Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 μg or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif⁺ plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his⁺ strain, and was also Nif^- on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.     

Histidine Mutants Requiring Adenine: Selection of Mutants with Reduced hisG Expression in SALMONELLA TYPHIMURIUM

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leaky types), and they map throughout the hisG gene.     

Genetic analysis of the histidine operon control region of Salmonella typhimurium

Mutants of the histidine operon control region (hisO) include two classes: (1) those completely unable to express the operon (His auxotrophs), and (2) prototrophs that are unable to achieve fully induced levels of operon expression (still His⁺ but sensitive to the drug amino-triazole). Using new, as well as previously existing hisO mutants, we constructed a fine-structure deletion map of hisO. Mutations that presumably alter the his promoter map at one end of hisO; mutations that alter the his attenuator map at the other end of hisO. Between the promoter and the attenuator lie a number of mutations that affect either the translation of the his leader peptide gene, or the formation and stability of his leader messenger RNA structures. All of the point mutations mapping in this central region revert to His⁺ at a very high frequency (10^?5 to 10^?6); this frequency is increased by both base substitution and frameshift-inducing mutagens. Many of the His^? mutants are suppressed by informational suppressors; all three types of nonsense mutations have been identified, demonstrating that translation of a region of hisO between the promoter and attenuator is essential for his operon expression. All of the hisO mutations tested are cis-dominant.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5′-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

Tandem duplications of the histidine operon observed following generalized transduction in Salmonella typhimurium

Unstable merodiploid transductants may be observed among the progeny of certain generalized transductional crosses between complementing mutations in the histidine operon of Salmonella typhimurium. In the presence of a functional recombination system, these transductants are unstable and they segregate His^? clones of both parental genotypes. The properties of these His⁺ transductants suggest that they contain tandem duplications of a region of DNA which includes the histidine operon, such that each copy of the duplication contains one of the two complementing mutations involved in the transduction. Transductional duplications have been observed from 14 pairs of his mutations, but only with complementing pairs of parental mutations. The length of duplicated material may be quite large: two duplications were found to include genetic markers ten minutes removed from the histidine operon on the Salmonella chromosomal map.These transductants appear to arise in a subpopulation of recipient cells which contain pre-existing tandem duplications of the histidine operon. As much as 0.01 to 0.1% of the cell population appears to be tandemly duplicated for a chromosomal region which includes the histidine operon.     

Deletion mutants of nitrogen fixation in Klebsiella pneumoniae: Mapping of a cluster of nif genes essential for nitrogenase activity

Deletions of the nitrogen fixation (nif) region of the Klebsiella genome were isolated by selecting for resistance to virulent phages whose resistance loci are adjacent to nif. The extent of the various deletions was monitored by assaying several different enzymes or gene products coded for by this segment of DNA. Three classes of deletion mutants were detected: (1) gluconate-6-phosphate dehydrogenase minus (gnd^?), histidine minus but histidinol dehydrogenase plus (his^?, his D⁺), nitrogenase plus (nif⁺), shikimate utilization plus (shu⁺); (2) gnd^?, his D^?, nif^?, shu⁺; (3) gnd^?, his D^?, nif^?, shu^?. From these studies we conclude that the cluster of nif genes essential for nitrogenase activity is located on the genetic linkage map of Klebsiella between his and shu; the gene order in this region in thus phage-resistance locus (rfb?), gnd, his operon, nif, shu. Genetic analysis substantiates the finding that the nif cluster is located proximally to the operator end of the his operon.     

Rearrangement of the Bacterial Chromosome Using Tn10 as a Region of Homology

The transposable tetracycline resistance element, Tn10, can serve as a region of homology to promote rec-dependent deletion, duplication and directed transposition of bacterial genes. Tn10 insertions in regions of the chromosome near the histidine operon (his) have been isolated and characterized in Salmonella typhimurium. When strains are constructed containing two Tn10 insertions flanking the his operon in the same orientation (Tn10-his-Tn10), recombination can occur between Tn10 sequences resulting in the deletion of the intervening his region. The sites of the Tn10 insertions determine the endpoints of the deletion. In crosses designed to construct strains carrying Tn10-his-Tn10, another class of unstable recombinants arises in which the his region exists in tandem duplication, with a Tn10 insertion joining the duplicated copies (his-Tn10-his). The sites of the parental Tn10 insertions mark the endpoints of the duplication. When a strain carrying Tn10-his-Tn10 is used as a donor of his⁺ in conjugation or P22-mediated transduction, recombinants can arise in which the his region has been transposed to the site of any Tn10 insertion, far from the normal location of his in the recipient chromosome. In this manner, the his operon has been moved to the site of a pyrB::Tn10 insertion and has been placed on F'' plasmids. At these new locations, the his⁺ character shows the rec-dependent deletion of his⁺ expected for a Tn10-his-Tn10 duplication. These methods should be generally useful for the manipulation of bacterial genes.     

