Growth of complete ammonia oxidizers on guanidine.

Guanidine is a chemically stable nitrogen compound that is excreted in human urine and is widely used in manufacturing of plastics, as a flame retardant and as a component of propellants, and is well known as a protein denaturant in biochemistry1-3. Guanidine occurs widely in nature and is used by several microorganisms as a nitrogen source, but microorganisms growing on guanidine as the only substrate have not yet been identified. Here we show that the complete ammonia oxidizer (comammox) Nitrospira inopinata and probably most other comammox microorganisms can grow on guanidine as the sole source of energy, reductant and nitrogen. Proteomics, enzyme kinetics and the crystal structure of a N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Incubation experiments with comammox-containing agricultural soil and wastewater treatment plant microbiomes suggested that guanidine serves as substrate for nitrification in the environment. The identification of guanidine as a growth substrate for comammox shows an unexpected niche of these globally important nitrifiers and offers opportunities for their isolation.

Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses.

Dehalobacter (Firmicutes) encompass obligate organohalide-respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2-trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane-bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane-bound b-type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.

Dual C-Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures.

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C-Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (-1.8 ± 0.2 and -19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (-1.22 ± 0.08 and -1.2 ± 0.5‰), leading to distinctly different trends (ΛC-Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C-Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC-Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.

Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M.

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146-242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron-sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas.

Bacterial interactions during sequential degradation of cyanobacterial necromass in a sulfidic arctic marine sediment.

Seafloor microorganisms impact global carbon cycling by mineralizing vast quantities of organic matter (OM) from pelagic primary production, which is predicted to increase in the Arctic because of diminishing sea ice cover. We studied microbial interspecies-carbon-flow during anaerobic OM degradation in arctic marine sediment using stable isotope probing. We supplemented sediment incubations with 13 C-labeled cyanobacterial necromass (spirulina), mimicking fresh OM input, or acetate, an important OM degradation intermediate and monitored sulfate reduction rates and concentrations of volatile fatty acids (VFAs) during substrate degradation. Sequential 16S rRNA gene and transcript amplicon sequencing and fluorescence in situ hybridization combined with Raman microspectroscopy revealed that only few bacterial species were the main degraders of 13 C-spirulina necromass. Psychrilyobacter, Psychromonas, Marinifilum, Colwellia, Marinilabiaceae and Clostridiales species were likely involved in the primary hydrolysis and fermentation of spirulina. VFAs, mainly acetate, produced from spirulina degradation were mineralized by sulfate-reducing bacteria and an Arcobacter species. Cellular activity of Desulfobacteraceae and Desulfobulbaceae species during acetoclastic sulfate reduction was largely decoupled from relative 16S rRNA gene abundance shifts. Our findings provide new insights into the identities and physiological constraints that determine the population dynamics of key microorganisms during complex OM degradation in arctic marine sediments.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Development and application of primers for the class Dehalococcoidia (phylum Chloroflexi) enables deep insights into diversity and stratification of subgroups in the marine subsurface.

Bacteria of the class Dehalococcoidia (DEH) (phylum Chloroflexi) are widely distributed in the marine subsurface and are especially prevalent in deep marine sediments. Nevertheless, little is known about the specific distributions of DEH subgroups at different sites and depths. This study therefore specifically examined the distributions of DEH through depths of various marine sediment cores by quantitative PCR and pyrosequencing using newly designed DEH 16S rRNA gene targeting primers. Quantification of DEH showed populations may establish in shallow sediments (i.e. upper centimetres), although as low relative proportions of total Bacteria, yet often became more prevalent in deeper sediments. Pyrosequencing revealed pronounced diversity co-exists within single biogeochemical zones, and that clear and sometimes abrupt shifts in relative proportions of DEH subgroups occur with depth. These shifts indicate varying metabolic properties exist among DEH subgroups. The distributional changes in DEH subgroups with depth may be related to a combination of biogeochemical factors including the availability of electron acceptors such as sulfate, the composition of organic matter and depositional regimes. Collectively, the results suggest DEH exhibit wider metabolic and genomic diversity than previously recognized, and this contributes to their widespread occurrence in the marine subsurface.

Divergent dual C-H isotopic fractionation pattern during anaerobic biodegradation of toluene within Aromatoleum species under nitrate-reducing conditions.

