Biosynthetic potential of the global ocean microbiome.

Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters ('Candidatus Eudoremicrobiaceae') that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.

Synthetic microbiota reveal priority effects and keystone strains in the Arabidopsis phyllosphere.

Multicellular organisms, including plants, are colonized by microorganisms, some of which are beneficial to growth and health. The assembly rules for establishing plant microbiota are not well understood, and neither is the extent to which their members interact. We conducted drop-out and late introduction experiments by inoculating Arabidopsis thaliana with synthetic communities from a resource of 62 native bacterial strains to test how arrival order shapes community structure. As a read-out we tracked the relative abundance of all strains in the phyllosphere of individual plants. Our results showed that community assembly is historically contingent and subject to priority effects. Missing strains could, to various degrees, invade an already established microbiota, which was itself resistant and remained largely unaffected by latecomers. Additionally, our results indicate that individual strains of Proteobacteria (Sphingomonas, Rhizobium) and Actinobacteria (Microbacterium, Rhodococcus) have the greatest potential to affect community structure as keystone species.

Genome-resolved metagenomics identifies genetic mobility, metabolic interactions, and unexpected diversity in perchlorate-reducing communities.

Dissimilatory perchlorate reduction is an anaerobic respiratory pathway that in communities might be influenced by metabolic interactions. Because the genes for perchlorate reduction are horizontally transferred, previous studies have been unable to identify uncultivated perchlorate-reducing populations. Here we recovered metagenome-assembled genomes from perchlorate-reducing sediment enrichments and employed a manual scaffolding approach to reconstruct gene clusters for perchlorate reduction found within mobile genetic elements. De novo assembly and binning of four enriched communities yielded 48 total draft genomes. In addition to canonical perchlorate reduction gene clusters and taxa, a new type of gene cluster with an alternative perchlorate reductase was identified. Phylogenetic analysis indicated past exchange between these gene clusters, and the presence of plasmids with either gene cluster shows that the potential for gene transfer via plasmid persisted throughout enrichment. However, a majority of genomes in each community lacked perchlorate reduction genes. Putative chlorate-reducing or sulfur-reducing populations were dominant in most communities, supporting the hypothesis that metabolic interactions might result from perchlorate reduction intermediates and byproducts. Other populations included a novel phylum-level lineage (Ca. Muirbacteria) and epibiotic prokaryotes with no known role in perchlorate reduction. These results reveal unexpected genetic diversity, suggest that perchlorate-reducing communities involve substantial metabolic interactions, and encourage expanded strategies to further understand the evolution and ecology of this metabolism.

Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway.

Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO32-) is oxidized to phosphate (PO43-), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2 reduction. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidation and for CO2 reduction to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO2 reduction to formate, which is then assimilated into biomass via the reductive glycine pathway.

Establishing Causality: Opportunities of Synthetic Communities for Plant Microbiome Research.

Plant microbiome research highlights the importance of indigenous microbial communities for host phenotypes such as growth and health. It aims to discover the molecular basis by which host-microbe and microbe-microbe interactions shape and maintain microbial communities and to understand the role of individual microorganisms, as well as their collective ecosystem function. Here, we discuss reductionist approaches to disentangle the inherent complexity of interactions in situ. Experimentally tractable, synthetic communities enable testing of hypotheses by targeted manipulation in gnotobiotic systems. Modifications of microbial, host, and environmental parameters allow for the quantitative assessment of host and microbe characteristics with dynamic and spatial resolution. We summarize first insights from this emerging field and discuss current challenges and limitations. Using multifaceted approaches to detect interactions and functions will provide new insights into the fundamental biology of plant-microbe interactions and help to harness the power of the microbiome.

Characterization of an anaerobic marine microbial community exposed to combined fluxes of perchlorate and salinity.

