Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

The diversity and evolution of microbial dissimilatory phosphite oxidation.

Phosphite is the most energetically favorable chemotrophic electron donor known, with a half-cell potential (Eo') of -650 mV for the PO43-/PO33- couple. Since the discovery of microbial dissimilatory phosphite oxidation (DPO) in 2000, the environmental distribution, evolution, and diversity of DPO microorganisms (DPOMs) have remained enigmatic, as only two species have been identified. Here, metagenomic sequencing of phosphite-enriched microbial communities enabled the genome reconstruction and metabolic characterization of 21 additional DPOMs. These DPOMs spanned six classes of bacteria, including the Negativicutes, Desulfotomaculia, Synergistia, Syntrophia, Desulfobacteria, and Desulfomonilia_A Comparing the DPO genes from the genomes of enriched organisms with over 17,000 publicly available metagenomes revealed the global existence of this metabolism in diverse anoxic environments, including wastewaters, sediments, and subsurface aquifers. Despite their newfound environmental and taxonomic diversity, metagenomic analyses suggested that the typical DPOM is a chemolithoautotroph that occupies low-oxygen environments and specializes in phosphite oxidation coupled to CO2 reduction. Phylogenetic analyses indicated that the DPO genes form a highly conserved cluster that likely has ancient origins predating the split of monoderm and diderm bacteria. By coupling microbial cultivation strategies with metagenomics, these studies highlighted the unsampled metabolic versatility latent in microbial communities. We have uncovered the unexpected prevalence, diversity, biochemical specialization, and ancient origins of a unique metabolism central to the redox cycling of phosphorus, a primary nutrient on Earth.

Tungstate Control of Microbial Sulfidogenesis and Souring of the Engineered Environment.

Sulfide accumulation in oil reservoir fluids (souring) from the activity of sulfate-reducing microorganisms (SRM) is of grave concern because of the associated health and facility failure risks. Here, we present an assessment of tungstate as a selective and potent inhibitor of SRM. Dose-response inhibitor experiments were conducted with a number of SRM isolates and enrichments at 30-80 °C and an increase in the effectiveness of tungstate treatment at higher temperatures was observed. To explore mixed inhibitor treatment modes, we tested synergy or antagonism between several inhibitors with tungstate, and found synergism between WO42- and NO2-, while additive effects were observed with ClO4- and NO3-. We also evaluated SRM inhibition by tungstate in advective upflow oil-sand-packed columns. Although 2 mM tungstate was initially sufficient to inhibit sulfidogenesis, subsequent temporal CaWO4 precipitation resulted in loss of the bioavailable inhibitor from solution and a concurrent increase in effluent sulfide. Mixing 4 mM sodium carbonate with the 2 mM tungstate was enough to promote tungstate solubility to reach inhibitory concentrations, without precipitation, and completely inhibit SRM activity. Overall, we demonstrate the effectiveness of tungstate as a potent SRM inhibitor, particularly at higher temperatures, and propose a novel carbonate-tungstate formulation for application to soured oil reservoirs.

Adaptation of Desulfovibrio alaskensis G20 to perchlorate, a specific inhibitor of sulfate reduction.

Hydrogen sulfide produced by sulfate-reducing microorganisms (SRM) poses significant health and economic risks, particularly during oil recovery. Previous studies identified perchlorate as a specific inhibitor of SRM. However, constant inhibitor addition to natural systems results in new selective pressures. Consequently, we investigated the ability of Desulfovibrio alaskensis G20 to evolve perchlorate resistance. Serial transfers in increasing concentrations of perchlorate led to robust growth in the presence of 100 mM inhibitor. Isolated adapted strains demonstrated a threefold increase in perchlorate resistance compared to the wild-type ancestor. Whole genome sequencing revealed a single base substitution in Dde_2265, the sulfate adenylyltransferase (sat). We purified and biochemically characterized the Sat from both wild-type and adapted strains, and showed that the adapted Sat was approximately threefold more resistant to perchlorate inhibition, mirroring whole cell results. The ability of this mutation to confer resistance across other inhibitors of sulfidogenesis was also assayed. The generalizability of this mutation was confirmed in multiple evolving G20 cultures and in another SRM, D. vulgaris Hildenborough. This work demonstrates that a single nucleotide polymorphism in Sat can have a significant impact on developing perchlorate resistance and emphasizes the value of adaptive laboratory evolution for understanding microbial responses to environmental perturbations.

