prevailing light conditions - green pigments at the surface and blue in deeper waters. Here, we show that PRs display high diversity in geographically distinct patterns despite similar physical water column properties such as mixing and light penetration. We compared summer and winter samples representing stratified and mixed conditions from both the Mediterranean and Sargasso Sea. As expected, in the Mediterranean Sea, green pigments were mainly confined to the surface and the percentage of blue pigments increased toward deeper samples; in the Sargasso Sea, unexpectedly, all PRs were of the blue type. As an additional result, both locations show seasonal dependence in the distribution of different PR families. Finally, spectral tuning was not restricted to a single PR family as previously reported but occurs across the sampled PR families from various microbial taxa. The distribution of tunable PRs across the PR tree suggests that ready adaptability has been distributed widely among microorganisms, and may be a reason that PRs are abundant and taxonomically widely dispersed.

  • CLUSTERER: extendable java application for sequence grouping and cluster analyses.

    Klepac-Ceraj V, Ceraj I, Polz MF
    2006 - Online J. Bioinf., 7: 15-21

    Abstract: 

    A Java application with a graphical user interface which assigns nucleic acid and protein sequences to clusters for each particular sequence divergence level, is described. The program provides graphical and tabular outputs of composition of clustered data, distributions of cluster sizes and rarefaction curves for all divergence levels. A summary of all sequence clusters for all sequence divergence levels is presented in a single graph, providing an overview of how sequences compose the clusters at a particular divergence level. Clusterer is available on UNIX, MacOS and Windows operating systems and is under MIT license. There are precompiled binaries for available for Java WebStart and as an applet as well as sources.

  • Patterns and mechanisms of genetic and phenotypic differentiation in marine microbes.

    Polz MF, Hunt DE, Preheim SP, Weinreich DM
    2006 - Philos. Trans. B., 1475: 2009-21

    Abstract: 

    Microbes in the ocean dominate biogeochemical processes and are far more diverse than anticipated. Thus, in order to understand the ocean system, we need to delineate microbial populations with predictable ecological functions. Recent observations suggest that ocean communities comprise diverse groups of bacteria organized into genotypic (and phenotypic) clusters of closely related organisms. Although such patterns are similar to metazoan communities, the underlying mechanisms for microbial communities may differ substantially. Indeed, the potential among ocean microbes for vast population sizes, extensive migration and both homologous and illegitimate genetic recombinations, which are uncoupled from reproduction, challenges classical population models primarily developed for sexually reproducing animals. We examine possible mechanisms leading to the formation of genotypic clusters and consider alternative population genetic models for differentiation at individual loci as well as gene content at the level of whole genomes. We further suggest that ocean bacteria follow at least two different adaptive strategies, which constrain rates and bounds of evolutionary processes: the 'opportunitroph', exploiting spatially and temporally variable resources; and the passive oligotroph, efficiently using low nutrient concentrations. These ecological lifestyle differences may represent a fundamental divide with major consequences for growth and predation rates, genome evolution and population diversity, as emergent properties driving the division of labour within microbial communities.

  • Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data.

    Marcelino LA, Backman V, Donaldson A, Steadman C, Thompson JR, Preheim SP, Lien C, Lim E, Veneziano D, Polz MF
    2006 - Proc. Natl. Acad. Sci. U.S.A., 37: 13629-34

    Abstract: 

    Microarrays have enabled the determination of how thousands of genes are expressed to coordinate function within single organisms. Yet applications to natural or engineered communities where different organisms interact to produce complex properties are hampered by theoretical and technological limitations. Here we describe a general method to accurately identify low-abundant targets in systems containing complex mixtures of homologous targets. We combined an analytical predictor of nonspecific probe-target interactions (cross-hybridization) with an optimization algorithm that iteratively deconvolutes true probe-target signal from raw signal affected by spurious contributions (cross-hybridization, noise, background, and unequal specific hybridization response). The method was capable of quantifying, with unprecedented specificity and accuracy, ribosomal RNA (rRNA) sequences in artificial and natural communities. Controlled experiments with spiked rRNA into artificial and natural communities demonstrated the accuracy of identification and quantitative behavior over different concentration ranges. Finally, we illustrated the power of this methodology for accurate detection of low-abundant targets in natural communities. We accurately identified Vibrio taxa in coastal marine samples at their natural concentrations (<0.05% of total bacteria), despite the high potential for cross-hybridization by hundreds of different coexisting rRNAs, suggesting this methodology should be expandable to any microarray platform and system requiring accurate identification of low-abundant targets amid pools of similar sequences.

  • Colonization dynamics of altered Schaedler flora is influenced by gender, aging, and Helicobacter hepaticus infection in the intestines of Swiss Webster mice.

    Ge Z, Feng Y, Taylor NS, Ohtani M, Polz MF, Schauer DB, Fox JG
    2006 - Appl. Environ. Microbiol., 7: 5100-3

    Abstract: 

    The distribution and colonization levels of the altered Schaedler flora (ASF) in their natural hosts are poorly understood. Intestinal colonization levels of the eight ASF strains in outbred Swiss Webster mice with or without Helicobacter hepaticus infection were characterized by real-time quantitative PCR. All ASF strains were detected in the cecum and colon, but some strains displayed significant variation in colonization levels with host age, gender, and H. hepaticus infection status.

  • Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of bacterial diversity.

    Hunt DE, Klepac-Ceraj V, Acinas SG, Gautier C, Bertilsson S, Polz MF
    2006 - Appl. Environ. Microbiol., 3: 2221-5

    Abstract: 

    The availability of a diverse set of 23S rRNA gene sequences enabled evaluation of the specificity of 39 previously published and 4 newly designed primers specific for bacteria. An extensive clone library constructed using an optimized primer pair resulted in similar gene richness but slightly differing coverage of some phylogenetic groups, compared to a 16S rRNA gene library from the same environmental sample.

  • Extensive variation in intracellular symbiont community composition among members of a single population of the wood-boring bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae).

    Luyten YA, Thompson JR, Morrill W, Polz MF, Distel DL
    2006 - Appl. Environ. Microbiol., 1: 412-7

    Abstract: 

    Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the gamma subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.

  • Extensive variation in intracellular symbiont community composition among members of a single population of the wood-boring bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae).

    Luyten YA, Thompson JR, Morrill W, Polz MF, Distel DL
    2006 - Appl. Environ. Microbiol., 1: 412-7

    Abstract: 

    Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the gamma subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.

  • Bacterial exoploymer and humic acid binding to calcite.

    Perry TD, Klepac-Ceraj V, Zhang XV, McNamara CJ, Polz MF, Martin ST, Berke N, Mitchell R
    2005 - Environ. Sci. Technol., 39: 8770-8775
  • PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample.

    Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF
    2005 - Appl. Environ. Microbiol., 12: 8966-9

    Abstract: 

    The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.

  • Illuminating reactive microbial transport in saturated porous media: demonstration of a visualization method and conceptual transport model.

    Oates PM, Castenson C, Harvey CF, Polz MF, Culligan P
    2005 - J. Contam. Hydrol., 4: 233-45

    Abstract: 

    We demonstrate a method to study reactive microbial transport in saturated translucent porous media using the bacteria Pseudomonas fluorescens 5RL genetically engineered to carry a plasmid with bioluminescence genes inducible by salicylate. Induced bacteria were injected into a cryolite grain filled chamber saturated with a sterile non-growth-promoting (phosphorus limited) chemical mixture containing salicylate as an aromatic hydrocarbon analogue. The amount of light produced by the bacteria serves as an estimator of the relative efficiency of aerobic biodegradation since bioluminescence is dependent on both salicylate and oxygen but only consumes oxygen. Bioluminescence was captured with a digital camera and analyzed to study the evolving spatial pattern of the bulk oxygen consuming reactions. As fluid flow transported the bacteria through the chamber, bioluminescence was observed to initially increase until an oxygen depletion zone developed behind the advective front. Bacterial transport was modeled with the advection dispersion equation and oxygen concentration was modeled assuming bacterial consumption via Monod kinetics with consideration of additional effects of rate-limited mass transfer from residual gas bubbles. Consistent with previous measurements, bioluminescence was considered proportional to oxygen consumed. Using the observed bioluminescence, model parameters were fit that were consistent with literature values and produced results in good agreement with the experimental data. These findings demonstrate potential for using this method to investigate the complex spatial and temporal dynamics of reactive microbial transport in saturated porous media.