The recent recognition of the environmental prevalence of perchlorate and its discovery on Mars, Earth's moon, and in meteorites, in addition to its novel application to controlling oil reservoir sulfidogenesis, has resulted in a renewed interest in this exotic ion and its associated microbiology. However, while plentiful data exists on freshwater perchlorate respiring organisms, information on their halophilic counterparts and microbial communities is scarce. Here, we investigated the temporal evolving structure of perchlorate respiring communities under a range of NaCl concentrations (1, 3, 5, 7, and 10 % wt/vol) using marine sediment amended with acetate and perchlorate. In general, perchlorate consumption rates were inversely proportional to NaCl concentration with the most rapid rate observed at 1 % NaCl. At 10 % NaCl, no perchlorate removal was observed. Transcriptional analysis of the 16S rRNA gene indicated that salinity impacted microbial community structure and the most active members were in families Rhodocyclaceae (1 and 3 % NaCl), Pseudomonadaceae (1 NaCl), Campylobacteraceae (1, 5, and 7 % NaCl), Sedimenticolaceae (3 % NaCl), Desulfuromonadaceae (5 and 7 % NaCl), Pelobacteraceae (5 % NaCl), Helicobacteraceae (5 and 7 % NaCl), and V1B07b93 (7 %). Novel isolates of genera Sedimenticola, Marinobacter, Denitromonas, Azoarcus, and Pseudomonas were obtained and their perchlorate respiring capacity confirmed. Although the obligate anaerobic, sulfur-reducing Desulfuromonadaceae species were dominant at 5 and 7 % NaCl, their enrichment may result from biological sulfur cycling, ensuing from the innate ability of DPRB to oxidize sulfide. Additionally, our results demonstrated enrichment of an archaeon of phylum Parvarchaeota at 5 % NaCl. To date, this phylum has only been described in metagenomic experiments of acid mine drainage and is unexpected in a marine community. These studies identify the intrinsic capacity of marine systems to respire perchlorate and significantly expand the known diversity of organisms capable of this novel metabolism.

(Per)chlorate-reducing bacteria can utilize aerobic and anaerobic pathways of aromatic degradation with (per)chlorate as an electron acceptor.

UNLABELLED: The pathways involved in aromatic compound oxidation under perchlorate and chlorate [collectively known as (per)chlorate]-reducing conditions are poorly understood. Previous studies suggest that these are oxygenase-dependent pathways involving O2 biogenically produced during (per)chlorate respiration. Recently, we described Sedimenticola selenatireducens CUZ and Dechloromarinus chlorophilus NSS, which oxidized phenylacetate and benzoate, two key intermediates in aromatic compound catabolism, coupled to the reduction of perchlorate or chlorate, respectively, and nitrate. While strain CUZ also oxidized benzoate and phenylacetate with oxygen as an electron acceptor, strain NSS oxidized only the latter, even at a very low oxygen concentration (1%, vol/vol). Strains CUZ and NSS contain similar genes for both the anaerobic and aerobic-hybrid pathways of benzoate and phenylacetate degradation; however, the key genes (paaABCD) encoding the epoxidase of the aerobic-hybrid phenylacetate pathway were not found in either genome. By using transcriptomics and proteomics, as well as by monitoring metabolic intermediates, we investigated the utilization of the anaerobic and aerobic-hybrid pathways on different electron acceptors. For strain CUZ, the results indicated utilization of the anaerobic pathways with perchlorate and nitrate as electron acceptors and of the aerobic-hybrid pathways in the presence of oxygen. In contrast, proteomic results suggest that strain NSS may use a combination of the anaerobic and aerobic-hybrid pathways when growing on phenylacetate with chlorate. Though microbial (per)chlorate reduction produces molecular oxygen through the dismutation of chlorite (ClO2(-)), this study demonstrates that anaerobic pathways for the degradation of aromatics can still be utilized by these novel organisms. IMPORTANCE: S. selenatireducens CUZ and D. chlorophilus NSS are (per)chlorate- and chlorate-reducing bacteria, respectively, whose genomes encode both anaerobic and aerobic-hybrid pathways for the degradation of phenylacetate and benzoate. Previous studies have shown that (per)chlorate-reducing bacteria and chlorate-reducing bacteria (CRB) can use aerobic pathways to oxidize aromatic compounds in otherwise anoxic environments by capturing the oxygen produced from chlorite dismutation. In contrast, we demonstrate that S. selenatireducens CUZ is the first perchlorate reducer known to utilize anaerobic aromatic degradation pathways with perchlorate as an electron acceptor and that it does so in preference over the aerobic-hybrid pathways, regardless of any oxygen produced from chlorite dismutation. D. chlorophilus NSS, on the other hand, may be carrying out anaerobic and aerobic-hybrid processes simultaneously. Concurrent use of anaerobic and aerobic pathways has not been previously reported for other CRB or any microorganisms that encode similar pathways of phenylacetate or benzoate degradation and may be advantageous in low-oxygen environments.

Phenotypic and genotypic description of Sedimenticola selenatireducens strain CUZ, a marine (per)chlorate-respiring gammaproteobacterium, and its close relative the chlorate-respiring Sedimenticola strain NSS.

Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30 °C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42 °C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.

Methane oxidation linked to chlorite dismutation.

We examined the potential for CH4 oxidation to be coupled with oxygen derived from the dissimilatory reduction of perchlorate, chlorate, or via chlorite (ClO(-) 2) dismutation. Although dissimilatory reduction of ClO(-) 4 and ClO(-) 3 could be inferred from the accumulation of chloride ions either in spent media or in soil slurries prepared from exposed freshwater lake sediment, neither of these oxyanions evoked methane oxidation when added to either anaerobic mixed cultures or soil enriched in methanotrophs. In contrast, ClO(-) 2 amendment elicited such activity. Methane (0.2 kPa) was completely removed within several days from the headspace of cell suspensions of Dechloromonas agitata CKB incubated with either Methylococcus capsulatus Bath or Methylomicrobium album BG8 in the presence of 5 mM ClO(-) 2. We also observed complete removal of 0.2 kPa CH4 in bottles containing soil enriched in methanotrophs when co-incubated with D. agitata CKB and 10 mM ClO(-) 2. However, to be effective these experiments required physical separation of soil from D. agitata CKB to allow for the partitioning of O2 liberated from chlorite dismutation into the shared headspace. Although a link between ClO(-) 2 and CH4 consumption was established in soils and cultures, no upstream connection with either ClO(-) 4 or ClO(-) 3 was discerned. This result suggests that the release of O2 during enzymatic perchlorate reduction was negligible, and that the oxygen produced was unavailable to the aerobic methanotrophs.

Physiological and genetic description of dissimilatory perchlorate reduction by the novel marine bacterium Arcobacter sp. strain CAB.

A novel dissimilatory perchlorate-reducing bacterium (DPRB), Arcobacter sp. strain CAB, was isolated from a marina in Berkeley, CA. Phylogenetically, this halophile was most closely related to Arcobacter defluvii strain SW30-2 and Arcobacter ellisii. With acetate as the electron donor, strain CAB completely reduced perchlorate (ClO4(-)) or chlorate (ClO3(-)) [collectively designated (per)chlorate] to innocuous chloride (Cl(-)), likely using the perchlorate reductase (Pcr) and chlorite dismutase (Cld) enzymes. When grown with perchlorate, optimum growth was observed at 25 to 30°C, pH 7, and 3% NaCl. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations were dominated by free-swimming straight rods with 1 to 2 polar flagella per cell. Strain CAB utilized a variety of organic acids, fructose, and hydrogen as electron donors coupled to (per)chlorate reduction. Further, under anoxic growth conditions strain CAB utilized the biogenic oxygen produced as a result of chlorite dismutation to oxidize catechol via the meta-cleavage pathway of aerobic catechol degradation and the catechol 2,3-dioxygenase enzyme. In addition to (per)chlorate, oxygen and nitrate were alternatively used as electron acceptors. The 3.48-Mb draft genome encoded a distinct perchlorate reduction island (PRI) containing several transposases. The genome lacks the pcrC gene, which was previously thought to be essential for (per)chlorate reduction, and appears to use an unrelated Arcobacter c-type cytochrome to perform the same function. IMPORTANCE The study of dissimilatory perchlorate-reducing bacteria (DPRB) has largely focused on freshwater, mesophilic, neutral-pH environments. This study identifies a novel marine DPRB in the genus Arcobacter that represents the first description of a DPRB associated with the Campylobacteraceae. Strain CAB is currently the only epsilonproteobacterial DPRB in pure culture. The genome of strain CAB lacks the pcrC gene found in all other DPRB tested, demonstrating a new variation on the (per)chlorate reduction pathway. The ability of strain CAB to oxidize catechol via the oxygenase-dependent meta-cleavage pathway in the absence of external oxygen by using the biogenic oxygen produced from the dismutation of chlorite provides a valuable model for understanding the anaerobic degradation of a broad diversity of xenobiotics which are recalcitrant to anaerobic metabolism but labile to oxygenase-dependent mechanisms.