Central carbon metabolism influences fidelity of DNA replication in Escherichia coli

Recent studies indicated that there is a direct link between central carbon metabolism (CCM) and initiation and elongation of DNA replication in Eschericha coli. Namely, effects of certain mutations in genes coding for replication proteins (dnaA, dnaB, dnaE, dnaG, and dnaN) could be specifically suppressed by deletions of some genes, whose products are involved in CCM reactions (pta, ackA, pgi, tktB, and gpmA). Here, we demonstrate that the link between CCM and DNA synthesis can be extended to the DNA replication fidelity, as we report changes of the mutator phenotypes of E. coli dnaQ49 and dnaX36 mutants (either suppression or enhancement) by dysfunctions of zwf, pta, ackA, acnB, and icdA genes. Overexpression of appropriate wild-type CCM genes in double mutants resulted in reversions to the initial mutator phenotypes, indicating that the effects were specific. Moreover, the observed suppression and enhancement effects were not caused by changes in bacterial growth rates. These results suggest that there is a genetic correlation between CCM and DNA replication fidelity in E. coli, apart from the already documented link between CCM and DNA replication initiation control and elongation efficiency.     

Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production

For the efficient production of poly(3-hydroxybutyrate) (PHB) using recombinant Escherichia coli, it is of primal importance to overproduce NADPH, which is necessary for the PHB synthetic pathway. In order to overproduce NADPH in the pentose phosphate (PP) pathway, a recombinant E. coli was constructed in which the phosphoglucose isomerase ( pgi) gene was knocked out to force the carbon flow into the PP pathway. The fermentation characteristics of the recombinant E. coli mutant lacking pgi were then investigated to determine the effect of overproduction of NADPH on efficient PHB production. It was found that, compared with the parent strain ( E. coli JM109), growth of the E. coli mutant lacking pgi ( E. coli DF11) is repressed due to NADPH overproduction in the PP pathway. Furthermore, repressed cell growth can be recovered to some extent by introducing a NADPH-consuming pathway, such as the PHB synthetic pathway. Efficient PHB production using such recombinant E. coli (DF11/pAeKG1) could be attained by appropriately controlling the glucose concentration in the fermentor. Total gene expression was investigated at the protein level by two-dimensional electrophoresis. Out of 22 differentially expressed proteins, 12 were identified with the aid of MALDI-TOF mass spectrometry. Variations in the accumulation of PHB in the recombinant pgi mutant carrying phb (E. coli DF11/pAeKG1) corresponded to the expression of proteins encoded by rpsA, znuA, fabD, potD, fkpA, gapA, ynaF and ibpA. The unfavorable conditions generated by PHB accumulation in the pgi mutant carrying phb resulted in the highest expression of 30S ribosomal protein S1, which ultimately caused a further increase in soluble protein synthesis.     

Cloning and characterization of the Escherichia coli lit gene, which blocks bacteriophage T4 late gene expression.

Escherichia coli lit mutations inhibit gene expression late in infection by bacteriophage T4. We cloned the lit gene from wild-type E. coli and three independent lit mutants. We present evidence that lit mutations [renamed lit(Con) mutations] cause overproduction of the lit gene product and that overproduction of this product causes the inhibition of gene expression. We also present evidence that the lit gene product is nonessential for E. coli growth, although the gene is common to most E. coli K-12 strains.     

Escherichia coli phosphoglucose isomerase can be substituted by members of the PGI family, the PGI/PMI family, and the cPGI family

The Escherichia coli strain Pgi-UdhA, a mutant of the strain MG1655, is deficient in both the pgi gene and the udhA gene and cannot grow on glucose as carbon and energy source. This strain was transformed with different pET-plasmids containing archaeal or bacterial pgi, cpgi or pgi/pmi genes from the three known PGI families (PGI, PGI/PMI, cPGI). Growth could be restored upon plasmid-based expression of pgi, pgi/pmi or cpgi genes indicating that these heterologous proteins can substitute for E. coli PGI. However, complete restoration of the growth rate could not be obtained by any of the PGIs, PGI/PMIs, or cPGIs used. The data indicate that the PGI function of the three PGI families is functionally exchangeable in glycolysis.     

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.     

Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity.

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.     

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.     

Ongoing phenotypic and genomic changes in experimental coevolution of RNA bacteriophage Qβ and Escherichia coli

According to the Red Queen hypothesis or arms race dynamics, coevolution drives continuous adaptation and counter-adaptation. Experimental models under simplified environments consisting of bacteria and bacteriophages have been used to analyze the ongoing process of coevolution, but the analysis of both parasites and their hosts in ongoing adaptation and counter-adaptation remained to be performed at the levels of population dynamics and molecular evolution to understand how the phenotypes and genotypes of coevolving parasite-host pairs change through the arms race. Copropagation experiments with Escherichia coli and the lytic RNA bacteriophage Qβ in a spatially unstructured environment revealed coexistence for 54 days (equivalent to 163-165 replication generations of Qβ) and fitness analysis indicated that they were in an arms race. E. coli first adapted by developing partial resistance to infection and later increasing specific growth rate. The phage counter-adapted by improving release efficiency with a change in host specificity and decrease in virulence. Whole-genome analysis indicated that the phage accumulated 7.5 mutations, mainly in the A2 gene, 3.4-fold faster than in Qβ propagated alone. E. coli showed fixation of two mutations (in traQ and csdA) faster than in sole E. coli experimental evolution. These observations suggest that the virus and its host can coexist in an evolutionary arms race, despite a difference in genome mutability (i.e., mutations per genome per replication) of approximately one to three orders of magnitude.     

NADPH-dependent pgi-gene knockout Escherichia coli metabolism producing shikimate on different carbon sources

We explored the physiological and metabolic effects of different carbon sources (glucose, fructose, and glucose/fructose mixture) in phosphoglucose isomerase (pgi) knockout Escherichia coli mutant producing shikimic acid (SA). It was observed that the pgi(-) mutant grown on glucose exhibited significantly lower cell growth compared with the pgi(+) strain and its mixed glucose/fructose fermentation grew well. Interestingly, when fructose was used as a carbon source, the pgi(-) mutant showed the enhanced SA production compared with the pgi(+) strain. In silico analysis of a genome-scale E. coli model was then conducted to characterize the cellular metabolism and quantify NAPDH regeneration, which allowed us to understand such experimentally observed attenuated cell growth and enhanced SA production in glucose- and fructose-consuming pgi(-) mutant, respectively with respect to cofactor regeneration.     

Isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12.

We describe the isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12. These mutations, designated spr(Am), were isolated and characterized in a lexA tif sfi genetic background. They abolished the sensitivity of the strain to UV light and resulted in high rates of synthesis of recA protein. Phage lambda+ failed to lysogenize the strains as observed with similar strains carrying non-amber spr mutations described previously, thereby indicating a constitutive expression of the phage induction pathway. Introduction of an amber suppressor mutation into a strain bearing the spr(Am) mutation restored expression of the LexA mutant phenotype. We conclude that spr mutations either inactivate or prevent synthesis of the lexA gene product and that loss of this product results in constitutive expression of the E. coli induction system in the tif sfi genetic background.     

Evidence for Frameshift Mutations in the Hish Gene of Escherichia Coli Causing Synthesis of a Partially Active Glutamine Amidotransferase

Among eight strains carrying acridine-induced mutations in hisH, five which mapped at four different sites in the promoter-distal region of the gene showed His+ phenotypes on media containing a purine. By complementation analysis, hisH enzyme was shown to be required for growth on purines. Purine-sensitive His+ revertants of strains able to grow on purines carried second-site mutations which in one case could be shown to map in hisG. Strains able to grow on purines were able to grow on 2-thiazolyl-DL-alanine, too. We conclude that frameshift mutations in the promoter-distal part of the hisH gene of E. coli do not completely abolish the activity of the gene product.     

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

A phylogenomic analysis of Escherichia coli / Shigella group: implications of genomic features associated with pathogenicity and ecological adaptation

ABSTRACT: BACKGROUND: The Escherichia coli species contains a variety of commensal and pathogenic strains, and its intraspecific diversity is extraordinarily high. With the availability of an increasing number of E. coli strain genomes, a more comprehensive concept of their evolutionary history and ecological adaptation can be developed using phylogenomic analyses. In this study, we constructed two types of whole-genome phylogenies based on 34 E. coli strains using collinear genomic segments. The first phylogeny was based on the concatenated collinear regions shared by all of the studied genomes, and the second phylogeny was based on the variable collinear regions that are absent from at least one genome. Intuitively, the first phylogeny is likely to reveal the lineal evolutionary history among these strains (i.e., an evolutionary phylogeny), whereas the latter phylogeny is likely to reflect the whole-genome similarities of extant strains (i.e., a similarity phylogeny). RESULTS: Within the evolutionary phylogeny, the strains were clustered in accordance with known phylogenetic groups and phenotypes. When comparing evolutionary and similarity phylogenies, a concept emerges that Shigella may have originated from at least three distinct ancestors and evolved into a single clade. By scrutinizing the properties that are shared amongst Shigella strains but missing in other E. coli genomes, we found that the common regions of the Shigella genomes were mainly influenced by mobile genetic elements, implying that they may have experienced convergent evolution via horizontal gene transfer. Based on an inspection of certain key branches of interest, we identified several collinear regions that may be associated with the pathogenicity of specific strains. Moreover, by examining the annotated genes within these regions, further detailed evidence associated with pathogenicity was revealed. CONCLUSIONS: Collinear regions are reliable genomic features used for phylogenomic analysis among closely related genomes while linking the genomic diversity with phenotypic differences in a meaningful way. The pathogenicity of a strain may be associated with both the arrival of virulence factors and the modification of genomes via mutations. Such phylogenomic studies that compare collinear regions of whole genomes will help to better understand the evolution and adaptation of closely related microbes and E. coli in particular.     

Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.     

Mutants of Salmonella typhimurium deficient in an endoprotease.

Three bands of hydrolytic activity toward the chromogenic protease substrate N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPNE) can be observed after gel electrophoresis of crude extracts of Salmonella typhimurium or Escherichia coli. Mutants deficient in one of these three activities have been isolated using a staining procedure that identifies colonies that show reduced ability to hydrolyze NAPNE. These mutants lack the strongest of the three bands of activity. The Salmonella mutations (designated apeA) are all co-transducible with purE, and the order (pro)-apeA-Hfr K17 origin-purE has been established. Strains carrying apeA mutations have wild-type doubling times. None of the apeA mutants isolated gains an auxotrophic requirement as a result of loss of the apeA gene product. The rates and extents of protein degradation during starvation for a carbon source or during growth after exposure to the amino acid analogue canavanine do not seem to be affected by apeA mutations. Revertants of apeA mutations (selected by screening for clones that have regained the ability to hydrolyze NAPNE) frequently contain a new enzymatic activity not found in wild-type cells.     

Description and interpretation of adaptive evolution of Escherichia coli K-12 MG1655 by using a genome-scale in silico metabolic model

Genome-scale in silico metabolic networks of Escherichia coli have been reconstructed. By using a constraint-based in silico model of a reconstructed network, the range of phenotypes exhibited by E. coli under different growth conditions can be computed, and optimal growth phenotypes can be predicted. We hypothesized that the end point of adaptive evolution of E. coli could be accurately described a priori by our in silico model since adaptive evolution should lead to an optimal phenotype. Adaptive evolution of E. coli during prolonged exponential growth was performed with M9 minimal medium supplemented with 2 g of alpha-ketoglutarate per liter, 2 g of lactate per liter, or 2 g of pyruvate per liter at both 30 and 37 degrees C, which produced seven distinct strains. The growth rates, substrate uptake rates, oxygen uptake rates, by-product secretion patterns, and growth rates on alternative substrates were measured for each strain as a function of evolutionary time. Three major conclusions were drawn from the experimental results. First, adaptive evolution leads to a phenotype characterized by maximized growth rates that may not correspond to the highest biomass yield. Second, metabolic phenotypes resulting from adaptive evolution can be described and predicted computationally. Third, adaptive evolution on a single substrate leads to changes in growth characteristics on other substrates that could signify parallel or opposing growth objectives. Together, the results show that genome-scale in silico metabolic models can describe the end point of adaptive evolution a priori and can be used to gain insight into the adaptive evolutionary process for E. coli.     

The OmpL porin does not modulate redox potential in the periplasmic space of Escherichia coli

The Escherichia coli DsbA protein is the major oxidative catalyst in the periplasm. Dartigalongue et al. (EMBO J., 19, 5980-5988, 2000) reported that null mutations in the ompL gene of E.coli fully suppress all phenotypes associated with dsbA mutants, i.e. sensitivity to the reducing agent dithiothreitol (DTT) and the antibiotic benzylpenicillin, lack of motility, reduced alkaline phosphatase activity and mucoidy. They showed that OmpL is a porin and hypothesized that ompL null mutations exert their suppressive effect by preventing efflux of a putative oxidizing-reducing compound into the medium. We have repeated these experiments using two different ompL null alleles in at least three different E.coli K-12 genetic backgrounds and have failed to reproduce any of the ompL suppressive effects noted above. Also, we show that, contrary to earlier results, ompL null mutations alone do not result in partial DTT sensitivity or partial motility, nor do they appreciably affect bacterial growth rates or block propagation of the male-specific bacteriophage M13. Thus, our findings clearly demonstrate that ompL plays no perceptible role in modulating redox potential in the periplasm of E.coli.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.