The recent discovery of complete ammonia oxidizers (comammox) contradicts the paradigm that chemolithoautotrophic nitrification is always catalyzed by two different microorganisms. However, our knowledge of the survival strategies of comammox in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Analyses of genomes and transcriptomes of four comammox organisms from two full-scale WWTPs revealed that comammox were active and showed a surprisingly high metabolic versatility. A gene cluster for the utilization of urea and a gene encoding cyanase suggest that comammox may use diverse organic nitrogen compounds in addition to free ammonia as the substrates. The comammox organisms also encoded the genomic potential for multiple alternative energy metabolisms, including respiration with hydrogen, formate, and sulfite as electron donors. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely help comammox survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. One of the comammox strains acquired from the anaerobic tank encoded and transcribed genes involved in homoacetate fermentation or in the utilization of exogenous acetate, both pathways being unexpected in a nitrifying bacterium. Surprisingly, this strain also encoded a respiratory nitrate reductase which has not yet been found in any other genome and might confer a selective advantage to this strain over other strains in anoxic conditions. The discovery of comammox in the genus changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and-among nitrifiers-unsurpassed ecophysiological versatility could make comammox an interesting target for optimizing nitrification in current and future bioreactor configurations.

Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes. Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages.

High-throughput sequencing has allowed unprecedented insight into the composition and function of complex microbial communities. With metatran- scriptomics, it is possible to interrogate the transcriptomes of multiple organisms si- multaneously to get an overview of the gene expression of the entire community. Studies have successfully used metatranscriptomics to identify and describe rela- tionships between gene expression levels and community characteristics. How- ever, metatranscriptomic data sets contain a rich suite of additional information that is just beginning to be explored. Here, we focus on antisense expression in meta- transcriptomics, discuss the different computational strategies for handling it, and highlight the strengths but also potentially detrimental effects on downstream anal- ysis and interpretation. We also analyzed the antisense transcriptomes of multiple genomes and metagenome-assembled genomes (MAGs) from five different data sets and found high variability in the levels of antisense transcription for individual spe- cies, which were consistent across samples. Importantly, we challenged the concep- tual framework that antisense transcription is primarily the product of transcriptional noise and found mixed support, suggesting that the total observed antisense RNA in complex communities arises from the combined effect of unknown biological and technical factors. Antisense transcription can be highly informative, including techni- cal details about data quality and novel insight into the biology of complex micro- bial communities.