  • Genotypic diversity within a natural coastal bacterioplankton population.

    Thompson JR, Pacocha S, Pharino C, Klepac-Ceraj V, Hunt DE, Benoit J, Sarma-Rupavtarm R, Distel DL, Polz MF
    2005 - Science, 5713: 1311-3

    Abstract: 

    The genomic diversity and relative importance of distinct genotypes within natural bacterial populations have remained largely unknown. Here, we analyze the diversity and annual dynamics of a group of coastal bacterioplankton (greater than 99% 16S ribosomal RNA identity to Vibrio splendidus). We show that this group consists of at least a thousand distinct genotypes, each occurring at extremely low environmental concentrations (on average less than one cell per milliliter). Overall, the genomes show extensive allelic diversity and size variation. Individual genotypes rarely recurred in samples, and allelic distribution did not show spatial or temporal substructure. Ecological considerations suggest that much genotypic and possibly phenotypic variation within natural populations should be considered neutral.

  • Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR.

    Randa MA, Polz MF, Lim E
    2004 - Appl. Environ. Microbiol., 9: 5469-76

    Abstract: 

    The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.

  • Fine-scale phylogenetic architecture of a complex bacterial community.

    Acinas SG, Klepac-Ceraj V, Hunt DE, Pharino C, Ceraj I, Distel DL, Polz MF
    2004 - Nature, 6999: 551-4

    Abstract: 

    Although molecular data have revealed the vast scope of microbial diversity, two fundamental questions remain unanswered even for well-defined natural microbial communities: how many bacterial types co-exist, and are such types naturally organized into phylogenetically discrete units of potential ecological significance? It has been argued that without such information, the environmental function, population biology and biogeography of microorganisms cannot be rigorously explored. Here we address these questions by comprehensive sampling of two large 16S ribosomal RNA clone libraries from a coastal bacterioplankton community. We show that compensation for artefacts generated by common library construction techniques reveals fine-scale patterns of community composition. At least 516 ribotypes (unique rRNA sequences) were detected in the sample and, by statistical extrapolation, at least 1,633 co-existing ribotypes in the sampled population. More than 50% of the ribotypes fall into discrete clusters containing less than 1% sequence divergence. This pattern cannot be accounted for by interoperon variation, indicating a large predominance of closely related taxa in this community. We propose that such microdiverse clusters arise by selective sweeps and persist because competitive mechanisms are too weak to purge diversity from within them.

  • Diversity and dynamics of a north atlantic coastal Vibrio community.

    Thompson JR, Randa MA, Marcelino LA, Tomita-Mitchell A, Lim E, Polz MF
    2004 - Appl. Environ. Microbiol., 7: 4103-10

    Abstract: 

    Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains. Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation. A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period. Quantitative PCR (QPCR) with primers specific for the genus Vibrio was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE). Vibrio populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations. Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction. It further showed that CDCE populations corresponded in most cases to approximately 98% rRNA similarity groups and suggested that the abundance of these follows temperature trends. Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples. Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend.

  • High overall diversity and dominance of microdiverse relationships in salt marsh sulphate-reducing bacteria.

