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1.
BMC Genomics ; 13: 138, 2012 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-22507456

RESUMO

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.


Assuntos
Biofilmes/crescimento & desenvolvimento , Carbono/metabolismo , Desulfovibrio vulgaris/crescimento & desenvolvimento , Desulfovibrio vulgaris/genética , Metabolismo Energético/genética , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Reatores Biológicos/microbiologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Análise por Conglomerados , Desulfovibrio vulgaris/efeitos dos fármacos , Desulfovibrio vulgaris/fisiologia , Metabolismo Energético/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácido Láctico/farmacologia , Microscopia Confocal , Modelos Biológicos , Plâncton/citologia , Plâncton/efeitos dos fármacos , Plâncton/microbiologia , Análise de Componente Principal , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sulfatos/farmacologia
2.
J Proteome Res ; 10(1): 339-48, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21105745

RESUMO

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid.


Assuntos
Autofagia/efeitos dos fármacos , Membranas Intracelulares/química , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/química , Fagossomos/metabolismo , Animais , Antígenos de Bactérias/farmacologia , Immunoblotting , Marcação por Isótopo , Camundongos , Microscopia de Fluorescência , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositóis/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Tripsina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
3.
Nucleic Acids Res ; 37(9): 2926-39, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19293273

RESUMO

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.


Assuntos
Desulfovibrio vulgaris/genética , Perfilação da Expressão Gênica , Genes Bacterianos , Proteínas de Bactérias/metabolismo , Desulfovibrio vulgaris/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Deleção de Sequência , Estresse Fisiológico
4.
Microb Cell Fact ; 7: 36, 2008 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19055772

RESUMO

BACKGROUND: Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol. RESULTS AND CONCLUSION: Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

5.
Curr Opin Biotechnol ; 19(3): 228-34, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18515068

RESUMO

Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systems-level studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.


Assuntos
Fontes Geradoras de Energia , Biologia de Sistemas , Fontes de Energia Bioelétrica , Biotecnologia , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Genômica
6.
J Proteome Res ; 7(6): 2320-31, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18416566

RESUMO

Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications (PTMs). Although PTMs are a critical aspect of cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase gamma-subunit (DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium, Desulfovibrio desulfuricans G20, also showed similar +42 Da modifications in the same pathway. Here, we discuss our methods and implications of potential trimethylation in the D. vulgaris sulfate reduction pathway.


Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio vulgaris/metabolismo , Processamento de Proteína Pós-Traducional , Sulfatos/metabolismo , Acetilação , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Cromatografia Líquida/métodos , Desulfovibrio desulfuricans/enzimologia , Desulfovibrio desulfuricans/genética , Desulfovibrio desulfuricans/metabolismo , Desulfovibrio vulgaris/enzimologia , Desulfovibrio vulgaris/genética , Sulfito de Hidrogênio Redutase/análise , Sulfito de Hidrogênio Redutase/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Redes e Vias Metabólicas , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/análise , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Conformação Proteica , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/metabolismo , Homologia de Sequência de Aminoácidos , Sulfato Adenililtransferase/análise , Sulfato Adenililtransferase/metabolismo , Sulfatos/química , Espectrometria de Massas em Tandem/métodos
7.
J Bacteriol ; 189(16): 5996-6010, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17545284

RESUMO

The responses of the anaerobic, sulfate-reducing organism Desulfovibrio vulgaris Hildenborough to low-oxygen exposure (0.1% O(2)) were monitored via transcriptomics and proteomics. Exposure to 0.1% O(2) caused a decrease in the growth rate without affecting viability. Concerted upregulation of the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O(2) exposure. Several of the candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c(3) complex. Other known oxidative stress response candidates remained unchanged during the low-O(2) exposure. To fully understand the results of the 0.1% O(2) exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O(2) exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were downregulated during air exposure. Our results highlight the differences in the cell-wide responses to low and high O(2) levels in D. vulgaris and suggest that while exposure to air is highly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O(2) levels in its environment.


Assuntos
Proteínas de Bactérias/análise , Desulfovibrio vulgaris/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Proteoma/análise , Proteínas de Bactérias/biossíntese , Desulfovibrio vulgaris/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Estresse Oxidativo , Transcrição Gênica
8.
Brief Funct Genomic Proteomic ; 5(2): 133-43, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16772278

RESUMO

The response of Desulfovibrio vulgaris Hildenborough (DvH), a sulphate-reducing bacterium, to nitrate stress was examined using quantitative proteomic analysis. DvH was stressed with 105 mM sodium nitrate (NaNO(3)), a level that caused a 50% inhibition in growth. The protein profile of stressed cells was compared with that of cells grown in the absence of nitrate using the iTRAQ peptide labelling strategy and tandem liquid chromatography separation coupled with mass spectrometry (quadrupole time-of-flight) detection. A total of 737 unique proteins were identified by two or more peptides, representing 22% of the total DvH proteome and spanning every functional category. The results indicate that this was a mild stress, as proteins involved in central metabolism and the sulphate reduction pathway were unperturbed. Proteins involved in the nitrate reduction pathway increased. Increases seen in transport systems for proline, glycine-betaine and glutamate indicate that the NaNO(3) exposure led to both salt stress and nitrate stress. Up-regulation observed in oxidative stress response proteins (Rbr, RbO, etc.) and a large number of ABC transport systems as well as in iron-sulphur-cluster-containing proteins, however, appear to be specific to nitrate exposure. Finally, a number of hypothetical proteins were among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress in DvH.


Assuntos
Desulfovibrio vulgaris/efeitos dos fármacos , Desulfovibrio vulgaris/metabolismo , Nitratos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteoma/análise , Proteômica/métodos , Desulfovibrio vulgaris/genética , Desulfovibrio vulgaris/crescimento & desenvolvimento
9.
J Bacteriol ; 188(11): 4068-78, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16707698

RESUMO

The ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA helicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.


Assuntos
Desulfovibrio vulgaris/genética , Genoma Bacteriano , Cloreto de Sódio/farmacologia , Proteínas de Bactérias/genética , Transporte Biológico , Meios de Cultura , Desulfovibrio vulgaris/efeitos dos fármacos , Desulfovibrio vulgaris/crescimento & desenvolvimento , Genômica , Análise de Sequência com Séries de Oligonucleotídeos , Óperon
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