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP.

Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 µm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

High-Throughput Screening To Identify Potent and Specific Inhibitors of Microbial Sulfate Reduction.

The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive, and corrosive. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to identify potent and selective inhibitors of SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Zinc pyrithione is the most potent inhibitor of sulfidogenesis that we identified, and is several orders of magnitude more potent than commonly used industrial biocides. Both zinc and copper pyrithione are also moderately selective against SRM. The high-throughput (HT) approach we present can be readily adapted to target SRM in diverse environments and similar strategies could be used to quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant to efforts to engineer environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms.

Despite the environmental and economic cost of microbial sulfidogenesis in industrial operations, few compounds are known as selective inhibitors of respiratory sulfate reducing microorganisms (SRM), and no study has systematically and quantitatively evaluated the selectivity and potency of SRM inhibitors. Using general, high-throughput assays to quantitatively evaluate inhibitor potency and selectivity in a model sulfate-reducing microbial ecosystem as well as inhibitor specificity for the sulfate reduction pathway in a model SRM, we screened a panel of inorganic oxyanions. We identified several SRM selective inhibitors including selenate, selenite, tellurate, tellurite, nitrate, nitrite, perchlorate, chlorate, monofluorophosphate, vanadate, molydate, and tungstate. Monofluorophosphate (MFP) was not known previously as a selective SRM inhibitor, but has promising characteristics including low toxicity to eukaryotic organisms, high stability at circumneutral pH, utility as an abiotic corrosion inhibitor, and low cost. MFP remains a potent inhibitor of SRM growing by fermentation, and MFP is tolerated by nitrate and perchlorate reducing microorganisms. For SRM inhibition, MFP is synergistic with nitrite and chlorite, and could enhance the efficacy of nitrate or perchlorate treatments. Finally, MFP inhibition is multifaceted. Both inhibition of the central sulfate reduction pathway and release of cytoplasmic fluoride ion are implicated in the mechanism of MFP toxicity.

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria.

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Phylogenetic distribution and experimental characterization of corrinoid production and dependence in soil bacterial isolates.

Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids-the vitamin B12 family of cofactors-are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microorganisms involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.

Fe(II) oxidation is an innate capability of nitrate-reducing bacteria that involves abiotic and biotic reactions.

Phylogenetically diverse species of bacteria can catalyze the oxidation of ferrous iron [Fe(II)] coupled to nitrate (NO(3)(-)) reduction, often referred to as nitrate-dependent iron oxidation (NDFO). Very little is known about the biochemistry of NDFO, and though growth benefits have been observed, mineral encrustations and nitrite accumulation likely limit growth. Acidovorax ebreus, like other species in the Acidovorax genus, is proficient at catalyzing NDFO. Our results suggest that the induction of specific Fe(II) oxidoreductase proteins is not required for NDFO. No upregulated periplasmic or outer membrane redox-active proteins, like those involved in Fe(II) oxidation by acidophilic iron oxidizers or anaerobic photoferrotrophs, were observed in proteomic experiments. We demonstrate that while "abiotic" extracellular reactions between Fe(II) and biogenic NO(2)(-)/NO can be involved in NDFO, intracellular reactions between Fe(II) and periplasmic components are essential to initiate extensive NDFO. We present evidence that an organic cosubstrate inhibits NDFO, likely by keeping periplasmic enzymes in their reduced state, stimulating metal efflux pumping, or both, and that growth during NDFO relies on the capacity of a nitrate-reducing bacterium to overcome the toxicity of Fe(II) and reactive nitrogen species. On the basis of our data and evidence in the literature, we postulate that all respiratory nitrate-reducing bacteria are innately capable of catalyzing NDFO. Our findings have implications for a mechanistic understanding of NDFO, the biogeochemical controls on anaerobic Fe(II) oxidation, and the production of NO(2)(-), NO, and N(2)O in the environment.

Surfaceomics and surface-enhanced Raman spectroscopy of environmental microbes: matching cofactors with redox-active surface proteins.

Trypsin shaving is a targeted proteomic method for identifying cell-surface exposed proteins on bacterial cells. For the identification of redox-active cell-surface proteins, trypsin-shaving datasets can be matched with surface-enhanced Raman spectra of intact cells to identify the cofactors associated with the cell-surface proteins. Together, these approaches could help resolve questions about the presence of cell-surface electron transport components in environmental microorganisms, especially microbes that oxidize and reduce metals and metalloids as electron donors and acceptors.

H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri.

The bioluminescent bacterium Vibrio fischeri initiates a specific, persistent symbiosis in the light organ of the squid Euprymna scolopes. During the early stages of colonization, V. fischeri is exposed to host-derived nitric oxide (NO). Although NO can be both an antimicrobial component of innate immunity and a key signaling molecule in eukaryotes, potential roles in beneficial host-microbe associations have not been described. V. fischeri hnoX encodes a heme NO/oxygen-binding (H-NOX) protein, a member of a family of bacterial NO- and/or O(2)-binding proteins of unknown function. We hypothesized that H-NOX acts as a NO sensor that is involved in regulating symbiosis-related genes early in colonization. Whole-genome expression studies identified 20 genes that were repressed in an NO- and H-NOX-dependent fashion. Ten of these, including hemin-utilization genes, have a promoter with a putative ferric-uptake regulator (Fur) binding site. As predicted, in the presence of NO, wild-type V. fischeri grew more slowly on hemin than a hnoX deletion mutant. Host-colonization studies showed that the hnoX mutant was also 10-fold more efficient in initially colonizing the squid host than the wild type; similarly, in mixed inoculations, it outcompeted the wild-type strain by an average of 16-fold after 24 h. However, the presence of excess hemin or iron reversed this dominance. The advantage of the mutant in colonizing the iron-limited light-organ tissues is caused, at least in part, by its greater ability to acquire host-derived hemin. Our data suggest that V. fischeri normally senses a host-generated NO signal through H-NOX(Vf) and modulates the expression of its iron uptake capacity during the early stages of the light-organ symbiosis.

Geochemical constraints on bacteriophage infectivity in terrestrial environments.

Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in the environment, we do not know how this variability influences the impact of phage predation on microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. Many alkaline earth metals and alkali metals enabled lytic infection but the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate-reducing microbiome, we found that the ability of lytic phages to influence nitrate reduction end-products depended upon the carbon source as well as ionic strength. For all phage:host pairs, the ion EC50s for phage infection exceeded the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where phages most influence terrestrial microbial functional ecology in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and nutrients are available to support the growth of specific phage hosts.

A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe's vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions.

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila.

UNLABELLED: Haem Nitric oxide/OXygen (H-NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H-NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H-NOXs in the regulation of biofilm formation. IN SUMMARY: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Δhnox1 strain displays a hyper-biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF-EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well-studied diguanylate cyclase, vca0956, results in a hyper-biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)-NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Δhnox1 background results in reversion of the hyper-biofilm phenotype back to wild-type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c-di-GMP production by lpg1057 and biofilm formation in response to NO.

Identification of a perchlorate reduction genomic island with novel regulatory and metabolic genes.

A comparative analysis of the genomes of four dissimilatory (per)chlorate-reducing bacteria has revealed a genomic island associated with perchlorate reduction. In addition to the characterized metabolic genes for perchlorate reductase and chlorite dismutase, the island contains multiple conserved uncharacterized genes possibly involved in electron transport and regulation.