The Environmental Stress Pathway Project (ESPP) focuses on the systems-biology and environmental activity of the sulfate-reducing bacterium (SRB) Desulfovibrio vulgaris Hildenborough (DvH). However, focusing on a single representative species of a genus offers little insight into the evolution, niche adaptation, and function of an entire microbial genus. Given the broad environmental significance of SRBs and their phylogenetic diversity, it is imperative that other representative species of the Desulfovibrio genus are investigated. Therefore, we are developing an integrated functional genomics platform in the SRB Desulfovibrio desulfuricans G20 (G20). The platform is compromised of phenotypic arrays, gene expression arrays, and a transposon mutant library. The ability of G20 to utilize carbon sources and electron acceptors for growth, survive stress conditions, and respond to growth inhibitors is measured on a high-throughput system to narrow the test conditions to be used in both gene expression and mutant profiling assays. Using high-density multiplex microarrays, we are monitoring gene expression under a number of diverse conditions in a global effort to elucidate G20 gene regulation. Our principle effort is the construction of a genome-wide, tagged transposon mutant library in G20. A hallmark of our transposon library is the ability to pool thousands of mutants for parallel phenotypic analysis. Profiling our transposon library under a large number of conditions will enable us to link a large fraction of the G20 genome to specific cellular phenotypes. Here we present our experimental approaches and preliminary data from each type of experiment. Functional genomic investigations into G20 will be used by the ESPP in a number of ways. First, G20 and DvH have a large number of orthologous genes. Therefore, functional insights made in G20 are a strong starting point for targeted experiments in DvH. Second, by expanding our experimental ?pan-genome? to include G20, we are now investigating more of the natural variation present in the genus Desulfovibrio. These data will be imperative for annotating and analyzing newly sequenced SRB genomes. Lastly, our integrated functional genomics data will be used to build a cellular model of G20 metabolism, regulation, and activity. This model can be used as a framework for the interpretation of many types of ESPP data generated in the laboratory and in the field.

The Environmental Stress Pathway Project (ESPP) aims to elucidate the molecular mechanisms by which microbial communities affect stress response and activity in the field and in the laboratory using sulfate reducing bacteria (SRB) as a model. To accomplish our goal of linking field observations to those in the laboratory, a systems-level understanding of SRB genome function is necessary. To meet this challenge, we are developing a mutagenesis and phenotyping strategy that is comprehensive across the genome and applicable to any microorganism amenable to transposon mutagenesis. Here we describe the application of our strategy to Shewanella oneidensis MR1 and the SRB Desulfovibrio desulfuricans G20. We have cloned and sequence-verified ~;;3000 barcode modules into a Gateway entry vector. Each module is a 175 base pair element containing two unique 20 base pair sequences, the UPTAG and DOWNTAG, flanked by common PCR priming sites. Each module can then be rapidly transferred in vitro to any DNA element, such as a transposon, that is made Gateway compatible. Transposon mutants marked by the modules will be sequenced to determine which of the ~;;3000 barcode modules was used and which gene was disrupted. Transposon mutants can be rapidly re-arrayed into a single pool containing ~;;3000 uniquely tagged, sequence-verified mutant strains. By sequencing saturating numbers of transposon mutants, we can identify and assay mutants in most nonessential genes in a given genome. The fitness of each mutant in the pool will be monitored in parallel by the hybridization of the barcodes to an Affymetrix microarray containing the barcode complements in a system identical to that used for the yeast deletion collection. Compared to other approaches for the parallel analysis of transposon mutants such as signature tagged mutagenesis, genetic footprinting, and transposon site hybridization, our approach offers much higher throughput, a single microarray design is universal for any organism, single mutational events are assayed, and mutant strains are archived for verification, distribution, and the systematic genetic interrogation of individual pathways. The successful completion of this project will enable the quantitative phenotypic analysis of thousands of mutants across a wide range of conditions. These data will be used to assign gene function on a global scale, aid in the identification of missing metabolic enzymes, and provide insight into the functional connectivity of different pathways. Our results in Desulfovibrio desulfuricans G20 will be extrapolated via comparative genomic analysis to other sequenced SRBs including Desulfovibrio vulgaris Hildenborough. Consequently, our findings will aid in the interpretation of both laboratory and field data collected by the ESPP.

The paradigm of large research communities collectively working on a small number of model bacteria such as Escherichia coli and Bacillus subtilis is changing. While these classic model bacteria will continue to be important for advanced systems biology and new technology development, we envision that increasingly small research teams will be deeply investigating their own favorite strains, for example as new hosts for metabolic engineering or as key members of a complex microbiome. Given the lack of a research community and the sheer number of possible bacteria to interrogate, the development and application of technologies to rapidly and inexpensively advance these unstudied strains to 'model-organism' status is imperative. Here, we discuss the minimal information and tools necessary to develop a new model bacterium and how existing approaches can bring this power into the hands of a single investigator.

Multidimensional gradients of inorganic compounds influence microbial activity in diverse pristine and anthropogenically perturbed environments. Here, we suggest that high-throughput cultivation and genetics can be systematically applied to generate quantitative models linking gene function, microbial community activity, and geochemical parameters. Metal resistance determinants represent a uniquely universal set of parameters around which to study and evaluate microbial fitness because they represent a record of the environment in which all microbial life evolved. By cultivating microbial isolates and enrichments in laboratory gradients of inorganic ions, we can generate quantitative predictions of limits on microbial range in the environment, obtain more accurate gene annotations, and identify useful strategies for predicting and engineering the trajectory of natural ecosystems.

The mineralization of organic matter in anoxic environments relies on the cooperative activities of hydrogen producers and consumers obligately linked by interspecies metabolite exchange in syntrophic consortia that may include sulfate reducing species such as Desulfovibrio. To evaluate the metabolic flexibility of syntrophic Desulfovibrio to adapt to naturally fluctuating methanogenic environments, we studied Desulfovibrio alaskensis strain G20 grown in chemostats under respiratory and syntrophic conditions with alternative methanogenic partners, Methanococcus maripaludis and Methanospirillum hungatei, at different growth rates. Comparative whole-genome transcriptional analyses, complemented by G20 mutant strain growth experiments and physiological data, revealed a significant influence of both energy source availability (as controlled by dilution rate) and methanogen on the electron transfer systems, ratios of interspecies electron carriers, energy generating systems, and interspecies physical associations. A total of 68 genes were commonly differentially expressed under syntrophic versus respiratory lifestyle. Under low-energy (low-growth-rate) conditions, strain G20 further had the capacity to adapt to the metabolism of its methanogenic partners, as shown by its differing gene expression of enzymes involved in the direct metabolic interactions (e.g., periplasmic hydrogenases) and the ratio shift in electron carriers used for interspecies metabolite exchange (hydrogen/formate). A putative monomeric [Fe-Fe] hydrogenase and Hmc (high-molecular-weight-cytochrome c3) complex-linked reverse menaquinone (MQ) redox loop become increasingly important for the reoxidation of the lactate-/pyruvate oxidation-derived redox pair, DsrC(red) and Fd(red), relative to the Qmo-MQ-Qrc (quinone-interacting membrane-bound oxidoreductase; quinone-reducing complex) loop. Together, these data underscore the high enzymatic and metabolic adaptive flexibility that likely sustains Desulfovibrio in naturally fluctuating methanogenic environments.

Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function. Importance: The first step of gene expression is the initiation of transcription from promoters, which have been traditionally thought to be located upstream of genes. Recently, studies showed that in diverse bacteria, promoters are often located inside genes. It has not been clear if these unexpected promoters are important to the organism or if they result from transcriptional noise. Here, we identify and examine promoters in eight related bacterial species. Promoters that lie within genes on the sense strand are often conserved as locations and in their sequences. Furthermore, these promoters often affect the bacteriums growth. Thus, many of these unexpected promoters are likely functional. Fewer promoters that lie within genes on the antisense strand are conserved, but the conserved ones seem to drive the expression of nearby genes.

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro , the characterized core synthases use methylcobalamin as a methyl donor, but in vivo , they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

The foliar plant pathogen Pseudomonas syringae can establish large epiphytic populations on leaf surfaces before apoplastic colonization. However, the bacterial genes that contribute to these lifestyles have not been completely defined. The fitness contributions of 4,296 genes in P. syringae pv. syringae B728a were determined by genome-wide fitness profiling with a randomly barcoded transposon mutant library that was grown on the leaf surface and in the apoplast of the susceptible plant Phaseolus vulgaris Genes within the functional categories of amino acid and polysaccharide (including alginate) biosynthesis contributed most to fitness both on the leaf surface (epiphytic) and in the leaf interior (apoplast), while genes involved in type III secretion system and syringomycin synthesis were primarily important in the apoplast. Numerous other genes that had not been previously associated with in planta growth were also required for maximum epiphytic or apoplastic fitness. Fourteen hypothetical proteins and uncategorized glycosyltransferases were also required for maximum competitive fitness in and on leaves. For most genes, no relationship was seen between fitness in planta and either the magnitude of their expression in planta or degree of induction in planta compared to in vitro conditions measured in other studies. A lack of association of gene expression and fitness has important implications for the interpretation of transcriptional information and our broad understanding of plant-microbe interactions.

Although most organisms synthesize methionine from homocysteine and methyl folates, some have "core" methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants.