Diagnosis of cellular states of microbial organisms using proteomics.

Two-dimensional (2-D) polyacrylamide gel electrophoresis has much to contribute to experimental analysis of the proteomes of microbial organisms, since this method separates most cellular proteins and allows synthesis rates to be determined quantitatively. Databases generated using 2-D gels can grow to be very large from even just a few experiments, since each sample provides the data for a field (or column) in the database for several hundreds to even thousands of records (or rows), each of which represents a single polypeptide species. The value of such databases for generating an encyclopedia of how each of the cell's proteins behave in different conditions (protein phenotypes) has been recognized for some time. The potential exists, however, to glean even more valuable information from such databases. Because the measurements of each protein are made in the context of all other proteins, a comprehensive glimpse of the cell's physiological state is theoretically achievable with each 2-D gel. By examining enough conditions (and 2-D gels), expression patterns of subsets of proteins (proteomic signatures) can be found that correlate with the cell's state. This type of information can provide a unique contribution to proteomic analysis, and should be a major focus of such analyses.

Effect of temperature on in vivo protein synthetic capacity in Escherichia coli.

In this report, we examine the effect of temperature on protein synthesis. The rate of protein accumulation is determined by three factors: the number of working ribosomes, the rate at which ribosomes are working, and the rate of protein degradation. Measurements of RNA/protein ratios and the levels of individual ribosomal proteins and rRNA show that the cellular amount of ribosomal machinery in Escherichia coli is constant between 25 and 37 degreesC. Within this range, in a given medium, temperature affects ribosomal function the same as it affects overall growth. Two independent methodologies show that the peptide chain elongation rate increases as a function of temperature identically to growth rate up to 37 degreesC. Unlike the growth rate, however, the elongation rate continues to increase up to 44 degreesC at the same rate as between 25 and 37 degreesC. Our results show that the peptide elongation rate is not rate limiting for growth at high temperature. Taking into consideration the number of ribosomes per unit of cell mass, there is an apparent excess of protein synthetic capacity in these cells, indicating a dramatic increase in protein degradation at high temperature. Temperature shift experiments show that peptide chain elongation rate increases immediately, which supports a mechanism of heat shock response induction in which an increase in unfolded, newly translated protein induces this response. In addition, we find that at low temperature (15 degreesC), cells contain a pool of nontranslating ribosomes which do not contribute to cell growth, supporting the idea that there is a defect in initiation at low temperature.

Escherichia coli proteome analysis using the gene-protein database.

The gene-protein database of Escherichia coli is a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two-dimensional (2-D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate the E. coli proteome including a project to localize on 2-D gels all of the open reading framed encoded by the E. coli chromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery are given.

Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli.

The pattern of proteins synthesized in Escherichia coli during steady-state growth in media with ample inorganic phosphate (Pi), upon limitation for Pi (without an alternative phosphorous compound), and during steady-state growth in media containing phosphonate (PHN) as the sole P source was examined by two-dimensional gel electrophoresis. Of 816 proteins monitored in these experiments, all those with differential synthesis rates greater than 2.0 or less than 0.5 upon phosphate limitation (P limitation) or during growth on PHN compared with their rates in the cultures with Pi were classified as belonging to the PL or PHN stimulon, respectively. The PL stimulon included 413 proteins, 208 showing induced synthesis and 205 showing repressed synthesis. The PHN stimulon was smaller: it included 257 proteins; 227 showed induced synthesis and 30 showed repressed synthesis. The overlap of the two stimulons included 137 proteins: most (118) were ones showing induced synthesis. The promoter regions of genes for several of the proteins with induced or repressed synthesis contained sequences which resembled the consensus sequence for PhoB binding. The aggregate mass of proteins responding to P limitation or growth on PHN was 30 to 40% of the cells' total mass. By comparing the proteins responding to P limitation with those responding to growth on PHN, one can speculate which proteins are likely involved in adapting cells to new P sources or in preparing cells to survive stationary phase.

Effects of overproducing the universal stress protein, UspA, in Escherichia coli K-12.

A plasmid with the structural uspA gene under the control of a tac promoter was used to study the effects of altering uspA expression levels under various growth conditions. We found that increasing UspA synthesis to levels corresponding to physiologically induced levels decreased the cell growth rate in minimal medium and reduced or abolished the cells' capacity to adapt to upshift conditions. As was demonstrated by two-dimensional gel electrophoresis, increased uspA expression caused global changes in the pattern of protein synthesis. In addition, electrophoretic analysis together with V8 protease peptide mapping demonstrated that the pIs of some specific proteins became more acidic as a result of the elevation of the levels of UspA.

Identification of a conditionally essential heat shock protein in Escherichia coli.

