Induction of osmoadaptive mechanisms and modulation of cellular physiology help Bacillus licheniformis strain SSA 61 adapt to salt stress.

Bacillus licheniformis strain SSA 61, originally isolated from Sambhar salt lake, was observed to grow even in the presence of 25 % salt stress. Osmoadaptive mechanisms of this halotolerant B. licheniformis strain SSA 61, for long-term survival and growth under salt stress, were determined. Proline was the preferentially accumulated compatible osmolyte. There was also increased accumulation of antioxidants ascorbic acid and glutathione. Among the different antioxidative enzymes assayed, superoxide dismutase played the most crucial role in defense against salt-induced stress in the organism. Adaptation to stress by the organism involved modulation of cellular physiology at various levels. There was enhanced expression of known proteins playing essential roles in stress adaptation, such as chaperones DnaK and GroEL, and general stress protein YfkM and polynucleotide phosphorylase/polyadenylase. Proteins involved in amino acid biosynthetic pathway, ribosome structure, and peptide elongation were also overexpressed. Salt stress-induced modulation of expression of enzymes involved in carbon metabolism was observed. There was up-regulation of a number of enzymes involved in generation of NADH and NADPH, indicating increased cellular demand for both energy and reducing power.

Comparative systems biology across an evolutionary gradient within the Shewanella genus.

To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology including the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of 10 closely related Shewanella strains and species to provide quantitative insights into this issue. Our analyses revealed that, despite extensive horizontal gene transfer within these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended on the degree of ecological specialization of the organisms evaluated. Using the gradient of evolutionary relatedness formed by these genomes, we were able to partly isolate the effect of ecology from that of evolutionary divergence and to rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms, when the organisms were grown under identical conditions, were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information toward beginning a systems-level understanding of bacterial species and genera.

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.

A combinatorial approach to studying protein complex composition by employing size-exclusion chromatography and proteome analysis.

The genome sequences of numerous organisms are available now, but gene sequences alone do not provide sufficient information to accurately deduce protein functions. Protein function is largely dependent on the association of multiple polypeptide chains into large structures with interacting subunits that regulate and support each other. Therefore, the mapping of protein interaction networks in a physiological context is conducive to deciphering protein functions, including those of hypothetical proteins. Although several high-throughput methods to globally identify protein interactions have been reported in recent years, these approaches often have a high rate of nonspecific or artificial interactions detected. For instance, the fraction of false positives of the protein interactions identified by yeast two-hybrid assay has been predicted to be of the order of 50%. We have developed a strategy to globally map Bacillus subtilis protein-protein interactions in a physiological context by fractionating the cell lysates using size-exclusion chromatography (SEC), followed by proteome analysis. Components of both known and unknown protein complexes, multisubunits and multiproteins, have been identified using this strategy. In one case, the partners of the B. subtilis protein complex have been coexpressed in Escherichia coli, and the formation of the overexpressed protein complex has been further confirmed by a pull-down assay.

Detection of in situ derivatized peptides in microbial biofilms by laser desorption 7.87 eV postionizaton mass spectrometry.

A novel analytical method based on laser desorption postionization mass spectrometry (LDPI-MS) was developed to investigate the competence and sporulation factor-a pentapeptide of amino acid sequence ERGMT-within intact Bacillus subtilis biofilms. Derivatization of the neat ERGMT peptide with quinoline- and anthracene-based tags was separately used to lower the peptide ionization potential and permit direct ionization by 7.87-eV vacuum ultraviolet radiation. The techniques of mass shifting and selective ionization of the derivatized peptide were combined here to permit detection of ERGMT peptide within intact biofilms by LDPI-MS, without any prior extraction or chromatographic separation. Finally, imaging MS specific to the derivatized peptide was demonstrated on an intact biofilm using LDPI-MS. The presence of ERGMT in the biofilms was verified by bulk extraction/LC-MS. However, MALDI imaging MS analyses were unable to detect ERGMT within intact biofilms.

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.

Tale of two metal reducers: comparative proteome analysis of Geobacter sulferreducens PCA and Shewanella oneidensis MR-1.

Geobacter sulfurreducens and S. oneidensis are the subjects of intense research efforts due to their potential applications to bioremediation. The characterization of their proteomes, being done in parallel with the analysis of their genome sequences, transcriptomes, and metabolomes, is providing valuable insights to both their similarities and their differences. A primary target of interest in the proteomes of both of these metal-reducing microbes is the characterization of their c-type cytochromes. The discovery of their full compliment of c-type cytochromes and the description of what growth conditions trigger their expression is central to harnessing their bioremediation potential. Proteome analyses thus far show that both G. sulfurreducens and S. oneidensis share the common location of a majority of their c-type cytochromes in their outer membranes. The c-type cytochromes of G. sulfurreducens, however, appear to be less soluble and therefore more difficult to isolate from the membranes than those expressed by S. oneidensis. The majority of the G. sulfurreducens c-type cytochromes also differ from those of S. oneidensis in that they have higher isoelectric points, most higher than pH 8.0. These characteristics of solubility and isoelectric point could be related and could indicate an underlying functional difference in the strategy for metal reduction between these two microbes. The global proteome results available for G. sulfurreducens and S. oneidensis at the time of this writing are primarily the result of 2DE analysis coupled to protein identification by LC-MS/MS of tryptic peptides from in-gel digests and represent the most abundant proteins detected by Coomassie blue or silver nitrate staining. Currently, several complimentary efforts utilising the 2D-LC-MS/MS approaches are in progress, promising a more complete protein inventory for these microbes in the near future. As these data are added to those already available, the intricate network of metabolic processes, regulation of protein synthesis and protein function, transport of nutrients, and signal transduction will be elucidated. The existing tools of proteomics will be complimented with newer methods such as protein chips and phage display to further characterize these microbial systems. The end result, in the not too distant future, will be predictive models of G. sulfurreducens and S. oneidensis behavior in their natural habitats under a variety of environmental conditions.

A database of unique protein sequence identifiers for proteome studies.

In proteome studies, identification of proteins requires searching protein sequence databases. The public protein sequence databases (e.g., NCBInr, UniProt) each contain millions of entries, and private databases add thousands more. Although much of the sequence information in these databases is redundant, each database uses distinct identifiers for the identical protein sequence and often contains unique annotation information. Users of one database obtain a database-specific sequence identifier that is often difficult to reconcile with the identifiers from a different database. When multiple databases are used for searches or the databases being searched are updated frequently, interpreting the protein identifications and associated annotations can be problematic. We have developed a database of unique protein sequence identifiers called Sequence Globally Unique Identifiers (SEGUID) derived from primary protein sequences. These identifiers serve as a common link between multiple sequence databases and are resilient to annotation changes in either public or private databases throughout the lifetime of a given protein sequence. The SEGUID Database can be downloaded (http://bioinformatics.anl.gov/SEGUID/) or easily generated at any site with access to primary protein sequence databases. Since SEGUIDs are stable, predictions based on the primary sequence information (e.g., pI, Mr) can be calculated just once; we have generated approximately 500 different calculations for more than 2.5 million sequences. SEGUIDs are used to integrate MS and 2-DE data with bioinformatics information and provide the opportunity to search multiple protein sequence databases, thereby providing a higher probability of finding the most valid protein identifications.

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.

Differential protein expression in the metal-reducing bacterium Geobacter sulfurreducens strain PCA grown with fumarate or ferric citrate.

Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.

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.

Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations.

The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.

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.

Shotgun proteomics of Methanococcus jannaschii and insights into methanogenesis.

Methanococcus jannaschii is an autotrophic hyperthermophilic archaeon isolated from an oceanic hydrothermal vent. Its primary pathway for energy production is methanogenesis from H2 and CO2. High-throughput Multidimensional Protein Identification Technology based on microcapillary LC/LC/ MS/MS was used to investigate the proteome of M. jannaschii and the methanogenesis pathway in cells grown in complex medium with high H2 supply. A total of 963 proteins have been unambiguously identified. The identified proteins represent approximately 54% of the whole genome of M. jannaschii. About 44% of the identified proteins are either conserved hypothetical or hypothetical proteins. We identified 83-95% of the proteins predicted to be involved in amino acid biosynthesis, cellular processes, central intermediary metabolism, energy metabolism, protein synthesis, transcription, and purine, pyridine, nucleoside, and nucleotide synthesis. Over 40% of these proteins have better than 50% sequence coverage. Approximately 90% of the predicted methanogenesis proteins were detected. In contrast, only 27-37% of predicted hypothetical proteins, proteins involved in transport and binding, and proteins with regulatory functions were identified. High peptide number, spectrum count, and sequence coverage have been used as indicators of high expression levels and are in good agreement with codon bias analysis. Predicted intein peptides were detected in MJ1043 (DNA-directed RNA polymerase, subunit A"), MJ0542 (phosphoenolpyruvate synthase), MJ0782 (transcription initiation factor IIB), and MJ1422 (putative replication factor C subunit). New peptides created by protein splicing were detected in MJ0885 (DNA dependent DNA polymerase), MJ0542, and MJ0782. The methanogenesis pathway and the enzymes involved are also discussed.

GELBANK: a database of annotated two-dimensional gel electrophoresis patterns of biological systems with completed genomes.

GELBANK is a publicly available database of two-dimensional gel electrophoresis (2DE) gel patterns of proteomes from organisms with known genome information (available at http://gelbank.anl.gov and ftp://bioinformatics.anl.gov/gelbank/). Currently it includes 131 completed, mostly microbial proteomes available from the National Center for Biotechnology Information. A web interface allows the upload of 2D gel patterns and their annotation for registered users. The images are organized by species, tissue type, separation method, sample type and staining method. The database can be queried based on protein or 2DE-pattern attributes. A web interface allows registered users to assign molecular weight and pH gradient profiles to their own 2D gel patterns as well as to link protein identifications to a given spot on the pattern. The website presents all of the submitted 2D gel patterns where the end-user can dynamically display the images or parts of images along with molecular weight, pH profile information and linked protein identification. A collection of images can be selected for the creation of animations from which the user can select sub-regions of interest and unlimited 2D gel patterns for visualization. The website currently presents 233 identifications for 81 gel patterns for Homo sapiens, Methanococcus jannaschii, Pyro coccus furiosus, Shewanella oneidensis, Escherichia coli and Deinococcus radiodurans.