Toluene is a pollutant frequently detected in contaminated groundwater, mostly due to leakage from underground gasoline storage tanks and pipeline ruptures. Multi-element compound-specific isotope analysis provides a framework to understand transformation processes and design efficient remediation strategies. In this study, we enriched an anaerobic bacterial culture derived from a BTEX-contaminated aquifer that couples toluene and phenol oxidation with nitrate reduction and the concomitant production of carbon dioxide and biomass. The 16S rRNA gene amplicon data indicated that the toluene-degrading consortium was dominated by an Aromatoleum population (87 ± 2 % relative abundance), and metagenome sequencing confirmed that the genome of this Aromatoleum sp. encoded glycyl-radical enzyme benzylsuccinate synthase (BssABC) and phenylphospate synthase (PpsA1BC) homologous genes involved in the first step of toluene and phenol transformation, respectively. Carbon and hydrogen isotopic fractionation were εbulk, C = - 3.5 ± 0.6 ‰ and εrp, H = - 85 ± 11 ‰, respectively, leading to a dual C-H isotope slope of ΛH/C = 26 ± 2. This value fits with a previously reported value for a consortium dominated by an Azoarcus species (ΛH/C = 19 ± 5) but differs from that reported for Aromatoleum aromaticum (ΛH/C = 14 ± 1), both of which grow with toluene under nitrate-reducing conditions. Overall, this suggests the existence of different BssABC enzymes with different mechanistic motifs even within the same Aromatoleum genus.

Benzene and sulfide removal from groundwater treated in a microbial fuel cell.

Sulfidic benzene-contaminated groundwater was used to fuel a two-chambered microbial fuel cell (MFC) over a period of 770 days. We aimed to understand benzene and sulfide removal processes in the anoxic anode chamber and describe the microbial community enriched over the operational time. Operated in batch feeding-like circular mode, supply of fresh groundwater resulted in a rapid increase in current production, accompanied by decreasing benzene and sulfide concentrations. The total electron recoveries for benzene and sulfide were between 18% and 49%, implying that benzene and sulfide were not completely oxidized at the anode. Pyrosequencing of 16S rRNA genes from the anode-associated bacterial community revealed the dominance of δ-Proteobacteria (31%), followed by ß-Proteobacteria, Bacteroidetes, ϵ-Proteobacteria, Chloroflexi, and Firmicutes, most of which are known for anaerobic metabolism. Two-dimensional compound-specific isotope analysis demonstrated that benzene degradation was initiated by monohydroxylation, probably triggered by small amounts of oxygen which had leaked through the cation exchange membrane into the anode chamber. Experiments with [(13)C(6) ]-benzene revealed incorporation of (13)C into fatty acids of mainly Gram-negative bacteria, which are therefore candidates for benzene degradation. Our study demonstrated simultaneous benzene and sulfide removal by groundwater microorganisms which use an anode as artificial electron acceptor, thereby releasing an electrical current.

Deciphering community interactions of sulfate-reducing microorganisms in complex microbial communities of marine sediments.

Sulfate-reducing microorganisms (SRM) are key players in global sulfur and carbon cycles, especially in anoxic marine sediments. They are critical in anaerobic food webs because they consume fermentation products like volatile fatty acids (VFAs) and/or hydrogen produced from other microbes that degrade organic matter. Apart from this, the interplay between SRM and other coexisting microorganisms is poorly understood. A recent study by Liang et al. provides intriguing new insights about how the activity of SRM influence microbial communities. Using an elegant combination of microcosm experiments, community ecology, genomics, and in vitro studies, they provide evidence that SRM are central in ecological networks and community assembly, and interestingly, that the control of pH by SRM activity has a substantial impact on other key bacteria, like members of the Marinilabiliales (Bacteroidota). This work has important implications for understanding how marine sediment microbes function together to provide important ecosystem services like recycling organic matter.

Proteogenomics of the novel Dehalobacterium formicoaceticum strain EZ94 highlights a key role of methyltransferases during anaerobic dichloromethane degradation.

Dichloromethane (DCM, methylene chloride) is a toxic, high-volume industrial pollutant of long-standing. Anaerobic biodegradation is crucial for its removal from contaminated environments, yet prevailing mechanisms remain unresolved, especially concerning dehalogenation. In this study, we obtained an assembled genome of a novel DCM-degrading strain, Dehalobacterium formicoaceticum strain EZ94, from a stable DCM-degrading consortium, and we analyzed its proteome during degradation of DCM. A gene cluster recently predicted to play a major role in anaerobic DCM catabolism (the mec cassette) was found. Methyltransferases and other proteins encoded by the mec cassette were among the most abundant proteins produced, suggesting their involvement in DCM catabolism. Reductive dehalogenases were not detected. Genes and corresponding proteins for a complete Wood-Ljungdahl pathway, which could enable further metabolism of DCM carbon, were also found. Unlike for the anaerobic DCM degrader "Ca. F. warabiya," no genes for metabolism of the quaternary amines choline and glycine betaine were identified. This work provides independent and supporting evidence that mec-associated methyltransferases are key to anaerobic DCM metabolism.