Protein D48.5 was recognized as a heat-inducible protein of Escherichia coli during the screening of a group of random, temperature-inducible Mud-Lac fusion mutants. Physiological and genetic analysis demonstrated that (i) the structural gene for this protein, designated htpI, is a member of the sigma 32-dependent heat shock regulon, (ii) at 37 degrees C the synthesis of protein D48.5 is nearly constitutive, increasing slightly with growth rate in media of different composition, and (iii) this protein is essential for growth at high temperature.

Expression and role of the universal stress protein, UspA, of Escherichia coli during growth arrest.

The synthesis of the small, cytoplasmic protein UspA universal stress protein A) of Escherichia coli is induced as soon as the cell growth rate falls below the maximal growth rate supported by the medium, regardless of the condition inhibiting growth. The increase in UspA synthesis appears to be the result of induction of the monocistronic uspA gene. Induction of this gene during a heat-shock treatment is demonstrated to be the result of transcriptional activation of a sigma 70-dependent promoter which has previously been shown to be activated also during carbon starvation-induced growth arrest. Mutant cells lacking UspA grow at rates indistinguishable from the isogenic parent at different temperatures and in the presence of different growth inhibitors but are impaired in their ability to survive prolonged periods of complete growth inhibition caused by a variety of diverse stresses, including CdCl2, H2O2, DNP, CCCP exposure, and osmotic shock. Moreover, the uspA mutation results in an increased sensitivity of cells to carbon-source starvation (i.e. glucose, glycerol or succinate depletion). Also, the mutation causes a marked alteration in the timing of starvation protein expression but protein expression during steady-state growth appears to be normal. The results presented have prompted us to postulate that UspA may have a general protective function related to the growth arrest state.

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol.

Escherichia coli was found to adapt to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol. The rates of synthesis of 53 proteins were increased following exposure to 2,4-dinitrophenol. Adaptation was accelerated when the cofactor pyrroloquinoline quinone was provided in the growth medium.

Stress response of Escherichia coli to elevated hydrostatic pressure.

The response of exponentially growing cultures of Escherichia coli to abrupt shifts in hydrostatic pressure was studied. A pressure upshift to 546 atm (55,304 kPa) of hydrostatic pressure profoundly perturbed cell division, nucleoid structure, and the total rate of protein synthesis. The number of polypeptides synthesized at increased pressure was greatly reduced, and many proteins exhibited elevated rates of synthesis relative to total protein synthesis. We designated the latter proteins pressure-induced proteins (PIPs). The PIP response was transient, with the largest induction occurring approximately 60 to 90 min postshift. Fifty-five PIPs were identified. Many of these proteins are also induced by heat shock or cold shock. The PIP demonstrating the greatest pressure induction was a basic protein of 15.6 kDa. High pressure inhibits growth but does not inhibit the synthesis of stringently controlled proteins. Cold shock is the only additional signal which has been found to elicit this type of response. These data indicate that elevated pressure induces a unique stress response in E. coli, the further characterization of which could be useful in delineating its inhibitory nature.

Expression analysis of cloned chromosomal segments of Escherichia coli.

The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. coli chromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels. Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, this approach can produce a definitive expression map of the E. coli genome.

Isolation and properties of a mutant of Escherichia coli with an insertional inactivation of the uspA gene, which encodes a universal stress protein.

Cells of Escherichia coli increase greatly the synthesis of a small cytoplasmic protein as soon as the cell growth rate falls below the maximal growth rate supported by the medium, regardless of the condition inhibiting growth. The gene, designated uspA (universal stress protein A), encoding this protein has been cloned and mapped, and its nucleotide sequence has been determined (T. Nyström and F.C. Neidhardt, Mol. Microbiol. 6:3187-3198, 1992). We now report the isolation of an E. coli mutant defective in UspA synthesis because of insertional inactivation of the corresponding gene. Analysis of such a mutant demonstrated that it grows at a rate indistinguishable from that of the isogenic parent but lags significantly when diluted into fresh medium, regardless of the carbon source included. In addition, the mutant exhibits a diauxic type of growth when grown on certain single substrates, such as glucose and gluconate. This growth phenotype was found to be the result of abnormal metabolism of the carbon source (e.g., glucose) accompanied by excretion into the medium of acetate. The diauxic type of growth may be attributed to the failure of cells to form acetyl coenzyme A synthetase and to form isocitrate lyase and malate synthase of the glyoxalate bypass, needed for the assimilation of the produced acetate, until glucose or gluconate has been completely exhausted. The uspA mutant appears to dissimilate glucose at an elevated rate that is not commensurate with its biosynthetic processes. These results suggest that the role of protein UspA may be to modulate and reorganize the flow of carbon in the central metabolic pathways of E. coli during growth arrest.

Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage.

