Glutathione production by efficient ATP-regenerating Escherichia coli mutants

There is an ongoing demand to improve the ATP-regenerating system for industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in Escherichia coli using the Permeable Cell Assay. This assay identified 40 single-gene deletion strains that had over 150% higher total cellular ATP synthetic activity relative to the parental strain. Most of them also showed higher ATP-driven glutathione synthesis. The deleted genes of the identified strains that showed increased efficiency of ATP regeneration for glutathione production could be divided into the following four groups: (1) glycolytic pathway-related genes, (2) genes related to degradation of ATP or adenosine, (3) global regulatory genes, and (4) genes whose contribution to the ATP regeneration is unknown. Furthermore, the high glutathione productivity of Δ nlpD , the highest glutathione-producing mutant strain, was due to its reduced sensitivity to the externally added ATP for ATP regeneration. This study showed that the Permeable Cell Assay was useful for improving the ATP-regenerating activity of E. coli for practical applications in various ATP-driven bioprocesses, much as that of glutathione production.     

An efficient method for quantitative determination of cellular ATP synthetic activity

The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. Under the used condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatment was used to make Escherichia coli cells permeable. ATP was discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.     

The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.     

ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification.

Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis. The major source of this endoprotease activity was found to be localized to the outer membrane of the cell. A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity. Using this assay, we identified some common laboratory strains that totally lack the protease. Genetic and Southern analyses of these null strains allowed us to conclude that the protease that cleaves T7 RNA polymerase is OmpT (formerly termed protein a), a known outer membrane endoprotease, and that the null phenotype results from deletion of the OmpT structural gene. A recombinant plasmid carrying the ompT gene enables these deletion strains to synthesize OmpT and converts them to a protease-positive phenotype. The plasmid led to overproduction of OmpT protein and protease activity in the E. coli K-12 and B strains we used, but only weak expression in the E. coli C strain, C1757. This strain-dependent difference in ompT expression was investigated with respect to the known influence of envZ on OmpT synthesis. A small deletion in the ompT region of the plasmid greatly diminishes the amount of OmpT protein and plasmid-encoded protease present in outer membranes. Use of ompT deletion strains for production of T7 RNA polymerase from the cloned gene has made purification of intact T7 RNA polymerase routine. Such strains may be useful for purification of other proteins expressed in E. coli.     

Carbon and energy metabolism of atp mutants of Escherichia coli.

The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis.     

The abundant class of nemis repeats provides RNA substrates for ribonuclease III in Neisseriae

About 2% of the Neisseria meningitidis genome is made up by nemis, short DNA sequences which feature long terminal inverted repeats (TIRs). Most nemis are interspersed with single-copy DNA and are found at close distance from cellular genes. In this work, we demonstrate than RNAs spanning nemis of different length and sequence compositions are specifically cleaved at hairpins formed by nemis termini by total cellular lysates derived from both Escherichia coli and Neisseria lactamica strains. The use of cellular extracts from E. coli strains impaired in the activity of known ribonucleases let to establish that cleavage at nemis TIRs is specifically mediated by the endoribonuclease RNase III. Data set the base for the identification of all of the neisserial genes that are regulated by RNase III because of their physical association with nemis DNA.     

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA

The ArcAB two-component system of Escherichia coli regulates the aerobic/anaerobic expression of genes that encode respiratory proteins whose synthesis is coordinated during aerobic/anaerobic cell growth. A genomic study of E. coli was undertaken to identify other potential targets of oxygen and ArcA regulation. A group of 175 genes generated from this study and our previous study on oxygen regulation (Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, G. W., and Gunsalus, R. P. (2003) J. Biol. Chem. 278, 29837-29855), called our gold standard gene set, have p values <0.00013 and a posterior probability of differential expression value of 0.99. These 175 genes clustered into eight expression patterns and represent genes involved in a large number of cell processes, including small molecule biosynthesis, macromolecular synthesis, and aerobic/anaerobic respiration and fermentation. In addition, 119 of these 175 genes were also identified in our previous study of the fnr allele. A MEME/weight matrix method was used to identify a new putative ArcA-binding site for all genes of the E. coli genome. 16 new sites were identified upstream of genes in our gold standard set. The strict statistical analyses that we have performed on our data allow us to predict that 1139 genes in the E. coli genome are regulated either directly or indirectly by the ArcA protein with a 99% confidence level.     

