Global proteomic analysis of the chromate response in Arthrobacter sp. strain FB24.

A global proteomic evaluation of the response of Arthrobacter sp. strain FB24 to 5 and 20 mM Cr(VI) was conducted using both two-dimensional gel electrophoresis (2-DGE) and liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS). The changes in protein expression found with 2-DGE indicate alterations in central metabolism and amino acid synthesis. Proteome coverage increased from 22% with 2-DGE to 71% with LC/LC-MS/MS. The proteins exhibiting the highest levels of expression under Cr(VI) stress suggest intracellular sulfur limitation, which could be driven by competition for the sulfate (SO4(2-)) transporter by the chromate (CrO4(2-)) ion. These results are consistent with the growth defects seen with strain FB24 when Cr(VI) concentrations exceeded 5 mM.

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression.

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that "aerobic" flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.

Steady state protein levels in Geobacter metallireducens grown with iron (III) citrate or nitrate as terminal electron acceptor.

Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.

Proteomic analysis of Psychrobacter cryohalolentis K5 during growth at subzero temperatures.

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4 degrees C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and -4 degrees C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (approximately 31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at -4 degrees C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at -4 degrees C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at -4 degrees C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to -4 degrees C.

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions.

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.

Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1.

BACKGROUND: Shewanella oneidensis MR-1 is a facultative, gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as oxygen, nitrate and metals, and has potential for bioremediation of heavy metal contaminated sites. The complete 5-Mb genome of S. oneidensis MR-1 was sequenced and standard sequence-comparison methods revealed approximately 42% of the MR-1 genome encodes proteins of unknown function. Defining the functions of hypothetical proteins is a great challenge and may need a systems approach. In this study, by using integrated approaches including whole genomic microarray and proteomics, we examined knockout effects of the gene encoding SO1377 (gi24372955), a member of the conserved, hypothetical, bacterial protein family COG2268 (Clusters of Orthologous Group) in bacterium Shewanella oneidensis MR-1, under various physiological conditions. RESULTS: Compared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein) profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR) demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin. CONCLUSION: Our results showed that the knock-out of SO1377 gene had pleiotropic effects and suggested that SO1377 may play a role in iron homeostasis and oxidative damage protection in S. oneidensis MR-1.

DNA microarray and proteomic analyses of the RpoS regulon in Geobacter sulfurreducens.

The regulon of the sigma factor RpoS was defined in Geobacter sulfurreducens by using a combination of DNA microarray expression profiles and proteomics. An rpoS mutant was examined under steady-state conditions with acetate as an electron donor and fumarate as an electron acceptor and with additional transcriptional profiling using Fe(III) as an electron acceptor. Expression analysis revealed that RpoS acts as both a positive and negative regulator. Many of the RpoS-dependent genes determined play roles in energy metabolism, including the tricarboxylic acid cycle, signal transduction, transport, protein synthesis and degradation, and amino acid metabolism and transport. As expected, RpoS activated genes involved in oxidative stress resistance and adaptation to nutrient limitation. Transcription of the cytochrome c oxidase operon, necessary for G. sulfurreducens growth using oxygen as an electron acceptor, and expression of at least 13 c-type cytochromes, including one previously shown to participate in Fe(III) reduction (MacA), were RpoS dependent. Analysis of a subset of the rpoS mutant proteome indicated that 15 major protein species showed reproducible differences in abundance relative to those of the wild-type strain. Protein identification using mass spectrometry indicated that the expression of seven of these proteins correlated with the microarray data. Collectively, these results indicate that RpoS exerts global effects on G. sulfurreducens physiology and that RpoS is vital to G. sulfurreducens survival under conditions typically encountered in its native subsurface environments.

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels.

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.

Low-temperature growth of Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of approximately 35 degrees C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature ( approximately 22 degrees C) MR-1 grows with a doubling time of about 40 min, but when moved from 22 degrees C to 3 degrees C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of approximately 67 h. In comparison to cells grown at 22 degrees C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22 degrees C.

Differentiation of human prostate cancer PC-3 cells induced by inhibitors of inosine 5'-monophosphate dehydrogenase.

To establish a system to study differentiation therapy drugs, we used the androgen-independent human prostate PC-3 tumor cell line as a target and mycophenolic acid (MPA), tiazofurin, or ribavirin, which are inhibitors of IMP dehydrogenase, as inducers. These inhibitors evoked replication arrest, caused an increase in cell size, and triggered vacuolization of the cytoplasm. By Northern and Western blotting and immunostaining, we demonstrated MPA-induced expression of 12 proteins reported to reside in prostasomes, organelles released by secretory luminal prostate cells. Additional MPA-induced proteins were identified by two-dimensional gel electrophoresis. Among these was keratin 17, a prostate cell differentiation marker. By Northern blotting, we also demonstrated the constitutive expression of keratins 8 and 18 and induced expression of keratin 19, three other prostate cell differentiation markers. In addition, we established that cells were committed to differentiate after the 2nd day of MPA treatment using guanosine, which can abrogate the effects of MPA. Based on the expression patterns of prostasomal proteins and keratins and the presence of tentative secretory vacuoles, we hypothesize that IMP dehydrogenase inhibitors induce androgen-independent PC-3 cells to mature into cells with a phenotype that resembles normal prostate luminal cells, but at their intermediate state of differentiation.

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry.

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions.

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.

Global analysis of a "simple" proteome: Methanococcus jannaschii.

The completed genome of Methanococcus jannaschii, including the main chromosome and two extra-chromosomal elements, predicts a proteome comprised of 1783 proteins. How many of those proteins are expressed at any given time and the relative abundance of the expressed proteins, however, cannot be predicted solely from the genome sequence. Two-dimensional gel electrophoresis coupled with peptide mass spectrometry is being used to identify the proteins expressed by M. jannaschii cells grown under different conditions as part of an effort to correlate protein expression with regulatory mechanisms. Here we describe the identification of 170 of the most abundant proteins found in total lysates of M. jannaschii grown under optimal fermentation conditions. To optimize the number of proteins detected, two different protein specific stains (Coomassie Blue R250 or silver nitrate) and two different first dimension separation methods (isoelectric focusing or nonequilibrium pH gradient electrophoresis) were used. Thirty-two percent of the proteins identified are annotated as hypothetical (21% conserved hypothetical and 11% hypothetical), 21% are enzymes involved in energy metabolism, 12% are proteins required for protein synthesis, and the remainder include proteins necessary for intermediary metabolism, cell division, and cell structure. Evidence of post-translational modification of numerous M. jannaschii proteins has been found, as well as indications of incomplete dissociation of protein-protein complexes. These results demonstrate the complexity of proteome analysis even when dealing with a relatively simple genome.

Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress.

The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems.