Signature-tagged mutagenesis (STM) is a powerful technique that can be used to identify genes expressed by bacteria during exposure to conditions in their natural environments. To date, there have been no reports of studies in which this approach was used to study organisms of environmental, rather than pathogenic, significance. We used a mini-Tn10 transposon-bearing plasmid, pBSL180, that efficiently and randomly mutagenized Desulfovibrio desulfuricans G20 in addition to Shewanella oneidensis MR-1. Using these organisms as model sediment-dwelling anaerobic bacteria, we developed a new screening system, modified from former STM procedures, to identify genes that are critical for sediment survival. The screening system uses microarray technology to visualize tags from input and output pools, allowing us to identify those lost during sediment incubations. While the majority of data on survival genes identified will be presented in future papers, we report here on chemotaxis-related genes identified by our STM method in both bacteria in order to validate our method. This system may be applicable to the study of numerous environmental bacteria, allowing us to identify functions and roles of survival genes in various habitats.

Environmental bacteria persist in various habitats, yet little is known about the genes that contribute to growth and survival in their respective ecological niches. Signature-tagged mutagenesis (STM) of Shewanella oneidensis MR-1 coupled with a screen involving incubations of mutant strains in anoxic aquifer sediments allowed us to identify 47 genes that enhance fitness in sediments. Gene functions inferred from annotations provide us with insight into physiological and ecological processes that environmental bacteria use while growing in sediment ecosystems. Identification of the mexF gene and other potential membrane efflux components by STM demonstrated that homologues of multidrug resistance genes present in pathogens are required for sediment fitness of nonpathogenic bacteria. Further studies with a mexF deletion mutant demonstrated that the multidrug resistance pump encoded by mexF is required for resistance to antibiotics, including chloramphenicol and tetracycline. Chloramphenicol-adapted cultures exhibited mutations in the gene encoding a TetR family regulatory protein, indicating a role for this protein in regulating expression of the mexEF operon. The relative importance of mexF for sediment fitness suggests that antibiotic efflux may be a required process for bacteria living in sediment systems.Ecology encompasses the study of organisms and their interactions with one another and with the surrounding environment. In the field of microbiology, observations of these relationships are far less direct than studies of macroscopic organisms, as we cannot readily observe, in a traditional sense, in situ processes that occur at molecular to microscopic levels. Some information can be ascertained from studies that monitor microbial communities (33) or dominant respiratory processes (23), but in situ molecular scale interactions often elude us. While pure culture studies can monitor physiological and molecular changes (e.g., through microarray and proteomic analyses) in response to defined components of a habitat, extrapolation of laboratory results to the real environment is often not possible. From an environmental standpoint, an in situ perspective of bacterial fitness would provide a greater understanding of processes that are of fundamental ecological importance, and the knowledge gained can then be applied toward enhancement of beneficial microbial activities (e.g., bioremediation) or prevention of disruptive activities (e.g., oil well souring).In this study, 47 genes that aid in bacterial ecological fitness in sediments were identified using in situ-based signaturetagged mutagenesis (STM) (12). STM is one technique by which whole genomes are screened for genes that enable bacteria to survive in their natural habitats. Genes involved in general cellular processes of DNA repair, transport, transcriptional regulation, and energy and amino acid metabolism, as well as genes encoding phage-related and transposon-related proteins, were identified as helpful for sediment fitness.Of all the mutants detected, ge...

The response of Desulfovibrio vulgaris to Cu(II) and Hg(II) was characterized. Both metals increased the lag phase, and Cu(II) reduced cell yield at concentrations as low as 50 M. mRNA expression was analyzed using random arbitrarily primed PCR, differential display, and quantitative PCR. Both Cu(II) and Hg(II) (50 M) caused upregulation of mRNA expression for an ATP binding protein (ORF2004) and an ATPase (ORF856) with four-to sixfold increases for Hg(II) and 1.4-to 3-fold increases with Cu(II). These results suggest that D. vulgaris uses an ATP-dependent mechanism for adapting to toxic metals in the environment.Sulfate-reducing bacteria (SRB) are known to be involved in metal bioremediation and for this purpose are often encouraged to grow in engineered systems. They can directly reduce and precipitate many metals, including uranium (11), technetium (9, 10), arsenate (12), and chromium (23), or precipitate metal cations, as metal sulfides (4, 24). Sulfide itself is also known to reduce certain oxidized metals (20), resulting in precipitation. Bioremediation processes may be conducted in bioreactors (4, 24, 25) or in situ (17). While SRB are capable of varied metal transformations, metals can also inhibit their growth (14-16). However, we are not aware of previous research aimed at addressing mechanisms of resistance to divalent metal ions by SRB.Differential display using random arbitrarily primed PCR (RAP-PCR) has been used to identify genes induced by growth under different conditions (1, 2, 8). These techniques were recently used by our group with Desulfovibrio desulfuricans subsp. aestuarii to identify differentially transcribed genes during growth on lactate or hydrogen (22). In the present study, we have used a similar approach to identify genes involved in metal resistance. After first determining metal concentrations needed to inhibit growth of Desulfovibrio vulgaris, we used RAP-PCR to identify genes whose transcription was enhanced in the presence of Cu 2ϩ or Hg 2ϩ . These transcripts were subsequently quantitated in metal-treated cultures and control cultures and were identified through genetic database analysis.Effects of metals on growth. Initial studies were aimed at determining suitable metal concentrations for differential expression experiments. D. vulgaris Hildenborough was grown anaerobically on lactate sulfate medium (LSM) and pyruvate medium (PM) without sulfide, bicarbonate, and CO 2 . LSM was prepared as previously described (7) and contained sodium DLlactate (50 mM), Na 2 SO 4 (50 mM), yeast extract (0.1%), vitamins, minerals, trace metals, and resazurin. D. vulgaris was also cultivated fermentatively in a similar pyruvate medium with pyruvate (20 mM) and MgCl 2 instead of MgSO 4 · 7H 2 O and no other sources of sulfate.For the preparation of inocula, cultures were grown to midexponential phase and harvested by centrifugation (4,650 ϫ g for 20 min). The cell pellet was washed twice and finally resuspended in fresh medium (without bicarbonate-sulfide). Cultures for metal inhibition and e...

Signature-tagged mutants of Desulfovibrio desulfuricans G20 were screened, and 97 genes crucial for sediment fitness were identified. These genes belong to functional categories including signal transduction, binding and transport, insertion elements, and others. Mutants with mutations in genes encoding proteins involved in amino acid biosynthesis, hydrogenase activity, and DNA repair were further characterized.Sulfate-reducing bacteria play important roles in a variety of anaerobic environments and have the potential to be used for bioremediation of metals and hydrocarbons (19,29,31). The vast majority of past studies have focused on growth in laboratory media. Yet with our current knowledge of the importance of changes in gene expression in response to environmental factors (14) and recent developments demonstrating the utility of in situ microbial studies (15,26), the limitations of studying microbial processes in the laboratory have become evident. Sediments provide unique habitats for microorganisms (12), and environmental bacteria must therefore contend with nutrient limitation, competition, osmotic changes, variation in redox potential, and other factors. Bacteria growing in sediments likely possess characteristics distinct from those grown in the laboratory under pure culture conditions (2). There have been limited efforts to prove that cellular functions observed in the laboratory are important for microorganisms growing in the natural environment. In order to address these issues, a modified signature-tagged mutagenesis technique was adopted and Desulfovibrio desulfuricans G20, a sulfate-reducing bacterium, and Shewanella oneidensis MR-1, an iron-reducing bacterium, were used as models for studying functions for in situ growth (9). Using S. oneidensis MR-1, 47 genes were identified that enhanced sediment fitness and it was further demonstrated that antibiotic efflux was a required process for bacteria in sediment (10). In this study, we identified D. desulfuricans G20 genes apparently necessary for fitness in aquifer sediments and further characterized several of them. G20 sediment (9), a G20 strain that had been adapted to sediment conditions, was used as a parent strain for construction of our tagged-transposon mutant library. Details on the generation of a mutant library were described in a previous study (9). Sediment fitness mutants were defined as mutants that were unable to grow in sediment or unable to compete with native microorganisms and were originally selected based on the fact that the oligonucleotide tag within the chromosome of the mutant was not recovered from untreated sediments after an 8-day incubation period. D. desulfuricans G20 increased in number roughly fivefold while growing in sediment during the 8-day incubation period (data not shown). Fitness mutants were retested and selected if cell numbers were less than 10% of the inoculum concentration after the 8-day sediment incubation. A total of 108 fitness mutants were identified. The transposon-inserted regions were sequenced, a...

No abstract