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.     

Invariance of the nucleoside triphosphate pools of Escherichia coli with growth rate

The ATP and GTP pools of Escherichia coli have recently been reported to increase approximately 10-fold with increasing growth rates in the range from 0.4 to 1.4 generations/hour (Gaal, T., Bartlett, M. S., Ross, W., Turnbough, C. L., and Gourse, R. L. (1997) Science 278, 2092-2097). Moreover, it was proposed that this variation of the nucleotide pools, particularly the ATP pool, might be responsible for the well known growth rate-dependent regulation of rRNA synthesis in E. coli. To test this hypothesis we have measured the nucleoside triphosphate pools as a function of growth rate for several E. coli strains. We found that the size of all four RNA precursor pools are essentially invariant with growth rate, in the range from 0.5 to 2.3 generations/hour. Nevertheless we observed the expected growth rate-dependent increase of RNA accumulation in these strains. In light of these results, it seems unlikely that nucleotide pool variations should be responsible for the growth rate-dependent regulation of rRNA synthesis.     

大肠杆菌PTS系统改造及重组菌生长性能测定

利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。     

Presence of rfe genes in Escherichia coli: their participation in biosynthesis of O antigen and enterobacterial common antigen.

In Salmonella, ilv-linked rfe genes participate in the biosynthesis of the enterobacterial common antigen (CA) as well as of certain types of O antigen (serogroups C1 and L). rff genes, probably in the same cluster with rfe, are required for CA synthesis (P.H. M?kel? et al., in preparation). Several Escherichia coli strains were studied to determine whether they also have rfe-rff genes that are involved in the synthesis of O antigen and CA, or of CA only. In a first approach, E, coli K-12 F-prime factors carrying the genes ilv and argH or argE and presumably rfe-rff genes were introduced into CA-negative Salmonella mutants that are blocked in CA synthesis because of mutated rfe or rff genes. All resulting ilv+ hybrids were CA positive. In recipients with group C1-derived rfb genes, the synthesis of O6,7-specific antigen was also restored. This result shows that E. coli K-12 has rfe and rff genes providing the functions required in the synthesis of CA and Salmonella 6,7-specific polysaccharide. By introduction of defective rfe regions from suitable Salmonella donors into E. coli O8, 09, and O100 strains, the synthesis of CA as well as of the O-specific polysaccharides was blocked. This indicates that in the E. coli strains tested the rfe genes are involved in the synthesis of both O antigen and CA. This suggestion was confirmed by the finding of E. coli rough mutants that had simultaneously become CA negative. In transduction experiments it could be shown that the appearance of the rough and CA- phenotype was due to a defect in the ilv-linked rfe region.     

Quantitative genome-wide analysis of yeast deletion strain sensitivities to oxidative and chemical stress

Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains (Winzeler et al., 1999). Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. WE carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multichemicalsensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen and 55 specific to mefloquine.We correlate these results with data from other large-scale studies to yield novel insights into cellular function.     