Microbial communities and processes in biofilters for post-treatment of ozonated wastewater treatment plant effluent.

Ozonation is an established solution for organic micropollutant (OMP) abatement in tertiary wastewater treatment. Biofiltration is the most common process for the biological post-treatment step, which is generally required to remove undesired oxidation products from the reaction of ozone with water matrix compounds. This study comparatively investigates the effect of filter media on the removal of organic contaminants and on biofilm properties for biologically activated carbon (BAC) and anthracite biofilters. Biofilms were analysed in two pilot-scale filters that have been operated for >50,000 bed volumes as post-treatment for ozonated wastewater treatment plant effluent. In parallel, the removal performance of bulk organics and OMP, including differentiation of adsorption and biotransformation through sodium azide inhibition, were carried out in bench-scale filter columns filled with material from the pilot filters. The use of BAC instead of anthracite resulted in an improved removal of organic bulk parameters, dissolved oxygen, and OMP. The OMP removal observed in the BAC filter but not in the anthracite filter was based on adsorption for most of the investigated compounds. For valsartan, however, biotransformation was found to be the dominant pathway, indicating that conditions for biotransformation of certain OMP are better on BAC than on anthracite. Adenosine triphosphate analyses in the media-attached biofilms of the pilot filters showed that biomass concentrations in the BAC filter were significantly higher than in the anthracite filter. The microbial communities (16S rRNA gene sequencing) appeared to be similar with respect to the types of organisms occurring on both filter materials. Alpha diversity also exhibited little variation between filter media. Beta diversity analysis, however, revealed that filter media and bed depth substantially influenced the biofilm composition. In practice, the impact of filter media on biofilm properties and biotransformation processes should be considered for the design of biofilters.

The Parkinson's drug entacapone disrupts gut microbiome homeostasis via iron sequestration.

Increasing evidence shows that many human-targeted drugs alter the gut microbiome, leading to implications for host health. However, much less is known about the mechanisms by which drugs target the microbiome and how drugs affect microbial function. Here we combined quantitative microbiome profiling, long-read metagenomics, stable isotope probing and single-cell chemical imaging to investigate the impact of two widely prescribed nervous system-targeted drugs on the gut microbiome. Ex vivo supplementation of physiologically relevant concentrations of entacapone or loxapine succinate to faecal samples significantly impacted the abundance of up to one third of the microbial species present. Importantly, we demonstrate that the impact of these drugs on microbial metabolism is much more pronounced than their impact on abundances, with low concentrations of drugs reducing the activity, but not the abundance of key microbiome members like Bacteroides, Ruminococcus or Clostridium species. We further demonstrate that entacapone impacts the microbiome due to its ability to complex and deplete available iron, and that microbial growth can be rescued by replenishing levels of microbiota-accessible iron. Remarkably, entacapone-induced iron starvation selected for iron-scavenging organisms carrying antimicrobial resistance and virulence genes. Collectively, our study unveils the impact of two under-investigated drugs on whole microbiomes and identifies metal sequestration as a mechanism of drug-induced microbiome disturbance.

Trichloromethane dechlorination by a novel Dehalobacter sp. strain 8M reveals a third contrasting C and Cl isotope fractionation pattern within this genus.

Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichloromethane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractionation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon (εC = -2.7 ± 0.1‰ for respiring cells, -3.1 ± 0.1‰ for cell suspensions, and - 4.1 ± 0.5‰ for crude protein extracts) and chlorine (εCl = -0.9 ± 0.1‰, -1.1 ± 0.1‰, and - 1.2 ± 0.2‰, respectively) isotope fractionation. A characteristic and consistent dual CCl isotope fractionation pattern was observed for the three systems (combined ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.

Long-Read Metagenome-Assembled Genomes Improve Identification of Novel Complete Biosynthetic Gene Clusters in a Complex Microbial Activated Sludge Ecosystem.

Microorganisms produce a wide variety of secondary/specialized metabolites (SMs), the majority of which are yet to be discovered. These natural products play multiple roles in microbiomes and are important for microbial competition, communication, and success in the environment. SMs have been our major source of antibiotics and are used in a range of biotechnological applications. In silico mining for biosynthetic gene clusters (BGCs) encoding the production of SMs is commonly used to assess the genetic potential of organisms. However, as BGCs span tens to over 200 kb, identifying complete BGCs requires genome data that has minimal assembly gaps within the BGCs, a prerequisite that was previously only met by individually sequenced genomes. Here, we assess the performance of the currently available genome mining platform antiSMASH on 1,080 high-quality metagenome-assembled bacterial genomes (HQ MAGs) previously produced from wastewater treatment plants (WWTPs) using a combination of long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies. More than 4,200 different BGCs were identified, with 88% of these being complete. Sequence similarity clustering of the BGCs implies that the majority of this biosynthetic potential likely encodes novel compounds, and few BGCs are shared between genera. We identify BGCs in abundant and functionally relevant genera in WWTPs, suggesting a role of secondary metabolism in this ecosystem. We find that the assembly of HQ MAGs using long-read sequencing is vital to explore the genetic potential for SM production among the uncultured members of microbial communities. IMPORTANCE Cataloguing secondary metabolite (SM) potential using genome mining of metagenomic data has become the method of choice in bioprospecting for novel compounds. However, accurate biosynthetic gene cluster (BGC) detection requires unfragmented genomic assemblies, which have been technically difficult to obtain from metagenomes until very recently with new long-read technologies. Here, we determined the biosynthetic potential of activated sludge (AS), the microbial community used in resource recovery and wastewater treatment, by mining high-quality metagenome-assembled genomes generated from long-read data. We found over 4,000 BGCs, including BGCs in abundant process-critical bacteria, with no similarity to the BGCs of characterized products. We show how long-read MAGs are required to confidently assemble complete BGCs, and we determined that the AS BGCs from different studies have very little overlap, suggesting that AS is a rich source of biosynthetic potential and new bioactive compounds.

Anaerobic bacterial degradation of protein and lipid macromolecules in subarctic marine sediment.

Microorganisms in marine sediments play major roles in marine biogeochemical cycles by mineralizing substantial quantities of organic matter from decaying cells. Proteins and lipids are abundant components of necromass, yet the taxonomic identities of microorganisms that actively degrade them remain poorly resolved. Here, we revealed identities, trophic interactions, and genomic features of bacteria that degraded 13C-labeled proteins and lipids in cold anoxic microcosms containing sulfidic subarctic marine sediment. Supplemented proteins and lipids were rapidly fermented to various volatile fatty acids within 5 days. DNA-stable isotope probing (SIP) suggested Psychrilyobacter atlanticus was an important primary degrader of proteins, and Psychromonas members were important primary degraders of both proteins and lipids. Closely related Psychromonas populations, as represented by distinct 16S rRNA gene variants, differentially utilized either proteins or lipids. DNA-SIP also showed 13C-labeling of various Deltaproteobacteria within 10 days, indicating trophic transfer of carbon to putative sulfate-reducers. Metagenome-assembled genomes revealed the primary hydrolyzers encoded secreted peptidases or lipases, and enzymes for catabolism of protein or lipid degradation products. Psychromonas species are prevalent in diverse marine sediments, suggesting they are important players in organic carbon processing in situ. Together, this study provides new insights into the identities, functions, and genomes of bacteria that actively degrade abundant necromass macromolecules in the seafloor.

Novel taxa of Acidobacteriota implicated in seafloor sulfur cycling.

Acidobacteriota are widespread and often abundant in marine sediments, yet their metabolic and ecological properties are poorly understood. Here, we examined metabolisms and distributions of Acidobacteriota in marine sediments of Svalbard by functional predictions from metagenome-assembled genomes (MAGs), amplicon sequencing of 16S rRNA and dissimilatory sulfite reductase (dsrB) genes and transcripts, and gene expression analyses of tetrathionate-amended microcosms. Acidobacteriota were the second most abundant dsrB-harboring (averaging 13%) phylum after Desulfobacterota in Svalbard sediments, and represented 4% of dsrB transcripts on average. Meta-analysis of dsrAB datasets also showed Acidobacteriota dsrAB sequences are prominent in marine sediments worldwide, averaging 15% of all sequences analysed, and represent most of the previously unclassified dsrAB in marine sediments. We propose two new Acidobacteriota genera, Candidatus Sulfomarinibacter (class Thermoanaerobaculia, "subdivision 23") and Ca. Polarisedimenticola ("subdivision 22"), with distinct genetic properties that may explain their distributions in biogeochemically distinct sediments. Ca. Sulfomarinibacter encode flexible respiratory routes, with potential for oxygen, nitrous oxide, metal-oxide, tetrathionate, sulfur and sulfite/sulfate respiration, and possibly sulfur disproportionation. Potential nutrients and energy include cellulose, proteins, cyanophycin, hydrogen, and acetate. A Ca. Polarisedimenticola MAG encodes various enzymes to degrade proteins, and to reduce oxygen, nitrate, sulfur/polysulfide and metal-oxides. 16S rRNA gene and transcript profiling of Svalbard sediments showed Ca. Sulfomarinibacter members were relatively abundant and transcriptionally active in sulfidic fjord sediments, while Ca. Polarisedimenticola members were more relatively abundant in metal-rich fjord sediments. Overall, we reveal various physiological features of uncultured marine Acidobacteriota that indicate fundamental roles in seafloor biogeochemical cycling.

