D-Tyrosine enhancement of microbiocide mitigation of carbon steel corrosion by a sulfate reducing bacterium biofilm.

Microbiocides are used to control problematic microorganisms. High doses of microbiocides cause environmental and operational problems. Therefore, using microbiocide enhancers to make microbiocides more efficacious is highly desirable. 2,2-dibromo-3-nitrilopropionamide (DBNPA) is a popular biodegradable microbiocide. D-Amino acids have been used in lab tests to enhance microbiocides to treat microbial biofilms. In this investigation, D-tyrosine was used to enhance DBNPA against Desulfovibrio vulgaris biofilm on C1018 carbon steel. After 7 days of incubation, the mass loss of coupons without treatment chemicals in the ATCC 1249 culture medium was found to be 3.1 ± 0.1 mg/cm2. With 150 ppm (w/w) DBNPA in the culture medium, the mass loss was reduced to 1.9 ± 0.1 mg/cm2 accompanied by a 1-log reduction in the sessile cell count. The 150 ppm DBNPA + 1 ppm D-tyrosine combination attained an extra 3-log reduction in sessile cell count and an additional 30% reduction in mass loss compared with 150 ppm DBNPA only treatment. The combination also led to a smaller maximum pit depth. Linear polarization resistance (LPR), electrochemical impedance spectrometry (EIS), and potentiodynamic polarization (PDP) tests corroborated the enhancement effects.

Effects of Genetic and Physiological Divergence on the Evolution of a Sulfate-Reducing Bacterium under Conditions of Elevated Temperature.

Adaptation via natural selection is an important driver of evolution, and repeatable adaptations of replicate populations, under conditions of a constant environment, have been extensively reported. However, isolated groups of populations in nature tend to harbor both genetic and physiological divergence due to multiple selective pressures that they have encountered. How this divergence affects adaptation of these populations to a new common environment remains unclear. To determine the impact of prior genetic and physiological divergence in shaping adaptive evolution to accommodate a new common environment, an experimental evolution study with the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) was conducted. Two groups of replicate populations with genetic and physiological divergence, derived from a previous evolution study, were propagated in an elevated-temperature environment for 1,000 generations. Ancestor populations without prior experimental evolution were also propagated in the same environment as a control. After 1,000 generations, all the populations had increased growth rates and all but one had greater fitness in the new environment than the ancestor population. Moreover, improvements in growth rate were moderately affected by the divergence in the starting populations, while changes in fitness were not significantly affected. The mutations acquired at the gene level in each group of populations were quite different, indicating that the observed phenotypic changes were achieved by evolutionary responses that differed between the groups. Overall, our work demonstrated that the initial differences in fitness between the starting populations were eliminated by adaptation and that phenotypic convergence was achieved by acquisition of mutations in different genes.IMPORTANCE Improving our understanding of how previous adaptation influences evolution has been a long-standing goal in evolutionary biology. Natural selection tends to drive populations to find similar adaptive solutions for the same selective conditions. However, variations in historical environments can lead to both physiological and genetic divergence that can make evolution unpredictable. Here, we assessed the influence of divergence on the evolution of a model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, in response to elevated temperature and found a significant effect at the genetic but not the phenotypic level. Understanding how these influences drive evolution will allow us to better predict how bacteria will adapt to various ecological constraints.

A comparative proteomic analysis of Desulfovibrio vulgaris Hildenborough in response to the antimicrobial agent free nitrous acid.

Sulfate reducing bacteria (SRB) can contribute to facilitating serious concrete corrosion through the production of hydrogen sulfide in sewers. Recently, free nitrous acid (FNA) was discovered as a promising antimicrobial agent to inhibit SRB activities thereby limiting hydrogen sulfide production in sewers. However, knowledge of the bacterial response to increasing levels of the antimicrobial agent is unknown. Here we report the proteomic response of Desulfovibrio vulgaris Hildenborough and reveal that the antimicrobial effect of FNA is multi-targeted and dependent on the FNA levels. This was achieved using a sequential window acquisition of all theoretical mass spectrometry analysis to determine protein abundance variations in D. vulgaris during exposure to different FNA concentrations. When exposed to 1.0 µg N/L FNA, nitrite reduction (nitrite reductase) related proteins and nitrosative stress related proteins, including the hybrid cluster protein, showed distinct increased abundances. When exposed to 4.0 and 8.0 µg N/L FNA, increased abundance was detected for proteins putatively involved in nitrite reduction. Abundance of proteins involved in the sulfate reduction pathway (from adenylylphophosulfate to sulfite) and lactate oxidation pathway (from pyruvate to acetate) were initially inhibited in response to FNA at 8 h incubation, and then recovered at 12 h incubation. Lowered ribosomal protein abundance in D. vulgaris was detected, however, total cellular protein levels were mostly constant in the presence or absence of FNA. In addition, this study indicates that proteins coded by genes DVU2543, DVU0772, and DVU3212 potentially participate in resisting oxidative stress with FNA exposure. These findings share new insights for understanding the dynamic responses of D. vulgaris to FNA and could be useful to guide and improve the practical applications of FNA-based technologies for control of sewer corrosion.

Key Metabolites and Mechanistic Changes for Salt Tolerance in an Experimentally Evolved Sulfate-Reducing Bacterium, Desulfovibrio vulgaris.

Rapid genetic and phenotypic adaptation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough to salt stress was observed during experimental evolution. In order to identify key metabolites important for salt tolerance, a clone, ES10-5, which was isolated from population ES10 and allowed to experimentally evolve under salt stress for 5,000 generations, was analyzed and compared to clone ES9-11, which was isolated from population ES9 and had evolved under the same conditions for 1,200 generations. These two clones were chosen because they represented the best-adapted clones among six independently evolved populations. ES10-5 acquired new mutations in genes potentially involved in salt tolerance, in addition to the preexisting mutations and different mutations in the same genes as in ES9-11. Most basal abundance changes of metabolites and phospholipid fatty acids (PLFAs) were lower in ES10-5 than ES9-11, but an increase of glutamate and branched PLFA i17:1ω9c under high-salinity conditions was persistent. ES9-11 had decreased cell motility compared to the ancestor; in contrast, ES10-5 showed higher cell motility under both nonstress and high-salinity conditions. Both genotypes displayed better growth energy efficiencies than the ancestor under nonstress or high-salinity conditions. Consistently, ES10-5 did not display most of the basal transcriptional changes observed in ES9-11, but it showed increased expression of genes involved in glutamate biosynthesis, cation efflux, and energy metabolism under high salinity. These results demonstrated the role of glutamate as a key osmolyte and i17:1ω9c as the major PLFA for salt tolerance in D. vulgaris The mechanistic changes in evolved genotypes suggested that growth energy efficiency might be a key factor for selection.IMPORTANCE High salinity (e.g., elevated NaCl) is a stressor that affects many organisms. Salt tolerance, a complex trait involving multiple cellular pathways, is attractive for experimental evolutionary studies. Desulfovibrio vulgaris Hildenborough is a model sulfate-reducing bacterium (SRB) that is important in biogeochemical cycling of sulfur, carbon, and nitrogen, potentially for bio-corrosion, and for bioremediation of toxic heavy metals and radionuclides. The coexistence of SRB and high salinity in natural habitats and heavy metal-contaminated field sites laid the foundation for the study of salt adaptation of D. vulgaris Hildenborough with experimental evolution. Here, we analyzed a clone that evolved under salt stress for 5,000 generations and compared it to a clone evolved under the same condition for 1,200 generations. The results indicated the key roles of glutamate for osmoprotection and of i17:1ω9c for increasing membrane fluidity during salt adaptation. The findings provide valuable insights about the salt adaptation mechanism changes during long-term experimental evolution.

Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation by Desulfovibrio vulgaris Hildenborough.

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered.IMPORTANCE The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production, resulting in product loss, a health hazard to workers, and ultimately abandonment of wells. Identification of the required genes is a critical step for determining the mechanism of biofilm formation by sulfate reducers. Here, the transporter by which putative biofilm structural proteins are exported from sulfate-reducing Desulfovibrio vulgaris Hildenborough cells was discovered, and a single nucleotide change within the gene coding for this transporter was found to be sufficient to completely stop formation of biofilm.

Dispersal and inhibitory roles of mannose, 2-deoxy-d-glucose and N-acetylgalactosaminidase on the biofilm of Desulfovibrio vulgaris.

Biofilms of sulfate-reducing bacteria (SRB) are often the major cause of microbiologically influenced corrosion. The representative SRB Desulfovibrio vulgaris has previously been shown to have a biofilm that consists primarily of protein. In this study, by utilizing lectin staining, we identified that the biofilm of D. vulgaris also consists of the matrix components mannose, fucose and N-acetylgalactosamine (GalNAc), with mannose predominating. Based on these results, we found that the addition of mannose and the nonmetabolizable mannose analog 2-deoxy-d-glucose inhibits the biofilm formation of D. vulgaris as well as that of D. desulfuricans; both compounds also dispersed the SRB biofilms. In addition, the enzyme N-acetylgalactosaminidase, which degrades GalNAc, was effective in dispersing D. vulgaris biofilms. Therefore, by determining composition of the SRB biofilm, effective biofilm control methods may be devised.

Metabolic dynamics of Desulfovibrio vulgaris biofilm grown on a steel surface.

In this study, a comparative metabolomics approach combining gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) was applied first between planktonic cells and biofilms and then between pure cultures and biofilms of Desulfovibrio vulgaris. The results revealed that the overall metabolic level of the biofilm cells was down-regulated, especially for metabolites related to the central carbon metabolism, compared to the planktonic cells and the pure culture of D. vulgaris. In addition, pathway enrichment analysis of the 58 metabolites identified by GC-MS showed that fatty acid biosynthesis in the biofilm cells was up-regulated, suggesting that fatty acids may be important for the formation, maintenance and function of D. vulgaris biofilm. This study offers a valuable perspective on the metabolic dynamics of the D. vulgaris biofilm.

Glyceryl trinitrate and caprylic acid for the mitigation of the Desulfovibrio vulgaris biofilm on C1018 carbon steel.

Microbiologically influenced corrosion (MIC), also known as biocorrosion, is caused by corrosive biofilms. MIC is a growing problem, especially in the oil and gas industry. Among various corrosive microbes, sulfate reducing bacteria (SRB) are often the leading culprit. Biofilm mitigation is the key to MIC mitigation. Biocide applications against biofilms promote resistance over time. Thus, it is imperative to develop new biodegradable and cost-effective biocides for large-scale field applications. Using the corrosive Desulfovibrio vulgaris (an SRB) biofilm as a model biofilm, this work demonstrated that a cocktail of glyceryl trinitrate (GTN) and caprylic acid (CA) was very effective for biofilm prevention and mitigation of established biofilms on C1018 carbon steel coupons. The most probable number sessile cell count data and confocal laser scanning microscope biofilm images proved that the biocide cocktail of 25 ppm (w/w) GTN + 0.1% (w/w) CA successfully prevented the D. vulgaris biofilm establishment on C1018 carbon steel coupons while 100 ppm GTN + 0.1% CA effectively mitigated pre-established D. vulgaris biofilms on C1018 carbon steel coupons. In both cases, the cocktails were able to reduce the sessile cell count from 10(6) cells/cm(2) to an undetectable level.

Extracellular Electron Transfer Is a Bottleneck in the Microbiologically Influenced Corrosion of C1018 Carbon Steel by the Biofilm of Sulfate-Reducing Bacterium Desulfovibrio vulgaris.

Carbon steels are widely used in the oil and gas industry from downhole tubing to transport trunk lines. Microbes form biofilms, some of which cause the so-called microbiologically influenced corrosion (MIC) of carbon steels. MIC by sulfate reducing bacteria (SRB) is often a leading cause in MIC failures. Electrogenic SRB sessile cells harvest extracellular electrons from elemental iron oxidation for energy production in their metabolism. A previous study suggested that electron mediators riboflavin and flavin adenine dinucleotide (FAD) both accelerated the MIC of 304 stainless steel by the Desulfovibrio vulgaris biofilm that is a corrosive SRB biofilm. Compared with stainless steels, carbon steels are usually far more prone to SRB attacks because SRB biofilms form much denser biofilms on carbon steel surfaces with a sessile cell density that is two orders of magnitude higher. In this work, C1018 carbon steel coupons were used in tests of MIC by D. vulgaris with and without an electron mediator. Experimental weight loss and pit depth data conclusively confirmed that both riboflavin and FAD were able to accelerate D. vulgaris attack against the carbon steel considerably. It has important implications in MIC failure analysis and MIC mitigation in the oil and gas industry.

Nutritional stress induces exchange of cell material and energetic coupling between bacterial species.

Knowledge of the behaviour of bacterial communities is crucial for understanding biogeochemical cycles and developing environmental biotechnology. Here we demonstrate the formation of an artificial consortium between two anaerobic bacteria, Clostridium acetobutylicum (Gram-positive) and Desulfovibrio vulgaris Hildenborough (Gram-negative, sulfate-reducing) in which physical interactions between the two partners induce emergent properties. Molecular and cellular approaches show that tight cell-cell interactions are associated with an exchange of molecules, including proteins, which allows the growth of one partner (D. vulgaris) in spite of the shortage of nutrients. This physical interaction induces changes in expression of two genes encoding enzymes at the pyruvate crossroads, with concomitant changes in the distribution of metabolic fluxes, and allows a substantial increase in hydrogen production without requiring genetic engineering. The stress induced by the shortage of nutrients of D. vulgaris appears to trigger the interaction.

Sulfur isotope fractionation during the evolutionary adaptation of a sulfate-reducing bacterium.

Dissimilatory sulfate reduction is a microbial catabolic pathway that preferentially processes less massive sulfur isotopes relative to their heavier counterparts. This sulfur isotope fractionation is recorded in ancient sedimentary rocks and generally is considered to reflect a phenotypic response to environmental variations rather than to evolutionary adaptation. Modern sulfate-reducing microorganisms isolated from similar environments can exhibit a wide range of sulfur isotope fractionations, suggesting that adaptive processes influence the sulfur isotope phenotype. To date, the relationship between evolutionary adaptation and isotopic phenotypes has not been explored. We addressed this by studying the covariation of fitness, sulfur isotope fractionation, and growth characteristics in Desulfovibrio vulgaris Hildenborough in a microbial evolution experiment. After 560 generations, the mean fitness of the evolved lineages relative to the starting isogenic population had increased by ∼ 17%. After 927 generations, the mean fitness relative to the initial ancestral population had increased by ∼ 20%. Growth rate in exponential phase increased during the course of the experiment, suggesting that this was a primary influence behind the fitness increases. Consistent changes were observed within different selection intervals between fractionation and fitness. Fitness changes were associated with changes in exponential growth rate but changes in fractionation were not. Instead, they appeared to be a response to changes in the parameters that govern growth rate: yield and cell-specific sulfate respiration rate. We hypothesize that cell-specific sulfate respiration rate, in particular, provides a bridge that allows physiological controls on fractionation to cross over to the adaptive realm.

Electron mediators accelerate the microbiologically influenced corrosion of 304 stainless steel by the Desulfovibrio vulgaris biofilm.

In the microbiologically influenced corrosion (MIC) caused by sulfate reducing bacteria (SRB), iron oxidation happens outside sessile cells while the utilization of the electrons released by the oxidation process for sulfate reduction occurs in the SRB cytoplasm. Thus, cross-cell wall electron transfer is needed. It can only be achieved by electrogenic biofilms. This work hypothesized that the electron transfer is a bottleneck in MIC by SRB. To prove this, MIC tests were carried out using 304 stainless steel coupons covered with the Desulfovibrio vulgaris (ATCC 7757) biofilm in the ATCC 1249 medium. It was found that both riboflavin and flavin adenine dinucleotide (FAD), two common electron mediators that enhance electron transfer, accelerated pitting corrosion and weight loss on the coupons when 10ppm (w/w) of either of them was added to the culture medium in 7-day anaerobic lab tests. This finding has important implications in MIC forensics and biofilm synergy in MIC that causes billions of dollars of damages to the US industry each year.

3D-fluorescence in situ hybridization of intact, anaerobic biofilm.

FISH (fluorescence in situ hybridization) is a valuable technique to visualize and quantify localization of different microbial species within biofilms. Biofilm conformation can be altered during typical sample preparation for FISH, which can impact observations in multispecies biofilms, including the relative positions of cells. Here, we describe methods to preserve 3-D structure during FISH for visualization of an anaerobic coculture biofilm of Desulfovibrio vulgaris Hildenborough and Methanococcus maripaludis.

Characterization of NaCl tolerance in Desulfovibrio vulgaris Hildenborough through experimental evolution.

Desulfovibrio vulgaris Hildenborough strains with significantly increased tolerance to NaCl were obtained via experimental evolution. A NaCl-evolved strain, ES9-11, isolated from a population cultured for 1200 generations in medium amended with 100 mM NaCl, showed better tolerance to NaCl than a control strain, EC3-10, cultured for 1200 generations in parallel but without NaCl amendment in medium. To understand the NaCl adaptation mechanism in ES9-11, we analyzed the transcriptional, metabolite and phospholipid fatty acid (PLFA) profiles of strain ES9-11 with 0, 100- or 250 mM-added NaCl in medium compared with the ancestral strain and EC3-10 as controls. In all the culture conditions, increased expressions of genes involved in amino-acid synthesis and transport, energy production, cation efflux and decreased expression of flagellar assembly genes were detected in ES9-11. Consistently, increased abundances of organic solutes and decreased cell motility were observed in ES9-11. Glutamate appears to be the most important osmoprotectant in D. vulgaris under NaCl stress, whereas, other organic solutes such as glutamine, glycine and glycine betaine might contribute to NaCl tolerance under low NaCl concentration only. Unsaturation indices of PLFA significantly increased in ES9-11. Branched unsaturated PLFAs i17:1 ω9c, a17:1 ω9c and branched saturated i15:0 might have important roles in maintaining proper membrane fluidity under NaCl stress. Taken together, these data suggest that the accumulation of osmolytes, increased membrane fluidity, decreased cell motility and possibly an increased exclusion of Na(+) contribute to increased NaCl tolerance in NaCl-evolved D. vulgaris.

Hybrid cluster proteins and flavodiiron proteins afford protection to Desulfovibrio vulgaris upon macrophage infection.

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.

Contribution of rubredoxin:oxygen oxidoreductases and hybrid cluster proteins of Desulfovibrio vulgaris Hildenborough to survival under oxygen and nitrite stress.

A genomic island (GEI) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, found to be able to migrate between two tRNA-Met loci of the genome, contains genes for rubredoxin:oxygen oxidoreductase-1 (roo1) and hybrid cluster protein-1 (hcp1) with additional copies for these genes (roo2 and hcp2) being found elsewhere on the chromosome. A suite of mutants was created in which roo2 and/or hcp2 and/or the GEI were either present or missing. The GEI and roo2 increased survival under microaerobic conditions and allowed growth in closer proximity to the air-water interface of soft agar tubes, two properties which appeared to be closely linked. When Hcp2(+) GEI(+) or Hcp2(-) GEI(+) cells, harbouring cytochrome c nitrite reductase (NrfHA) and growing on lactate and sulfate, were amended with 10 mM nitrite at mid-log phase (8-10 mM sulfide), all nitrite was reduced within 30 h with a rate of 3.0 mmol (g biomass)(-1) h(-1) after which sulfate reduction resumed. However, Hcp2(+) GEI(-) or Hcp2(-) GEI(-) cells were unable to use lactate, causing sulfide to be used as electron donor for nitrite reduction at a sixfold lower rate. Complementation studies indicated that hcp1, not roo1, enhanced the rate of nitrite reduction under these conditions. Hcp2 enhanced the rate of nitrite reduction when, in addition to lactate, hydrogen was also present as an electron donor. These results indicate a critical role of Hcps in alleviating nitrite stress in D. vulgaris Hildenborough by maintaining the integrity of electron transport chains from lactate or H(2) to NrfHA through removal of reactive nitrogen species. It thus appears that the GEI contributes considerably to the fitness of the organism, allowing improved growth in microaerobic environments found in sulfide-oxygen gradients and in environments, containing both sulfide and nitrite, through the action of Roo1 and Hcp1 respectively.

A synergistic D-tyrosine and tetrakis hydroxymethyl phosphonium sulfate biocide combination for the mitigation of an SRB biofilm.

Microbiologically influenced corrosion (MIC) is a major problem in various industries such as oil and gas, and water utilities. Billions of dollars are lost to microbiologically influenced corrosion (MIC) each year in the US. The key to MIC control is biofilm mitigation. Sulfate-reducing bacteria (SRB) are often the culprits. They are also involved in souring and biofouling. SRB biofilms are notoriously difficult to eradicate. Due to environmental concerns and increasing costs, better biocide treatment strategies are desired. Recent studies suggested that D-tyrosine and some other D-amino acids may signal biofilm dispersal. Experimental results in this work indicated that D-tyrosine is an effective biocide enhancer for tetrakis hydroxymethyl phosphonium sulfate (THPS) that is a green biocide. Desulfovibrio vulgaris (ATCC 7757) was used in biofilm prevention and biofilm removal tests. It was found that 100 ppm D-tyrosine alone and 50 ppm THPS alone were both ineffective against the SRB biofilm. However, when 1 ppm D-tyrosine was combined with 50 ppm THPS, the synergy between the two chemicals successfully prevented the establishment of the SRB biofilm on C1018 mild steel coupon surfaces in batch treatment tests. It also eradicated established SRB biofilms from coupon surfaces in both 1 and 3-h shock treatment tests.

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

BACKGROUND: Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. RESULTS: The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. CONCLUSIONS: Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Sustainable syntrophic growth of Dehalococcoides ethenogenes strain 195 with Desulfovibrio vulgaris Hildenborough and Methanobacterium congolense: global transcriptomic and proteomic analyses.

Dehalococcoides ethenogenes strain 195 (DE195) was grown in a sustainable syntrophic association with Desulfovibrio vulgaris Hildenborough (DVH) as a co-culture, as well as with DVH and the hydrogenotrophic methanogen Methanobacterium congolense (MC) as a tri-culture using lactate as the sole energy and carbon source. In the co- and tri-cultures, maximum dechlorination rates of DE195 were enhanced by approximately three times (11.0±0.01 µmol per day for the co-culture and 10.1±0.3 µmol per day for the tri-culture) compared with DE195 grown alone (3.8±0.1 µmol per day). Cell yield of DE195 was enhanced in the co-culture (9.0±0.5 × 10(7) cells per µmol Cl(-) released, compared with 6.8±0.9 × 10(7) cells per µmol Cl(-) released for the pure culture), whereas no further enhancement was observed in the tri-culture (7.3±1.8 × 10(7) cells per µmol Cl(-) released). The transcriptome of DE195 grown in the co-culture was analyzed using a whole-genome microarray targeting DE195, which detected 102 significantly up- or down-regulated genes compared with DE195 grown in isolation, whereas no significant transcriptomic difference was observed between co- and tri-cultures. Proteomic analysis showed that 120 proteins were differentially expressed in the co-culture compared with DE195 grown in isolation. Physiological, transcriptomic and proteomic results indicate that the robust growth of DE195 in co- and tri-cultures is because of the advantages associated with the capabilities of DVH to ferment lactate to provide H(2) and acetate for growth, along with potential benefits from proton translocation, cobalamin-salvaging and amino acid biosynthesis, whereas MC in the tri-culture provided no significant additional benefits beyond those of DVH.

Functional characterization of Crp/Fnr-type global transcriptional regulators in Desulfovibrio vulgaris Hildenborough.

Crp/Fnr-type global transcriptional regulators regulate various metabolic pathways in bacteria and typically function in response to environmental changes. However, little is known about the function of four annotated Crp/Fnr homologs (DVU0379, DVU2097, DVU2547, and DVU3111) in Desulfovibrio vulgaris Hildenborough. A systematic study using bioinformatic, transcriptomic, genetic, and physiological approaches was conducted to characterize their roles in stress responses. Similar growth phenotypes were observed for the crp/fnr deletion mutants under multiple stress conditions. Nevertheless, the idea of distinct functions of Crp/Fnr-type regulators in stress responses was supported by phylogeny, gene transcription changes, fitness changes, and physiological differences. The four D. vulgaris Crp/Fnr homologs are localized in three subfamilies (HcpR, CooA, and cc). The crp/fnr knockout mutants were well separated by transcriptional profiling using detrended correspondence analysis (DCA), and more genes significantly changed in expression in a ΔDVU3111 mutant (JW9013) than in the other three paralogs. In fitness studies, strain JW9013 showed the lowest fitness under standard growth conditions (i.e., sulfate reduction) and the highest fitness under NaCl or chromate stress conditions; better fitness was observed for a ΔDVU2547 mutant (JW9011) under nitrite stress conditions and a ΔDVU2097 mutant (JW9009) under air stress conditions. A higher Cr(VI) reduction rate was observed for strain JW9013 in experiments with washed cells. These results suggested that the four Crp/Fnr-type global regulators play distinct roles in stress responses of D. vulgaris. DVU3111 is implicated in responses to NaCl and chromate stresses, DVU2547 in nitrite stress responses, and DVU2097 in air stress responses.