    Klepac-Ceraj V, Bahr M, Crump BC, Teske AP, Hobbie JE, Polz MF
    2004 - Environ. Microbiol., 7: 686-98

    Abstract: 

    The biogeochemistry of North Atlantic salt marshes is characterized by the interplay between the marsh grass Spartina and sulphate-reducing bacteria (SRB), which mineralize the diverse carbon substrates provided by the plants. It was hypothesized that SRB populations display high diversity within the sediment as a result of the rich spatial and chemical structuring provided by Spartina roots. A 2000-member 16S rRNA gene library, prepared with delta-proteobacterial SRB-selective primers, was analysed for diversity patterns and phylogenetic relationships. Sequence clustering detected 348 16S rRNA sequence types (ribotypes) related to delta-proteobacterial SRB, and it was estimated that a total of 623 ribotypes were present in the library. Similarity clustering showed that approximately 46% of these sequences fell into groups with < 1% divergence; thus, microheterogeneity accounts for a large portion of the observable genetic diversity. Phylogenetic comparison revealed that sequences most frequently recovered were associated with the Desulfobacteriaceae and Desulfobulbaceae families. Sequences from the Desulfovibrionaceae family were also observed, but were infrequent. Over 80% of the delta-proteobacterial ribotypes clustered with cultured representatives of Desulfosarcina, Desulfococcus and Desulfobacterium genera, suggesting that complete oxidizers with high substrate versatility dominate. The large-scale approach demonstrates the co-existence of numerous SRB-like sequences and reveals an unexpected amount of microdiversity.

  • Spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract.

    Sarma-Rupavtarm RB, Ge Z, Schauer DB, Fox JG, Polz MF
    2004 - Appl. Environ. Microbiol., 5: 2791-800

    Abstract: 

    The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant Lactobacillus murinus ASF361 present at 10(5) to 10(7) cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10(8) to 10(10) cells/g of tissue. The variation between the three mice was small for most ASF strains, except for Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.

  • Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons.

    Acinas SG, Marcelino LA, Klepac-Ceraj V, Polz MF
    2004 - J. Bacteriol., 9: 2629-35

    Abstract: 

    The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but approximately 40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to approximately 2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.

  • A(r)Ray of hope in analysis of the function and diversity of microbial communities.

    Polz MF, Bertilsson S, Acinas SG, Hunt D
    2003 - Biol. Bull., 2: 196-9

    Abstract: 

    The vast majority of microorganisms in the environment remain uncultured, and their existence is known only from sequences retrieved by PCR. As a consequence, our understanding of the ecological function of dominant microbial populations in the environment is limited. We will review microbial diversity studies and show that these may have moved from an extreme underestimation to a potentially severe overestimation of diversity. The latter results from a simple PCR-generated artifact: the cloning of heteroduplex molecules followed by Escherichia coli mismatch repair, which may generate an exponential increase in observed sequence diversity. However, simple modifications to current PCR amplification protocols minimize such artifactual sequences and may bring within our reach estimation of bacterial diversity in environmental samples. Such estimates may spur new culture-independent approaches based on genomic and microarray technology, allowing correlation of phylogenetic identity with the ecological function of unculturable organisms. In particular, we are developing a DNA microarray that enables identification of individual populations active in utilization of specific organic substrates. The array consists of 16S and 23S rDNA-targeted oligonucleotides and is hybridized to RNA extracted from samples incubated with (14)C-labeled organic substrates. Populations that metabolize the substrate can be identified by the radiolabel incorporated in their rRNA after only one to two cell doublings, ensuring realistic preservation of community structure. Thus, the microarray approach may provide a powerful means to link microbial community structure with in situ function of individual populations.

  • Solar disinfection (SODIS): simulation of solar radiation for global assessment and application for point-of-use water treatment in Haiti.

    Oates PM, Shanahan P, Polz MF
    2003 - Water Res., 1: 47-54

    Abstract: 

    Haiti and other developing countries do not have sufficient meteorological data to evaluate if they meet the solar disinfection (SODIS) threshold of 3-5 h of solar radiation above 500 W/m2, which is required for adequate microbial inactivation in drinking water. We have developed a mathematical model based on satellite-derived daily total energies to simulate monthly mean, minimum, and maximum 5-h averaged peak solar radiation intensities. This model can be used to assess if SODIS technology would be applicable anywhere in the world. Field measurements were made in Haiti during January 2001 to evaluate the model and test SODIS efficacy as a point-of-use treatment option. Using the total energy from a measured solar radiation intensity profile, the model recreated the intensity profile with 99% agreement. NASA satellite data were then used to simulate the mean, minimum, and maximum 5-h averaged peak intensities for Haiti in January, which were within 98.5%, 62.5%, and 86.0% agreement with the measured values, respectively. Most of the discrepancy was attributed to the heterogeneous nature of Haiti's terrain and the spatial resolution of the NASA data. Additional model simulations suggest that SODIS should be effective year-round in Haiti. Actual SODIS efficacy in January was tested by the inactivation of total coliform, E. coli, and H2S-producing bacteria. Exposure period proved critical. One-day exposure achieved complete bacterial inactivation 52% of the time, while a 2-day exposure period achieved complete microbial inactivation 100% of the time. A practical way of providing people with cold water every morning that has undergone a 2-day exposure would be to rotate three groups of bottles every morning, so two groups are out in the sun and one is being used for consumption.

  • Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers.

    Bertilsson S, Cavanaugh CM, Polz MF
    2002 - Appl. Environ. Microbiol., 12: 6077-86

    Abstract: 

    Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.

  • Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'.

    Thompson JR, Marcelino LA, Polz MF
    2002 - Nucleic Acids Res., 9: 2083-8

    Abstract: 

    Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to 'reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.

  • Population Dynamics of Two Toluene Degrading Bacterial Species in a Contaminated Stream.

    Tay ST, Hemond FH, Krumholz LR, Cavanaugh CM, Polz MF
    2001 - Microb. Ecol., 2: 124-131

    Abstract: 

    Toluene uptake by a benthic biofilm community was previously shown to vary seasonally from 0.03 m hr?1 in winter to 0.2 m hr?1 in summer in a solvent-contaminated stream of the Aberjona watershed. We used quantitative PCR to estimate the population dynamics of previously isolated species of toluene-degrading Xanthobacter autotrophicus and Mycobacterium sp. in both toluene-contaminated and uncontaminated reaches of the stream, and to estimate their relative roles in overall biodegradation rate. Quantification using specific 16S rDNA primers forX. autotrophicus and Mycobacterium sp. showed that populations of both species were much larger in the toluene-contaminated than the toluene-free reach, in agreement with earlier culture-based investigations. A relatively brief bloom of X. autotrophicus occurred in the contaminated reach in the summer, while Mycobacterium sp. populations occurred at elevated densities for more than 5 months. Calculations showed that Mycobacterium, previously thought to be less important than Xanthobacter in annual toluene degradation based on single time-point CFU estimates, appears actually more important because of this longer persistence.

  • Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

    Kuai L, Nair AA, Polz MF
    2001 - Appl. Environ. Microbiol., 7: 3168-73

    Abstract: 

    A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.

  • When bacteria hitch a ride.

    Polz MF, Ott JA, Bright M, Cavanaugh CM
    2000 - ASM News., 66: 531-539
  • Diversity and heterogeneity of epibiotic bacterial communities on the marine nematode Eubostrichus dianae.

    Polz MF, Harbison C, Cavanaugh CM
    1999 - Appl. Environ. Microbiol., 9: 4271-5

    Abstract: 

    The diversity of a microbial community covering the surface of a marine nematode was analyzed by performing a 16S ribosomal DNA (rDNA) restriction cutting and sequencing analysis. In two clone libraries constructed by using individual nematodes, 54 and 85 restriction patterns were identified, and only 13 of these patterns were common to both libraries. Sequence analysis indicated that the common patterns belonged to four groups related to sequences of cytophagas, sulfate-reducing bacteria, members of the gamma subclass of the class Proteobacteria, and caulobacters. At least two groups appeared to be permanent members of the community as they were also detected in a 16S rDNA library constructed 3 years previously by using 100 pooled nematode specimens. A surprising outcome was that very dominant filamentous bacteria were apparently not represented in the clone libraries, as quantitative probing showed that none of the common operational taxonomic unit groups displayed the expected overwhelming dominance. Nevertheless, our analysis revealed both an unexpectedly high level of bacterial diversity and heterogeneity in samples representing presumably very similar microenvironments.

  • Importance of Xanthobacter autotrophicus in toluene biodegradation within a contaminated stream.

    Tay ST, Hemond HF, Polz MF, Cavanaugh CM, Krumholz LR
    1999 - Syst. Appl. Microbiol., 1: 113-8

    Abstract: 

    Toluene-degrading strains T101 and T102 were isolated from rock surface biomass in a toluene-contaminated freshwater stream. These organisms were present at a density of 5.5 x 10(6) cells/g of rock surface biomass. Both are aerobic, rod-shaped, Gram-negative, non-motile, catalase-positive, oxidase-positive, with yellow pigments, and can grow on benzene. Phylogenetic analyses show that strains T101 and T102 have 16S rDNA sequences identical to Xanthobacter autotrophicus. Fatty acid analyses indicate that they are different strains of the same species Xanthobacter autotrophicus, and that they have high levels of cis-11-octadecenoic acid and cis-9-hexadecenoic acid; 3-hydroxyhexadecanoic acid is the major hydroxy fatty acid present. Strains T101 and T102 had maximal velocities (Vmax) for toluene biodegradation of 3.8 +/- 0.5 and 28.3 +/- 2.2 mumoles toluene/mgprotein-hr, and half-saturation constants (Ks) of 0.8 +/- 0.5 and 11.5 +/- 2.4 microM, respectively. Strain T102 has a higher capacity than strain T101 to degrade toluene, and kinetic calculations suggest that strain T102 may be a major contributor to toluene biodegradation in the stream.

  • Bias in template-to-product ratios in multitemplate PCR.

    Polz MF, Cavanaugh CM
    1998 - Appl. Environ. Microbiol., 10: 3724-30

    Abstract: 

    Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

  • Two new Mycobacterium strains and their role in toluene degradation in a contaminated stream.

    Tay ST, Hemond HF, Polz MF, Cavanaugh CM, Dejesus I, Krumholz LR
    1998 - Appl. Environ. Microbiol., 5: 1715-20

    Abstract: 

    Two toluene-degrading strains, T103 and T104, were isolated from rock surface biomass in a freshwater stream contaminated with toluene. The strains exhibit different capacities for degradation of toluene and other aromatic compounds and have characteristics of the genus Mycobacterium. Both are aerobic, rod-shaped, gram-positive, nonmotile, and acid-alcohol fast and produce yellow pigments. They have mainly straight-chain saturated and monounsaturated fatty acids with 10 to 20 carbon atoms and large amounts of tuberculostearic acid that are typical of mycobacteria. Fatty acid analyses indicate that T103 and T104 are different mycobacterial strains that are related at the subspecies level. Their identical 16S rDNA sequences are most similar to Mycobacterium aurum and Mycobacterium komossense, and they constitute a new species of fast-growing mycobacteria. Ecological studies reveal that toluene contamination has enriched for toluene-degrading bacteria in the epilithic microbial community. Strains T103 and T104 play only a small role in toluene degradation in the stream, although they are present in the habitat and can degrade toluene. Other microorganisms are consequently implicated in the biodegradation.

Book chapters and other publications

4 Publications found
  • Population Genomics: Microorganisms

    Polz MF, Rajora O
    2019 - in Population Genomics: Microorganisms. (Polz MF, Rajora O). Nature Springer
  • Overview: Quantitative and theoretical microbial population biology.

    Polz MF, Hanage WP
    2012 - in The Prokaryotes, 4th Edition. . (Rosenberg E, Delong EF, Thompson F, Lory S, Stackebrandt E). Springer-Verlag, Berlin Heidelberg
  • Dynamics of vibrio populations and their role in environmental nutrient cycling.

    Thompson JT, Polz MF
    2006 - 190-203. in The Biology of Vibrios. (Thompson FL, Austin B, Swings J). ASM Press, Washington DC
  • Diversity and sources of human bacterial pathogens and overview of methods of their detection and quantification.

    Thompson JT, Marcelino L, Polz MF
    2005 - in Oceans and Health: Pathogens in the Marine Environment. (Belkin S, Colwell R). Springer