A high-resolution electron microscopy study of nucleosomes from simian virus 40 chromatin

The DNA sequence changes of 31 mutations altering the attenuation control mechanism of the histidine operon are presented. These mutations are discussed in terms of a model for operon regulation that involves a his leader peptide gene whose translation regulates formation of alternative stem-loop structures in the his leader messenger RNA. Three suppressible mutations generate nonsense codons (ochre and UGA) in the his leader peptide gene, demonstrating that translation of this gene is essential for operon expression. Eight mutations presumably reduce the efficiency of translation initiation of the his leader peptide gene, causing reduced levels of operon expression. Five of these mutations directly alter the leader peptide gene initiator codon (AUG). Three mutations alter sequences just in front of the initiator codon and presumably alter the ribosome recognition site. Fourteen mutations reduce the stability of the his leader mRNA stem-loop structures that are alternatives to the attenuator stem. The properties of these mutations provide support for the role of these stem-loop structures in preventing formation of the attenuator stem. Finally, we show that mutations that alter the attenuator stem suppress hisO mutations. This lends support to the proposal that these hisO mutations cause reduced levels of operon expression due to excessive attenuator stem formation. The properties of these 31 mutations provide substantial support for the model of his operon regulation described in this paper.     

Tandem chromosomal duplications in Salmonella typhimurium: fusion of histidine genes to novel promoters.

Salmonella strains harboring tandem chromosomal duplications have been identified following selection for expression of a histidine biosynthetic gene whose promoter is deleted. In such strains, tandem duplications fuse the selected his gene to “foreign” regulatory elements, thereby allowing gene expression. Selection is made for hisD⁺ activity in deletion strain hisOG203. Among the revertants, strains harboring tandem chromosomal duplications have been identified by a number of their properties. (1) Their HisD⁺ phenotype is genetically unstable. (2) Such instability is dependent on recombination (recA) activity. (3) Genetic tests demonstrate that these strains are merodiploid for large regions (up to 25%) of the Salmonella genome. (4) Recipient strains that inherit the HisD⁺ phenotype of these duplication-carrying revertants also inherit the donor's merodiploid state. (5) In certain revertants the functional hisD⁺ gene and the sequence which promotes merodiploid transductant formation are linked to chromosomal markers located far from the normal his region.Previous reports have concluded that the instability of strains isolated by this selection is due to translocation of the hisD⁺ gene to an extrachromosomal element (the pi-histidine factor). We believe that in all strains we have tested (33 independent isolates) instability can better be accounted for as due to tandem duplication events which permit expression of hisD. At least two mechanisms are responsible for duplication formation. One mechanism is dependent on recombination function and generates identical revertants having a duplication of 16% of the chromosome. A second mechanism operates independently of recombination activity; individual duplications produced by this process have variable endpoints.     

Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability

The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP⁺-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB _Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB _Av ). In comparison with the wild type strain expressing pPHB _Av , the specific accumulation of PHB (g_PHB/g_DCW) in MG1655 Δack-pta/pTrcgapN/pPHB _Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.     

Adaptation of Escherichia coli to Elevated Sodium Concentrations Increases Cation Tolerance and Enables Greater Lactic Acid Production

Adaptive evolution was employed to generate sodium (Na⁺)-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na⁺ concentrations. The isolates also showed increased tolerance of K⁺, although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na⁺ tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na⁺. Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na⁺ tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.     

Genetic fusions that place the lactose genes under histidine operon control

The genes of the Salmonella histidine operon (his) have been placed on an F′ pro lac plasmid using genetic methods that rely on recombinational homology provided by Tn10 transposon insertions. The position and orientation of the transposed his genes permit subsequent deletion mutations to form operon fusions that put the lac genes under his operon control. Strains carrying such fusions show co-ordinate regulation of histidinol dehydrogenase and beta-galactosidase expression. While all of the operon fusions have an intact hisD gene, complementation testing and deletion mapping reveal that the genes downstream of hisD are deleted to varying extents. The beta-galactosidase produced by these operon fusions is itself a fused protein containing the amino terminus of one or another of the his enzymes. Two of the operon fusions having join-points in the hisB gene retain histidinol phosphate phosphatase activity and may produce a bifunctional protein having beta-galactosidase as well as the phosphatase activity. The methods that have been used to isolate these his-lac fusions should be applicable to other genetic systems.     

Vanillin Resistance Induced by BssS Overexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Overexpression of the BssS gene, a biofilm formation regulator, in planktonic Escherichia coli cells has been shown to confer the vanillin-resistant phenotype Van^r to the bacteria. The MG1655P_L-tac-bssS strain started growing in liquid aerated LB medium with 2 g/L vanillin after a lag phase of 17 ± 2 h, whereas the original MG1655 strain did not grow under these conditions. The role of aldehyde reductase YqhD, a vanillin- degrading enzyme, in Van^r phenotype formation has been assessed. However, the Van^r trait in the MG1655P_L-tac-bssS strain primarily depended on autoinducer-2 (AI-2), which formed in E. coli cells with an intact luxS gene. We supposed that BssS acts together with autoinducer-2 (which presumably accumulated during the prolonged lag phase) to induce vanillin resistance determined by changes in the expression of a range of genes.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

First Enzyme of Histidine Biosynthesis and Repression Control of Histidyl-Transfer Ribonucleic Acid Synthetase of Salmonella typhimurium

The regulation of formation of histidyl-transfer ribonucleic acid (tRNA) synthetase was examined in strains of Salmonella typhimurium. When the first of the histidine-forming enzymes was wild type, the presence of 2-thiazolealanine in the growth medium prevented repression of histidyl-tRNA synthetase formation elicited by the addition of 1, 2, 4-triazole-3-alanine to these cultures. Conversely, thiazolealanine had no effect on repression of histidyl-tRNA synthetase formation by triazolealanine in hisG mutant strains. These data suggest a relationship between the control of histidyl-tRNA synthetase formation and the functional state of the histidine operon.     

Purification and characterization of a mutant ATP phosphoribosyltransferase hypersensitive to histidine feedback inhibition.

A mutant form of ATP phosphoribosyltranferase (EC 2.4.2.17), hisG1708c, which results in abnormally slow growth of Salmonella typhimurium at 20 °C was purified to homogeneity and kinetic and chemical behavior were characterized. Initial velocity steady-state substrate kinetics of wild-type and mutant enzymes at 37 °C were consistent with sequential kinetics and demonstrated that standard assay concentrations of substrates were sufficient to substantially saturate both enzymes. Nearly time-independent inhibition by histidine at 37 °C could be obtained only after incubation in the presence of product and histidine. Studies at 37 °C showed that the mutant enzyme is 24 times more sensitive to histidine than the wild type in a negatively cooperative manner instead of the positively cooperative manner seen for wild type. Pure mutant enzyme exhibits two major electrophoretic species of native enzyme. Although one less cysteine is titratable in native mutant enzyme, the amino acid compositions of mutant and wild-type enzymes are similar. Histidine produces an ultraviolet difference spectrum in mutant enzyme closely resembling that produced in wild type. Binding of histidyl-tRNA to mutant enzyme is substantially inhibited by histidine. It is concluded that the hisG1708c mutation alters some conformational processes coupled to the histidine binding site while not affecting others.     

Role of a hisU gene in the control of stable RNA synthesis in Salmonella typhimurium

We have previously reported a fivefold reduction in expression of the ilvGEDA operon in a hisU mutant (hisU1820) originally isolated as a histidine regulatory mutant that exhibited derepressed (deattenuated) expression of the his operon. More recently, we have reported that a unitary explanation of the effect of this mutant on amino acid control is complicated by the observation of relaxed control of stable RNA synthesis during carbon/energy source downshifts. In the present study, we report the results of an analysis of the relaxation in control of RNA synthesis in relation to the accumulation of the guanosine polyphosphates, ppGpp and pppGpp. Unexpectedly, we observed that, despite the inability to restrict RNA accumulation upon carbon/energy downshifts, this mutant formed ppGpp at the normal rate. Further, the evidence clearly indicates that the defective control of RNA in this hisU mutant is not owing to an alteration in the spoT gene and that the relA-mediated RNA control is unaltered. However, relaxed RNA synthesis in hisU is suppressed by hyper-elevated levels of ppGpp; thus, an inverse correlation between RNA accumulation and ppGpp level during carbon/energy downshifts is still demonstrable in the hisU mutant. These data led us to the observation that the increased accumulation of stable RNA upon a carbon/energy downshift is apparently the consequence of a hisU-conferred increase in RNA stability.     

Quantification and analysis of metabolic characteristics of aerobic succinate-producing Escherichia coli under different aeration conditions

The production of succinate by engineered Escherichia coli strains has been widely investigated. In this study, quantitative comparison of metabolic fluxes was carried out for the wild-type E. coli strain and a quintuple mutant strain QZ1111 that was designed for the production of succinate aerobically by knocking out five genes (ptsG, poxB, pta, sdhA, iclR) of the wild-type E. coli MG1655. Metabolic flux distributions of both strains were quantified by ¹³C-labeling experiments, together with the determination of physiological parameters and the expression level of key genes. The experimental results indicated that under the same aeration condition the fraction of oxaloacetate molecules originating from phosphoenolpyruvate was increased in E. coli QZ1111 compared to that in the wild-type E. coli MG1655. The glyoxylate shunt was likely activated in E. coli QZ1111 only under high aeration condition but repressed in other conditions, indicating that the deletion of the iclR gene could not completely remove the repression of the glyoxylate shunt with limited oxygen supply. Our results also suggested further genetic manipulation strategies to enhance the production yield of succinate.     

Emergence of Biofilm-Forming Subpopulations upon Exposure of Escherichia coli to Environmental Bacteriophages

Exposure of Escherichia coli MG1655 to environmental bacteriophages results in rapid selection for phage-tolerant subpopulations displaying increased biofilm formation. Analysis of one phage-tolerant strain revealed large amounts of the DNA-binding Dps protein in the outer membrane protein and production of fimbria-like structures. In dps and fimA mutant derivatives of MG1655, no selection of phage-tolerant bacteria upon exposure to bacteriophages occurred, suggesting a role for Dps and type I pili in bacteriophage tolerance.     

操纵子前导区的修饰及PRPP合成途径的加强对大肠杆菌积累L-组氨酸的影响

目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。