一种新的基于Red重组及I-Sec I体内切割的大肠杆菌基因组无痕删减方法

目前常用的基因修饰方法是在Red同源重组介导下,电转线性PCR片段替换染色体上指定序列。因PCR过程错误掺入,该方法常常会在同源序列部位产生一些突变。为了避免此类突变,我们建立了一种新的无痕删除方法。首先将含有抗性标记(两侧带有I-Sec I识别位点)的线性DNA电转到Red重组感受态细胞内,用抗性基因替换基因组上指定序列;然后,将携带融合同源臂(两侧带有I-Sec I位点)的供体质粒导入上述细胞,诱导表达I-Sec I内切酶切割供体质粒释放同源片段,同时切除染色体上抗性基因产生双链断裂,通过分子间同源重组实现无痕删除。我们应用该方法连续删除了大肠杆菌DH1基因组上11个非必需区,使基因组减小10.59%。PCR测序证明所有删减区域同源臂未发生突变,基因组重测序证明指定区域被删除。删减菌的生长变化不大,但耐酸能力有所改变,并对番茄红素合成有不同影响。     

PCR-mediated one-step deletion of targeted chromosomal regions in haploid <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event. In this paper, we show that the PCD method efficiently deletes internal regions in a single transformation. Of the six chromosomal regions targeted for deletion by this method, five regions (16 to 38 kb in length) containing 10 to 19 nonessential genes were successfully eliminated at high efficiency. The one targeted region on chromosome XIII that was not deleted was subsequently found to contain sequences essential for yeast growth. While 14 individual genes in this region have been reported to be nonessential, synthetic lethal interactions may occur among these nonessential genes. Phenotypic analysis showed that four deletion strains still exhibited normal growth while possible synthetic growth defects were observed in another strain harboring a 19-gene deletion on chromosome XV. These results demonstrate that the PCD method is a useful tool for deleting genes and for analyzing their functions in defined chromosomal regions.     

The proteins encoded by the rbs operon of Escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA.

The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.     

A genome rearrangement has orphaned the Escherichia coli K-12 AcpT phosphopantetheinyl transferase from its cognate Escherichia coli O157:H7 substrates

Phosphopantetheinyl transferases (PPTases) are enzymes that catalyse the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of a carrier protein. These carrier proteins use the 4'-phosphopantetheine thiol to shuttle intermediates between the active sites of biosynthetic enzymes involved in fatty acid, non-ribosomal peptide and polyketide synthesis. Three PPTases have been previously been identified in Escherichia coli K-12 and other E. coli strains by homology searches and are encoded by the genes acpS, entD and acpT. Both AcpS and EntD have been well studied whereas the function of AcpT has been an enigma because no carrier protein substrate could be found. We report genetic and biochemical evidence that AcpT modifies two carrier proteins encoded in O-island 138, a cluster of fatty acid biosynthesis-like genes located adjacent to acpT in the genome of the pathogenic E. coli strain O157:H7 (E. coli K-12 and several other sequenced E. coli and Shigella strains lack O-island 138). The two carrier proteins of O-island 138 of strain O157:H7 are not modified (or only very poorly modified) by AcpS, the PPTase responsible for 4'-phosphopantetheine attachment to the acyl carrier protein (AcpP) of fatty acid synthesis. We demonstrate that AcpT cannot functionally replace AcpS in E. coli K-12 either in its native chromosomal location or upon insertion of acpT into the acpS chromosomal location. However, in the absence of AcpS activity AcpT does allow very slow growth thus providing a rationale for its retention in the absence of its cognate substrates. These results together with phylogenetic analyses and comparisons of the E. coli and Shigella strains of known genome sequence strongly argue that AcpT has been orphaned from its cognate substrates by a deletion event that occurred in a common ancestor of these organisms. This seems one of the few cases where a chromosomal rearrangement has been functionally demonstrated to be a deletion event rather than an insertion event in the reference organism. We also show that the previously reported suppression of an acpS mutation by the deletion of Lon protease is an artifact of the increased capsular polysaccharide production of lon strains.     

大肠杆菌最小基因组分析和删减进展

大肠杆菌是基础研究最透彻、应用广泛的微生物,构建含减小甚至是最小基因组的大肠杆菌将为合成生物学的研究和应用提供理想的底盘生物。介绍了大肠杆菌最小基因组的生长与繁殖必需基因的生物信息学分析和实验鉴定,基因组敲除技术,以及删减基因组的大肠杆菌菌株的构建和应用等方面的研究进展。     

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.