Genomic insights into diverse bacterial taxa that degrade extracellular DNA in marine sediments.

Extracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments. 13C-DNA added to sediment microcosms was largely degraded within 10 d and mineralized to 13CO2. SIP probing of DNA revealed diverse 'Candidatus Izemoplasma', Lutibacter, Shewanella and Fusibacteraceae incorporated DNA-derived 13C-carbon. NanoSIMS confirmed incorporation of 13C into individual bacterial cells of Fusibacteraceae sorted from microcosms. Genomes of the 13C-labelled taxa all encoded enzymatic repertoires for catabolism of DNA or subcomponents of DNA. Comparative genomics indicated that diverse 'Candidatus Izemoplasmatales' (former Tenericutes) are exceptional because they encode multiple (up to five) predicted extracellular nucleases and are probably specialized DNA-degraders. Analyses of additional sediment metagenomes revealed extracellular nuclease genes are prevalent among Bacteroidota at diverse sites. Together, our results reveal the identities and functional properties of microorganisms that may contribute to the key ecosystem function of degrading and recycling DNA in the seabed.

Woeseiales transcriptional response to shallow burial in Arctic fjord surface sediment.

Distinct lineages of Gammaproteobacteria clade Woeseiales are globally distributed in marine sediments, based on metagenomic and 16S rRNA gene analysis. Yet little is known about why they are dominant or their ecological role in Arctic fjord sediments, where glacial retreat is rapidly imposing change. This study combined 16S rRNA gene analysis, metagenome-assembled genomes (MAGs), and genome-resolved metatranscriptomics uncovered the in situ abundance and transcriptional activity of Woeseiales with burial in four shallow sediment sites of Kongsfjorden and Van Keulenfjorden of Svalbard (79°N). We present five novel Woeseiales MAGs and show transcriptional evidence for metabolic plasticity during burial, including sulfur oxidation with reverse dissimilatory sulfite reductase (dsrAB) down to 4 cm depth and nitrite reduction down to 6 cm depth. A single stress protein, spore protein SP21 (hspA), had a tenfold higher mRNA abundance than any other transcript, and was a hundredfold higher on average than other transcripts. At three out of the four sites, SP21 transcript abundance increased with depth, while total mRNA abundance and richness decreased, indicating a shift in investment from metabolism and other cellular processes to build-up of spore protein SP21. The SP21 gene in MAGs was often flanked by genes involved in membrane-associated stress response. The ability of Woeseiales to shift from sulfur oxidation to nitrite reduction with burial into marine sediments with decreasing access to overlying oxic bottom waters, as well as enter into a dormant state dominated by SP21, may account for its ubiquity and high abundance in marine sediments worldwide, including those of the rapidly shifting Arctic.

Rational design of a microbial consortium of mucosal sugar utilizers reduces Clostridiodes difficile colonization.

Many intestinal pathogens, including Clostridioides difficile, use mucus-derived sugars as crucial nutrients in the gut. Commensals that compete with pathogens for such nutrients are therefore ecological gatekeepers in healthy guts, and are attractive candidates for therapeutic interventions. Nevertheless, there is a poor understanding of which commensals use mucin-derived sugars in situ as well as their potential to impede pathogen colonization. Here, we identify mouse gut commensals that utilize mucus-derived monosaccharides within complex communities using single-cell stable isotope probing, Raman-activated cell sorting and mini-metagenomics. Sequencing of cell-sorted fractions reveals members of the underexplored family Muribaculaceae as major mucin monosaccharide foragers, followed by members of Lachnospiraceae, Rikenellaceae, and Bacteroidaceae families. Using this information, we assembled a five-member consortium of sialic acid and N-acetylglucosamine utilizers that impedes C. difficile's access to these mucosal sugars and impairs pathogen colonization in antibiotic-treated mice. Our findings underscore the value of targeted approaches to identify organisms utilizing key nutrients and to rationally design effective probiotic mixtures.

Novel alkane hydroxylase gene (alkB) diversity in sediments associated with hydrocarbon seeps in the Timor Sea, Australia.

Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem's response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation.