Virtual 2-D gel electrophoresis: visualization and analysis of the E. coli proteome by mass spectrometry.

Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis. This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated. Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage. Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains. Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels. In the 5.7-6.0 pH range, E. coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels. Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E. coli proteins.

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.

Mapping regulatory networks in microbial cells.

Genome sequences are the blueprints of diverse life forms but they reveal little information about how cells make coherent responses to environmental changes. The combined use of gene fusions, gene chips, 2-D polyacrylamide gel electrophoresis, mass spectrometry and 'old-fashioned' microbial physiology will provide the means to reveal a cell's regulatory networks and how those networks are integrated.

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.

Unique identification of proteins from small genome organisms: theoretical feasibility of high throughput proteome analysis.

We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two-dimensional (2-D) gels as well as the distribution and clustering of proteins in the predicted proteome of E. coli. We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2-D gels, based on the current level of accuracy. We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy. In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms.

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.

Application of two-dimensional protein gels in biotechnology.

The optimal use of biological systems for technologically developed products will not be achieved until biological systems are completely defined in biochemical terms. Two-dimensional polyacrylamide gel electrophoresis, 2-D gels, are contributing to this goal. These gels separate complex mixtures of proteins into individual polypeptide species. The ultimate use of 2-D gels is the construction of cellular 2-D gel databases which identify the proteins on the gels and catalog their responses to different environmental conditions. In addition to these global analyses, many applications for 2-D gels in basic, applied and clinical research have been shown.

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.

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.

The gene-protein database of Escherichia coli: edition 4.

The gene-protein database of Escherichia coli has as its core an index that links each of the protein spots from a two-dimensional polyacrylamide gel to the gene that encodes the protein. Additional information about each protein and its gene is generated from two-dimensional gel analysis or collated from the literature to form the database. Earlier editions of the database have provided periodic updates of information. The current edition does this, but also introduces a new reference gel image produced by an electrophoresis system recently adopted in this laboratory. The new gel system was chosen because it offers an improved opportunity for other investigations to produce close replicas of the reference gel pattern, thereby allowing easier access to the information of the database and encouraging independent contribution to the database. The new gel format also is larger and hence more compatible with computer assisted image analysis, which has become essential for a project of this magnitude. This edition continues the use of the former reference gel images, but adds a reference image of an equilibrium gel of E. coli strain W3110 produced by the new standardized gel system. At this time, 55% of the protein spots annotated on the previous equilibrium reference gel for this organism have been located on the new reference image, and these identifications are included in the tables of the database.

Gene-protein database of Escherichia coli K-12: edition 3.

The first two editions of the E. coli Gene-Protein Index were published to provide identifications of protein spots resolved by two-dimensional gel electrophoresis as the products of known genes. This third edition has been expanded to include information about genes and proteins gained directly from two-dimensional gel analysis--including information about protein spots not yet characterized genetically or biochemically--and is therefore more properly called a cellular protein database. An alpha-numeric designation has been uniquely assigned to each of the 616 polypeptide spots in the current database. To this, information is linked about the polypeptide's identification (protein name, gene name, Enzyme Commission--EC number), location on reference gels (x-y coordinates), genetics (Genbank code, DNA sequence reference), biochemistry (molecular weight, isoelectric point), and physiology (steady state level of the protein as a function of media and temperature, membership in various regulons and stimulons).

Ribosomes as sensors of heat and cold shock in Escherichia coli.

Nearly all cells respond to an increase in temperature by inducing a set of proteins, called heat shock proteins (HSPs). Because a large number of other stress conditions induce the HSPs (or at least the most abundant ones), this response is often termed the universal stress response. However, a careful study of conditions that truly mimic a temperature shift suggested that these proteins are induced in response to a change in the translational capacity of the cell. To test this directly, Escherichia coli cells were treated with antibiotics that target the prokaryotic ribosome. Two-dimensional gels were used to evaluate the ability of these drugs to alter the rate of synthesis of the HSPs. One group of antibiotics induced the HSPs, whereas a second group repressed the HSPs and induced another set of proteins normally induced in response to a cold shock. Depending on the concentration used, the induction of the heat or cold shock proteins mimicked a mild or severe temperature shift. In addition, antibiotics of the cold shock-inducing group were found to block high temperature induction of the HSPs. The results implicate the ribosome as a prokaryotic sensor for the heat and cold shock response networks, a role it may serve in eukaryotes as well.

Loss of 4.5S RNA induces the heat shock response and lambda prophage in Escherichia coli.

During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth. Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response. Several hours later, lambda prophage was induced and the cells lysed.

Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli.

The addition of isopropyl thio-beta-D-galactoside (IPTG) to Escherichia coli cells containing multiple copies of the heat shock regulatory gene htpR (rpoH) under the control of an IPTG-inducible promoter (P-tac) induced 15 of the 17 polypeptides of the heat shock (HTP) regulon. The time course and magnitude of the induction closely resembled that caused by a shift to 42 degrees C. Nevertheless the two means of inducing the heat shock regulon differed in outcome. Cultures grown at 28 degrees C and induced by incubation at 42 degrees C for 15 min gave significant protection against a challenge temperature of 50 degrees C, but no protection was afforded by a 15-min IPTG treatment at 28 degrees C. It could be shown that there was no interference by IPTG with the development of thermotolerance at 42 degrees C. Also, treatment of a wild strain of E. coli with various toxic agents revealed no correlation between the development of thermotolerance and the induction of any subset of the heat shock proteins. Thermotolerance appears to develop by processes other than the htpR-dependent induction of heat shock proteins.

Induction of proteins in response to low temperature in Escherichia coli.

When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10 degrees C, growth stops for several hours before a new rate of growth is established. During this growth lag the number of proteins synthesized is dramatically reduced, and at one point only about two dozen proteins are made; 13 of these are made at differential rates that are 3 to 300 times increased over the rates at 37 degrees C. The protein with the highest rate of synthesis during the lag is not detectably made at 37 degrees C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.

Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli.

Heat and various inhibitory chemicals were tested in Escherichia coli for the ability to cause accumulation of adenylylated nucleotides and to induce proteins of the heat shock (htpR-controlled), the oxidation stress (oxyR-controlled), and the SOS (lexA-controlled) regulons. Under the conditions used, heat and ethanol initiated solely a heat shock response, hydrogen peroxide and 6-amino-7-chloro-5,8-dioxoquinoline (ACDQ) induced primarily an oxidation stress response and secondarily an SOS response, nalidixic acid and puromycin induced primarily an SOS and secondarily a heat shock response, isoleucine restriction induced a poor heat shock response, and CdCl2 strongly induced all three stress responses. ACDQ, CdCl2, and H2O2 each stimulated the synthesis of approximately 35 proteins by factors of 5- to 50-fold, and the heat shock, oxidation stress, and SOS regulons constituted a minor fraction of the overall cellular response. The pattern of accumulation of adenylylated nucleotides during these treatments was inconsistent with a simple role for these nucleotides as alarmones sufficient for triggering the heat shock response, but was consistent with a role in the oxyR-mediated response.

Heat shock response in Escherichia coli influences cell division.

Analysis of a mutant in fam, a pleiotropic gene affecting cell division in Escherichia coli, revealed that this gene is probably identical to the heat shock regulatory gene htpR. The fam-715 mutant and different htpR mutants were found to share the following three characteristics: temperature-sensitive growth, faulty cell division, and inability to induce the normal cellular heat shock response. These defects were all corrected in fam and htpR mutants by complementation with plasmids carrying intact htpR+ or by recombination between these mutant alleles and a plasmid carrying only a portion of htpR. These results implicate the E. coli heat shock system in the regulation of cell division and raise the question of a similar role in other organisms.

Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor.

We have sequenced a cloned segment of E. coli chromosomal DNA that includes the heat shock regulatory gene htpR. This segment contains an 852 nucleotide open reading frame bounded by transcriptional and translational signals. Both in vivo and in vitro the cloned segment produces a single protein that migrates in gels with the cellular protein (F33.4) implicated as the htpR product. Properties of a cloned fragment of the coding sequence truncated at the promoter-distal end are consistent with this assignment. The htpR gene product appears homologous to the sigma factor of RNA polymerase, and the two proteins are predicted to have similar secondary structure. In addition, two regions of the predicted htpR product resemble protein-DNA contact points conserved in known DNA-binding proteins.