Salmonella typhimurium is a facultative intracellular pathogen, able both to invade and to survive within eukaryotic cells and to grow in various extracellular environments. To compare the bacterial responses to these disparate environments and to shed light on the nature of the intracellular environment, we have examined the pattern of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The levels of approximately 40 proteins were observed to increase during growth within macrophage-like U937 cells, while approximately 100 proteins exhibited levels that were repressed relative to those of an extracellular control culture. To aid in the interpretation of these results, the patterns of proteins made by S. typhimurium exposed to various environmental conditions in the laboratory were determined. The intracellular protein pattern was then compared with each of these benchmark protein patterns. This analysis revealed that, as expected, the intracellular environment appears to impose numerous stresses on the bacteria, but unexpectedly, the macrophage-induced response was not a simple sum of the individual stress responses displayed during extracellular growth.

Growth rate paradox of Salmonella typhimurium within host macrophages.

The growth rate of Salmonella typhimurium U937 within host macrophages was estimated by two independent methods. The extent to which ribosomal protein L12 is acetylated to produce ribosomal protein L7 changes markedly with the growth rate. By this measure, the intracellular bacteria appeared to be growing rapidly. Measurements of viable bacteria, however, indicated that the bacteria were growing slowly. A solution of this apparent growth rate paradox was sought by treating U937 cells infected with S. typhimurium X3306 with ampicillin or chloramphenicol to help determine the number of bacteria that were actively growing and dividing in the intracellular condition. Use of these antibiotics showed that by 2 h after invasion, the intracellular bacteria consisted of at least two populations, one static and the other rapidly dividing. This finding implies that previously described changes in the gene expression of S. typhimurium are important for the survival and/or multiplication of the bacteria within the macrophage.

Modulation of the heat shock response by one-carbon metabolism in Escherichia coli.

A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol. This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium. The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium. The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.

The gene-protein database of Escherichia coli: edition 5.

The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.

Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Escherichia coli.

The response of non-differentiating bacteria to nutrient starvation is complex and includes the sequential synthesis of starvation-inducible proteins. Although starvation for different individual nutrients generally provokes unique and individual patterns of protein expression, some starvation stimulons share member proteins. Two-dimensional polyacrylamide gel electrophoresis revealed that the synthesis of a small (13.5 kDa) cytoplasmic protein in Escherichia coli was greatly increased during growth inhibition caused by the exhaustion of any of a variety of nutrients (carbon, nitrogen, phosphate, sulphate, required amino acid) or by the presence of a variety of toxic agents including heavy metals, oxidants, acids and antibiotics. To determine further the mode of regulation of the protein designated UspA (universal stress protein A) we cloned the gene encoding the protein by the technique of reverse genetics. We isolated the protein from a preparative two-dimensional polyacrylamide gel, determined its N-terminal amino acid sequence, and used this sequence to construct a degenerate oligonucleotide probe. Two phages of the Kohara library were found to contain the gene which then was subcloned from the DNA in the overlapping region of these two clones. The amino acid sequence, deduced from the nucleotide sequence of the uspA gene, shows no significant homology with any other known protein. The uspA gene maps at 77 min on the E. coli W3110 chromosome, and is transcribed in a clockwise direction. The increase in the level of UspA during growth arrest was found to be primarily a result of transcriptional activation of the corresponding gene. The induction was independent of the RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB and H-NS proteins during stress conditions that are known to induce or activate these global regulators. The -10 and -35 regions upstream of the transcriptional start site of the uspA gene are characteristic of a sigma 70-dependent promoter.

Function of a relaxed-like state following temperature downshifts in Escherichia coli.

Temperature downshifts of Escherichia coli throughout its growth range resulted in transient growth inhibition and a cold shock response consisting of transient induction of several proteins, repression of heat shock proteins, and, despite the growth lag, continued synthesis of proteins involved in transcription and translation. The paradoxical synthesis of the latter proteins, which are normally repressed when growth is arrested, was explored further. First, by means of a nutritional downshift, a natural stringent response was induced in wild-type cells immediately prior to a shift from 37 to 10 degrees C. These cells displayed decreased synthesis of transcriptional and translational proteins and decreased induction of cold shock proteins; also, adaptation for growth at 10 degrees C was delayed, even after restoration of the nutrient supplementation. Next, the contribution of guanosine 5'-triphosphate-3'-diphosphate and guanosine 5'-diphosphate-3'-diphosphate, collectively abbreviated (p)ppGpp, to the alteration in cold shock response was studied with the aid of a mutant strain in which overproduction of these nucleotides can be artificially induced. Induction of (p)ppGpp synthesis immediately prior to shifting this strain from 37 to 10 degrees C produced results differing only in a few details from those described above for nutritional downshift of the wild-type strain. Finally, shifting a relA spoT mutant, which cannot synthesize (p)ppGpp, from 24 to 10 degrees C resulted in a greater induction of the cold shock proteins, increased synthesis of transcriptional and translational proteins, decreased synthesis of a major heat shock protein, and faster adaptation to growth than for the wild-type strain. Our results indicate that the previously reported decrease in the (p)ppGpp level following temperature downshift plays a physiological role in the regulation of gene expression and adaptation for growth at low temperature.

The lrp gene product regulates expression of lysU in Escherichia coli K-12.

In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion. A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein. The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA.