Regulation of menaquinone production in Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans strain G20 [abstract]

Abstract only availableMenaquinones are cytoplasmic membrane components that have important roles in electron transport for energy generation in anaerobic bacteria. A simple and sensitive protocol for the purification and quantification of menaquinones from the sulfate-reducing anaerobes Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans strain G20 has been established. This method was used to determine whether differences occurred in menaquinone production in cells grown by fermentation when compared with those grown by respiration. Purified menaquinone extracts from both species were analyzed using thin-layer chromatography, UV absorption spectroscopy, and high-performance liquid chromatography. It was observed that each species possesses the same two menaquinones but the ratio between the two menaquinones differed in each species. The effects of alternate growth modes on the menaquinone content of the cells is being explored. Methods to further characterize and identify the two menaquinones is currently in progress

Salt Stress in Desulfovibrio Vulgaris Hildenborough: An Integrated Genomics Approach

I-017Recent interest in the ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste, has made all factors that affect its physiology of great interest. Increased salinity constitutes an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in a striking cell elongation in D. vulgaris. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we undertook a systems approach to explore the effects of excess NaCl on D. vulgaris. This study demonstrates that import of osmoprotectants such as glycine betaine and ectoine constitute the primary mechanism used by D. vulgaris to counter hyper-ionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increase in both RNA and DNA helicases suggested that salt stress had affected the stability of nucleic acid base pairing. An overall increase in branched fatty acids indicated changes in cell wall fluidity. An immediate response to salt stress included upregulation of chemotaxis genes though flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The extensive NaCl stress analysis was compared with microarray data from KCl stress and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods has allowed us to present a conceptual model for salt stress response in D. vulgaris that can be compared to other microorganisms.This work was part of the Virtual Institute for Microbial Stress and Survival supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics Program:GTL through contract DE-AC03- 76SF00099 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy

Analysis of a Ferric Uptake Regulator (Fur) Mutant ofDesulfovibrio vulgaris Hildenborough

Previous experiments examining the transcriptional profileof the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of theFur regulon in response to various environmental stressors. To test theinvolvement of Fur in the growth response and transcriptional regulationof D. vulgaris, a targeted mutagenesis procedure was used for deletingthe fur gene. Growth of the resulting ?fur mutant (JW707) was notaffected by iron availability, but the mutant did exhibit increasedsensitivity to nitrite and osmotic stresses compared to the wild type.Transcriptional profiling of JW707 indicated that iron-bound Fur acts asa traditional repressor for ferrous iron uptake genes (feoAB) and othergenes containing a predicted Fur binding site within their promoter.Despite the apparent lack of siderophore biosynthesis genes within the D.vulgaris genome, a large 12-gene operon encoding orthologs to TonB andTolQR also appeared to be repressed by iron-bound Fur. While other genespredicted to be involved in iron homeostasis were unaffected by thepresence or absence of Fur, alternative expression patterns that could beinterpreted as repression or activation by iron-free Fur were observed.Both the physiological and transcriptional data implicate a globalregulatory role for Fur in the sulfate-reducing bacterium D.vulgaris

Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses

<p>Abstract</p> <p>Background</p> <p><it>Neocallimastix patriciarum</it> is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (<it>GHs</it>) produced by this anaerobic fungus.</p> <p>Results</p> <p>We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to <it>N. patriciarum </it>to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative <it>GH </it>contigs and classified them into 25 <it>GH</it> families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the <it>GH1</it>, <it>GH3</it>, <it>GH5</it>, <it>GH6</it>, <it>GH9</it>, <it>GH18</it>, <it>GH43 </it>and <it>GH48 </it>gene families, which were highly expressed in <it>N. patriciarum </it>cultures grown on different feedstocks.</p> <p>Conclusions</p> <p>These <it>GH </it>genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.</p

Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation

Hypothetical (HyP) and conserved HyP genes account for >30% of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 347 of the 3634 genes were annotated as conserved HyP (9.5%) along with 887 HyP genes (24.4%). Given the large fraction of the genome, it is plausible that some of these genes serve critical cellular roles. The study goals were to determine which genes were expressed and provide a more functionally based annotation. To accomplish this, expression profiles of 1234 HyP and conserved genes were used from transcriptomic datasets of 11 environmental stresses, complemented with shotgun LC–MS/MS and AMT tag proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. One thousand two hundred and twelve of these genes were transcribed with 786 producing detectable proteins. There was no evidence for expression of 17 predicted genes. Except for the latter, monocistronic gene annotation was expanded using the above criteria along with matching Clusters of Orthologous Groups. Polycistronic genes were annotated in the same manner with inferences from their proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the relevance of the inferred functional annotations

Impact of elevated nitrate on sulfate-reducing bacteria: A comparative study of Desulfovibrio vulgaris

Sulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70 mM NaNO{sub 3} but not by 70 mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level

Complementation with and without hypothetical protein DVU0851 in a qmoABC, hypothetical protein (DVU0851) Deletion Mutation of Desulfovibrio vulgaris Hildenborough

Deletion of the qmoABC and a hypothetical protein (HP) (DVU0848-51) in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough resulted in an inability to reduce sulfate. No suppressor mutations appeared in cultures of the deletion incubated in the presence of sulfate. Curiously, the D(qmoABC, HP) mutant was also unable to ferment pyruvate. Respiration of sulfate and fermentation of pyruvate was restored by supplying the qmoABC, HP genes. In order to verify if the hypothetical protein at the end of the operon contributes to the ability of this organism to reduce sulfate and ferment pyruvate, two complemented strains were constructed: both with the qmoABC genes, one with and one without the hypothetical protein. These two strains are compared for their ability to grow on sulfate as the sole electron-donor. Latest results suggest an integral role of the hypothetical protein in sulfate reduction

Effect of the Deletion of qmoABC and the Promoter-Distal Gene Encoding a Hypothetical Protein on Sulfate Reduction in Desulfovibrio vulgaris Hildenborough▿ †

The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion in Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo (quinone-interacting membrane-bound oxidoreductase) complex is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5′-phosphosulfate reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for the DVU0851 protein. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as the terminal electron acceptor. Complementation of the Δ(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking

Re-evaluation of CO 2 laser myringotomy for managing children with persistent acute otitis media

Abstract Background: This study aimed to re-evaluate the utility of CO 2 laser myringotomy in children with persistent acute otitis media (PAOM). Methods: From September 2002 to April 2008, 40 children with PAOM received CO 2 laser myringotomy. PAOM was defined as continuing symptoms and signs after systemic antibiotic treatment. Before laser myringotomy, the eardrums were checked under videotelescopy, pure tone audiometry or behavior audiometry and tympanometry. Middle ear effusions were collected using our own designed bottle culture device after laser myringotomy. The patients were followed up at outpatient clinics for 3 months. Cultured middle ear pathogens, healing time of the eardrums, hospital course (for admitted patients), and the development of middle ear effusions were recorded. Results: Forty children with 53 ears with PAOM were enrolled. Eight patients (20%) had bilateral disease and underwent bilateral laser myringotomy. The overall culture rate was 28.3%, and Streptococcus pneumoniae was the most common pathogen. Eight patients (20%) were admitted for intravenous antibiotics. The average eardrum healing time was 22 days in those with positive cultures, and 16.4 days in those with negative cultures ( p ¼ 0.125). All eardrums healed in 1 month. The resolution rate was 62.5% at 1 month. Patients with bilateral PAOM were prone to have positive middle ear culture (5/8) compared with those with unilateral PAOM (8/32) ( p ¼ 0.086). Conclusion: CO 2 laser myringotomy is an applicable means of treating PAOM. S pneumoniae is the most common pathogen in PAOM. CO 2 laser myringotomy allows for the drainage and culture sampling of middle ear effusion, relieving the need of taking prolonged medication