Wastewater treatment plants (WWTPs) are a source and reservoir for subsequent spread of various antibiotic resistance genes (ARGs). However, little is known about the activity and hosts of ARGs in WWTPs. Here, we utilized both metagenomic and metatranscriptomic approaches to comprehensively reveal the diversity, abundance, expression and hosts of ARGs in activated sludge (AS) from three conventional WWTPs in Taiwan. Based on deep sequencing data and a custom-made ARG database, a total of 360 ARGs associated with 24 classes of antibiotics were identified from the three AS metagenomes, with an abundance range of 7.06 × 10-1-1.20 × 10-4 copies of ARG/copy of 16S rRNA gene. Differential coverage binning analysis revealed that >22 bacterial phyla were the putative hosts of the identified ARGs. Surprisingly, genus Mycobacterium and family Burkholderiaceae were observed as multi-drug resistant harboring 14 and 50 ARGs. Metatranscriptome analysis showed 65.8% of the identified ARGs were being expressed, highlighting that ARGs were not only present, but also transcriptionally active in AS. Remarkably, 110 identified ARGs were annotated as plasmid-associated and displayed a close to two-fold increased likelihood of being transcriptionally expressed compared to those ARGs found exclusively within bacterial chromosomes. Further analysis showed the transcript abundance of aminoglycoside, sulfonamide, and tetracycline resistance genes was mainly contributed by plasmid-borne ARGs. Our approach allowed us to specifically link ARGs to their transcripts and genetic context, providing a comprehensive insight into the prevalence, expression and hosts of ARGs in AS. Overall, results of this study enhance our understanding of the distribution and dissemination of ARGs in WWTPs, which benefits environmental risk assessment and management of ARB and ARGs.

Marine Benthic Group D (MBG-D) archaea, discovered by 16S rRNA gene survey decades ago, are ecologically important, yet understudied and uncultured sedimentary archaea. In this study, a comprehensive meta-analysis based on the 16S rRNA genes of MBG-D archaea showed that MBG-D archaea are one of the most frequently found archaeal lineages in global sediment with widespread distribution and high abundance, including 16 subgroups in total. Interestingly, some subgroups show significant segregations toward salinity and methane seeps. Co-occurrence analyses indicate significant non-random association of MBG-D archaea with Lokiarchaeota (in both saline and freshwater sediments) and Hadesarchaea, suggesting potential interactions among these archaeal groups. Meanwhile, based on four nearly complete metagenome-assembled genomes (MAGs) and corresponding metatranscriptomes reconstructed from mangrove and intertidal mudflat sediments, we provide insights on metabolic potentials and ecological functions of MBG-D archaea. MBG-D archaea appear to be capable of transporting and assimilating peptides and generating acetate and ethanol through fermentation. Metatranscriptomic analysis suggests high expression of genes for acetate and amino acid utilization and for peptidases, especially the M09B-type extracellular peptidase (collagenase) showing high expression levels in all four mangrove MAGs. Beyond heterotrophic central carbon metabolism, the MBG-D genomes include genes that might encode two autotrophic pathways: Wood–Ljundahl (WL) pathways using both H₄MPT and H₄folate as C₁ carriers, and an incomplete dicarboxylate/4-hydroxybutyrate cycle with alternative bypasses from pyruvate to malate/oxaloacetate during dicarboxylation. These findings reveal MBG-D archaea as an important ubiquitous benthic sedimentary archaeal group with specific mixotrophic metabolisms, so we proposed the name Thermoprofundales as a new Order within the Class Thermoplasmata. Globally, Thermoprofundales and other benthic archaea might synergistically transform benthic organic matter, possibly playing a vital role in sedimentary carbon cycle.

Background: Increasing attention has recently been devoted to the anaerobic ammonium oxidation (anammox) in eutrophic lakes due to its potential key functions in nitrogen (N) removal for eutrophication control. However, successful enrichment of anammox bacteria from lake sediments is still challenging, partly due to the ecological interactions between anammox and denitrifying bacteria across such enrichment with lake sediments remain unclear. Results: This study thus designed to fill such knowledge gaps using bioreactors to enrich anammox bacteria with eutrophic lake sediments for more than 365 days. We continuously monitored the influent and effluent water, measured the anammox and denitrification efficiencies, quantified the anammox and denitrifying bacteria, as well as the related N cycling genes. We found that the maximum removal efficiencies of NH₄⁺ and NO₂⁻ reached up to 85.92% and 95.34%, respectively. Accordingly, the diversity of anammox and denitrifying bacteria decreased significantly across the enrichment, and the relative dominant anammox (e.g., Candidatus Jettenia) and denitrifying bacteria (e.g., Thauera, Afipia) shifted considerably. The ecological cooperation between anammox and denitrifying bacteria tended to increase the microbial community stability, indicating a potential coupling between anammox and denitrifying bacteria. Moreover, the nirS-type denitrifiers showed stronger coupling with anammox bacteria than that of nirK-type denitrifiers during the enrichment. Functional potentials as depicted by metagenome sequencing confirmed the ecological interactions between anammox and denitrification. Metagenome-assembled genomes-based ecological model indicated that the most dominant denitrifiers could provide various materials such as amino acid, cofactors, and vitamin for anammox bacteria. Cross-feeding in anammox and denitrifying bacteria highlights the importance of microbial interactions for increasing the anammox N removal in eutrophic lakes. Conclusions: This study greatly expands our understanding of cooperation mechanisms among anammox and denitrifying bacteria during the anammox enrichment with eutrophic lake sediments, which sheds new insights into N removal for controlling lake eutrophication.

The discovery of comammox in the genus Nitrospira 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 Nitrospira an interesting target for optimizing nitrification in current and future bioreactor configurations.