Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.     

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.     

Comprehensive proteome profiling of the Fe(III)‐reducing myxobacterium Anaeromyxobacter dehalogenans 2CP‐C during growth with fumarate and ferric citrate

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta‐proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2‐D reference map of the soluble A. dehalogenans proteome in order to provide a sound basis for further proteomic studies as well as to gain first global insights into the metabolic activities of this bacterium. Using a combination of 2‐DE and MALDI‐TOF‐MS, a total of 720 proteins spots were identified, representing 559 unique protein species. Using the proteome data, altogether 50 metabolic pathways were found to be expressed during growth with fumarate as primary electron acceptor. An analysis of the pathways revealed an extensive display of enzymes involved in the catabolism and anabolism of a variety of amino acids, including the unexpected fermentation of lysine to butyrate. Moreover, using the reference gel as basis, a semi‐quantitative analysis of protein expression changes of A. dehalogenans during growth with ferric citrate as electron acceptor was conducted. The adaptation to Fe(III) reducing conditions involved the expression changes of a total of 239 proteins. The results suggest that the adaptation to Fe(III) reductive conditions involves an increase in metabolic flux through the tricarboxylic acid cycle, which is fueled by an increased catabolism of amino acids.     

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.     

Mass spectrometric mapping of the enzymes involved in the phenol degradation of an indigenous soil pseudomonad

The enzymes involved in the degradation of phenol by a new soil bacterium referred as Pseudomonas sp. strain phDV1 were characterized. The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol 2,3-dioxygenase (C23O), was isolated using sucrose density centrifugation and anion exchange chromatography. The purified C23O was detected and identified by absorption spectroscopy and peptide mapping. Further, the Pseudomonas sp. strain phDV1 proteome was monitored under different growth substrate conditions, using glucose or phenol as sole carbon and energy source. Sucrose density centrifugation was used to collect and concentrate the cell fraction exhibiting C23O activity and to reduce the complexity of the total protein mixture. 1-DE Tricine PAGE electrophoresis separation in combination with MALDI-TOF MS was attempted for the identification of the proteins involved in the metabolic pathway. We found a different expression of 19 proteins depending on the growth substrate (phenol or glucose) and 10 were identified as enzymes involved in the phenol degradation.     

Proteome analysis of the phenotypic variation process in Photorhabdus luminescens

Photorhabdus luminescens is an insect pathogen associated with specific soil nematodes. The bacterium has a complex life cycle with a symbiotic stage in which bacteria colonize the intestinal tract of the nematodes, and a pathogenic stage against susceptible larval-stage insect. Symbiosis-"deficient" phenotypic variants (known as secondary forms) arise during prolonged incubation. Correspondence analysis of the in silico proteome translated from the genome sequence of strain TT01 identified two major biases in the amino acid composition of the proteins. We analyzed the proteome, separating three classes of extracts: cellular, extracellular, and membrane-associated proteins, resolved by 2-DE. Approximately 450 spots matching the translation products of 231 different coding DNA sequences were identified by PMF. A comparative analysis was performed to characterize the protein content of both variants. Differences were evident during stationary growth phase. Very few proteins were found in variant II supernatants, and numerous proteins were lacking in the membrane-associated fraction. Proteins up-regulated by the phenotypic variation phenomenon were involved in oxidative stress, energy metabolism, and translation. The transport and binding of iron, sugars and amino acids were also affected and molecular chaperones were strongly down-regulated. A potential role for H-NS in phenotypic variation control is discussed.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Proteomic characterization of the sulfur-reducing hyperthermophilic archaeon Thermococcus onnurineus NA1 by 2-DE/MS–MS

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, was isolated from a deep-sea hydrothermal vent area in Papua New Guinea. The strain requires elemental sulfur as a terminal electron acceptor for heterotrophic growth on peptides, amino acids and sugars. Recently, genome sequencing of Thermococcus onnurineus NA1 was completed. In this study, 2-DE/MS–MS analysis of the cytosolic proteome was performed to elucidate the metabolic characterization of Thermococcus onnurineus NA1 at the protein level. Among the 1,136 visualized protein spots, 110 proteins were identified. Enzymes related to metabolic pathways of amino acids utilization, glycolysis, pyruvate conversion, ATP synthesis, and protein synthesis were identified as abundant proteins, highlighting the fact that these are major metabolic pathways in Thermococcus onnurineus NA1. Interestingly, multiple spots of phosphoenolpyruvate synthetase and elongation factor Tu were found on 2D gels generated by truncation at the N-terminus, implicating the cellular regulatory mechanism of this key enzyme by protease degradation. In addition to the proteins involved in metabolic systems, we also identified various proteases and stress-related proteins. The proteomic characterization of abundantly induced proteins using 2-DE/MS–MS enables a better understanding of Thermococcus onnurineus NA1 metabolism.     

Quantitative proteome profiling during the fermentation process of pleiotropic Bacillus subtilis mutants

Using a combined quantitative proteomic and bioinformatic approach, we monitored the cytoplasmic proteome profile of the Gram-positive bacterium Bacillus subtilis during a fermentation process in complex medium. Proteome signatures were applied to elucidate the physiological changes occurring in the gene expression profile during growth. Furthermore, we determined the significance level of quantitative proteome changes, identified relative to the threshold of scatter in replicated samples and developed a statistically rigorous method that allowed us to determine significant fold-changes at 95% confidence between different proteomes. Different functional groups of proteins were clustered according to their pattern of significant expression changes. The largest group is induced by the exhaustion of glucose and the presence of alternative carbon and nitrogen sources. Furthermore, depletion of glucose caused the induction of the trichloroacetic acid (TCA) cycle enzymes and the downregulation of glycolytic enzymes. The onset of the transition phase may be provoked by amino acid starvation, resulting in the RelA-dependent repression of proteins involved in the translation process and in the induction of amino acid biosynthetic pathways. Comparisons between the parental strain and two subtilisin-hypersecreting strains revealed only small cytoplasmic differences in the main metabolic pathways. Instead, the overproduction of degradative enzymes in both of these mutants was reflected in the extracellular proteome.     

Pseudomonas putida Response to Cadmium: Changes in Membrane and Cytosolic Proteomes

Pseudomonas putida is a saprophytic bacterium with remarkable environmental adaptability and the capacity to tolerate high concentrations of heavy metals. The strain P. putida-Cd001 was isolated from soil contaminated with Cd, Zn and Pb. Membrane-associated and cytosolic proteomes were analyzed to identify proteins whose expression was modulated in response to 250 μM CdSO(4). We identified 44 protein spots in the membrane and 21 in the cytosolic fraction differentially expressed in Cd-treated samples compared to untreated controls. Outer membrane porins from the OprD and OprI families were less abundant in bacteria exposed to Cd, whereas those from the OprF and OprL, OprH and OprB families were more abundant, reflecting the increased need to acquire energy sources, the need to maintain membrane integrity and the process of adaptation. Components of the efflux system, such as the CzcB subunit of the CBA system, were also induced by Cd. Analysis of the cytosolic proteome revealed that proteins involved in protein synthesis, degradation and folding were induced along with enzymes that combat oxidative stress, showing that the entire bacterial proteome is modulated by heavy metal exposure. This analysis provides new insights into the adaptation mechanisms used by P. putida-Cd001 to survive in Cd-polluted environments.     

Identification of Lactobacillus plantarum genes that are induced in the gastrointestinal tract of mice

Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of environmental niches, including the human gastrointestinal (GI) tract. Moreover, this lactic acid bacterium can survive passage through the human or mouse stomach in an active form. To investigate the genetic background of this persistence, resolvase-based in vivo expression technology (R-IVET) was performed in L. plantarum WCFS1 by using the mouse GI tract as a model system. This approach identified 72 L. plantarum genes whose expression was induced during passage through the GI tract as compared to laboratory media. Nine of these genes encode sugar-related functions, including ribose, cellobiose, sucrose, and sorbitol transporter genes. Another nine genes encode functions involved in acquisition and synthesis of amino acids, nucleotides, cofactors, and vitamins, indicating their limited availability in the GI tract. Four genes involved in stress-related functions were identified, reflecting the harsh conditions that L. plantarum encounters in the GI tract. The four extracellular protein encoding genes identified could potentially be involved in interaction with host specific factors. The rest of the genes are part of several functionally unrelated pathways or encode (conserved) hypothetical proteins. Remarkably, a large number of the functions or pathways identified here have previously been identified in pathogens as being important in vivo during infection, strongly suggesting that survival rather than virulence is the explanation for the importance of these genes during host residence.     

Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.     

A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.     

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction

The effect of heat stress on hexaploid wheat grain proteome was recently analyzed in our previous works. Proteomic tools allowed the characterization of heat-responsive proteins of total endosperm, composed mainly of prolamins. The present work completes this study; our aim was to analyze the effect of heat stress on the water-soluble fraction, composed essentially of albumins and globulins. These proteins were separated by two-dimensional electrophoresis (2-DE), visualized by Coomassie Brilliant Blue (CBB) staining and analyzed by Melanie-3 software. Of the 43 heat-changed proteins, 24 were found to be up-regulated whereas 19 spot proteins were down-regulated. All of these proteins were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 42 spots. Of these, some were enzymes involved in different metabolic pathways of plants, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase, involved in the starch synthesis pathway; beta-amylase, involved in carbohydrate metabolism, and the ATP synthase beta-chain that was related to four heat-decreased proteins. Moreover, five heat up-regulated proteins showed similarities with small heat shock proteins while three other spots were related to elongation factors or eucaryotic translation initiation factors. Proteins involved in abiotic stresses or in plant defense mechanism were also identified and are discussed.     

Linking proteome responses with physiological and biochemical effects in herbicide-exposed Chlamydomonas reinhardtii

Exposure to a toxicant causes proteome alterations in an organism. In ecotoxicology, analysis of these changes may allow linking them to physiological and biochemical endpoints, providing insights into subcellular exposure effects and responses and, ultimately mechanisms of action. Based on this, useful protein markers of exposure can be identified. We investigated the proteome changes induced by the herbicides paraquat, diuron, and norflurazon in the green alga Chlamydomonas reinhardtii. Shotgun proteome profiling and spectral counting quantification in combination with G-test statistics revealed significant changes in protein abundance. Functional enrichment analysis identified protein groups that responded to the exposures. Significant changes were observed for 149-254 proteins involved in a variety of metabolic pathways. While some proteins and functional protein groups responded to several tested exposure conditions, others were affected only in specific cases. Expected as well as novel candidate markers of herbicide exposure were identified, the latter including the photosystem II subunit PsbR or the VIPP1 protein. We demonstrate that the proteome response to toxicants is generally more sensitive than the physiological and biochemical endpoints, and that it can be linked to effects on these levels. Thus, proteome profiling may serve as a useful tool for ecotoxicological investigations in green algae.     

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.