Identification of secondary metabolite gene clusters in the Pseudovibrio genus reveals encouraging biosynthetic potential toward the production of novel bioactive compounds
Naughton LM, Romano S, O'Gara F, Dobson ADW
2017 - Front Microbiol, 8: 1494
Increased incidences of antimicrobial resistance and the emergence of pan-resistant 'superbugs' have provoked an extreme sense of urgency amongst researchers focusing on the discovery of potentially novel antimicrobial compounds. A strategic shift in focus from the terrestrial to the marine environment has resulted in the discovery of a wide variety of structurally and functionally diverse bioactive compounds from numerous marine sources, including sponges. Bacteria found in close association with sponges and other marine invertebrates have recently gained much attention as potential sources of many of these novel bioactive compounds. Members of the genus Pseudovibrio are one such group of organisms. In this study, we interrogate the genomes of 21 Pseudovibrio strains isolated from a variety of marine sources, for the presence, diversity and distribution of biosynthetic gene clusters (BGCs). We expand on results obtained from antiSMASH analysis to demonstrate the similarity between the Pseudovibrio-related BGCs and those characterized in other bacteria and corroborate our findings with phylogenetic analysis. We assess how domain organization of the most abundant type of BGCs present among the isolates (Non-ribosomal peptide synthetases and Polyketide synthases) may influence the diversity of compounds produced by these organisms and highlight for the first time the potential for novel compound production from this genus of bacteria, using a genome guided approach.
In situ architecture, function, and evolution of a contractile injection system.
Böck D, Medeiros JM, Tsao HF, Penz T, Weiss GL, Aistleitner K, Horn M, Pilhofer M
2017 - Science, 6352: 713-717
Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.
Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments.
'Candidatus Cochliophilus cryoturris' (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus.
Tsao HF, Scheikl U, Volland JM, Köhsler M, Bright M, Walochnik J, Horn M
2017 - Sci Rep, 1: 3394
Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as 'Candidatus Cochliophilus cryoturris'. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.
Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont.
König L, Siegl A, Penz T, Haider S, Wentrup C, Polzin J, Mann E, Schmitz-Esser S, Domman D, Horn M
2017 - mSystems, 2: e00202-16
Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as the carbon source during initial stages and a postreplicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes. IMPORTANCE Chlamydiae are known as major bacterial pathogens of humans, causing the ancient disease trachoma, but they are also frequently found in the environment where they infect ubiquitous protists such as amoebae. All known chlamydiae require a eukaryotic host cell to thrive. Using the environmental chlamydia Protochlamydia amoebophila within its natural host, Acanthamoeba castellanii, we investigated gene expression dynamics in vivo and throughout the complete chlamydial developmental cycle for the first time. This allowed us to infer how a major virulence mechanism, the type III secretion system, is regulated and employed, and we show that the physiology of chlamydiae undergoes a complete shift regarding carbon metabolism and energy generation. This study provides comprehensive insights into the infection strategy of chlamydiae and reveals a unique adaptation to life within a eukaryotic host cell.
HuR small-molecule inhibitor elicits differential effects in adenomatosis polyposis and colorectal carcinogenesis
Lang M, Berry D, Passecker K, Mesteri I, Bhuju S, Ebner F, Sedlyarov V, Evstatiev R, Dammann K, Loy A, Kuzyk O, Kovarik P, Khare V, Beibel M, Roma G, Meisner-Kober N, Gasche C
2017 - Cancer Res., 77: 2424-2438
HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3' untranslated region of mRNAs encoding oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APCMin mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of Prevotella, Akkermansia, and Lachnospiraceae Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD.
Giant viruses with an expanded complement of translation system components.
Schulz F, Yutin N, Ivanova NN, Ortega DR, Lee TK, Vierheilig J, Daims H, Horn M, Wagner M, Jensen GJ, Kyrpides NC, Koonin EV, Woyke T
2017 - Science, 6333: 82-85
The discovery of giant viruses blurred the sharp division between viruses and cellular life. Giant virus genomes encode proteins considered as signatures of cellular organisms, particularly translation system components, prompting hypotheses that these viruses derived from a fourth domain of cellular life. Here we report the discovery of a group of giant viruses (Klosneuviruses) in metagenomic data. Compared with other giant viruses, the Klosneuviruses encode an expanded translation machinery, including aminoacyl transfer RNA synthetases with specificities for all 20 amino acids. Notwithstanding the prevalence of translation system components, comprehensive phylogenomic analysis of these genes indicates that Klosneuviruses did not evolve from a cellular ancestor but rather are derived from a much smaller virus through extensive gain of host genes.
Variant profiling of evolving prokaryotic populations.
Zojer M, Schuster LN, Schulz F, Pfundner A, Horn M, Rattei T
2017 - PeerJ, e2997
Genomic heterogeneity of bacterial species is observed and studied in experimental evolution experiments and clinical diagnostics, and occurs as micro-diversity of natural habitats. The challenge for genome research is to accurately capture this heterogeneity with the currently used short sequencing reads. Recent advances in NGS technologies improved the speed and coverage and thus allowed for deep sequencing of bacterial populations. This facilitates the quantitative assessment of genomic heterogeneity, including low frequency alleles or haplotypes. However, false positive variant predictions due to sequencing errors and mapping artifacts of short reads need to be prevented. We therefore created VarCap, a workflow for the reliable prediction of different types of variants even at low frequencies. In order to predict SNPs, InDels and structural variations, we evaluated the sensitivity and accuracy of different software tools using synthetic read data. The results suggested that the best sensitivity could be reached by a union of different tools, however at the price of increased false positives. We identified possible reasons for false predictions and used this knowledge to improve the accuracy by post-filtering the predicted variants according to properties such as frequency, coverage, genomic environment/localization and co-localization with other variants. We observed that best precision was achieved by using an intersection of at least two tools per variant. This resulted in the reliable prediction of variants above a minimum relative abundance of 2%. VarCap is designed for being routinely used within experimental evolution experiments or for clinical diagnostics. The detected variants are reported as frequencies within a VCF file and as a graphical overview of the distribution of the different variant/allele/haplotype frequencies. The source code of VarCap is available at https://github.com/ma2o/VarCap. In order to provide this workflow to a broad community, we implemeted VarCap on a Galaxy webserver, which is accessible at http://galaxy.csb.univie.ac.at.
Mucispirillum schaedleri is an abundant inhabitant of the intestinal mucus layer of rodents and other animals and has been suggested to be a pathobiont, a commensal that plays a role in disease. In order to gain insights into its lifestyle, we analyzed the genome and transcriptome of M. schaedleri ASF 457 and performed physiological experiments to test traits predicted by its genome. Although described as a mucus inhabitant, M. schaedleri has limited capacity for degrading host-derived mucosal glycans and other complex polysaccharides. Additionally, M. schaedleri reduces nitrate and expresses systems for scavenging oxygen and reactive oxygen species in vivo, which may account for its localization close to the mucosal tissue and expansion during inflammation. Also of note, M. schaedleri harbors a type VI secretion system and putative effector proteins and can modify gene expression in mucosal tissue, suggesting intimate interactions with its host and a possible role in inflammation. The M. schaedleri genome has been shaped by extensive horizontal gene transfer, primarily from intestinal Epsilon- and Deltaproteobacteria, indicating that horizontal gene transfer has played a key role in defining its niche in the gut ecosystem.
Convergent patterns in the evolution of mealybug symbioses involving different intrabacterial symbionts.
Szabó G, Schulz F, Toenshoff ER, Volland JM, Finkel OM, Belkin S, Horn M
2017 - ISME J, 3: 715-726
Mealybugs (Insecta: Hemiptera: Pseudococcidae) maintain obligatory relationships with bacterial symbionts, which provide essential nutrients to their insect hosts. Most pseudococcinae mealybugs harbor a unique symbiosis setup with enlarged betaproteobacterial symbionts ('Candidatus Tremblaya princeps'), which themselves contain gammaproteobacterial symbionts. Here we investigated the symbiosis of the manna mealybug, Trabutina mannipara, using a metagenomic approach. Phylogenetic analyses revealed that the intrabacterial symbiont of T. mannipara represents a novel lineage within the Gammaproteobacteria, for which we propose the tentative name 'Candidatus Trabutinella endobia'. Combining our results with previous data available for the nested symbiosis of the citrus mealybug Planococcus citri, we show that synthesis of essential amino acids and vitamins and translation-related functions partition between the symbiotic partners in a highly similar manner in the two systems, despite the distinct evolutionary origin of the intrabacterial symbionts. Bacterial genes found in both mealybug genomes and complementing missing functions in both symbioses were likely integrated in ancestral mealybugs before T. mannipara and P. citri diversified. The high level of correspondence between the two mealybug systems and their highly intertwined metabolic pathways are unprecedented. Our work contributes to a better understanding of the only known intracellular symbiosis between two bacteria and suggests that the evolution of this unique symbiosis included the replacement of intrabacterial symbionts in ancestral mealybugs.
Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large-scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts' morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica, some have undergone symbiont replacement by other phylogenetically-distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage-specific. We propose a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont's tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host-symbiont interactions.
Metabolic and physiological interdependencies in the Bathymodiolus azoricus symbiosis.
Ponnudurai R, Kleiner M, Sayavedra L, Petersen JM, Moche M, Otto A, Becher D, Takeuchi T, Satoh N, Dubilier N, Schweder T, Markert S
2017 - ISME J, 11: 463–477
The hydrothermal vent mussel Bathymodiolus azoricus lives in an intimate symbiosis with two types of chemosynthetic Gammaproteobacteria in its gills: a sulfur oxidizer and a methane oxidizer. Despite numerous investigations over the last decades, the degree of interdependence between the three symbiotic partners, their individual metabolic contributions, as well as the mechanism of carbon transfer from the symbionts to the host are poorly understood. We used a combination of proteomics and genomics to investigate the physiology and metabolism of the individual symbiotic partners. Our study revealed that key metabolic functions are most likely accomplished jointly by B. azoricus and its symbionts: (1) CO2 is pre-concentrated by the host for carbon fixation by the sulfur-oxidizing symbiont, and (2) the host replenishes essential biosynthetic TCA cycle intermediates for the sulfur-oxidizing symbiont. In return (3), the sulfur oxidizer may compensate for the host's putative deficiency in amino acid and cofactor biosynthesis. We also identified numerous 'symbiosis-specific' host proteins by comparing symbiont-containing and symbiont-free host tissues and symbiont fractions. These proteins included a large complement of host digestive enzymes in the gill that are likely involved in symbiont digestion and carbon transfer from the symbionts to the host.The ISME Journal advance online publication, 1 November 2016; doi:10.1038/ismej.2016.124.