Studies on the modes of action of azaserine in Escherichia coli. Mechanism of resistance to azaserine.

Growth of wildtype Escherichia coli was inhibited by azaserine. There was an inverse relationship between the initial rate of uptake of phenylalanine and the azaserine concentration. Moderately azaserine-resistant mutants exhibited an initial rate that was similar to that of an aroP mutant, but highly azaserine-resistant mutants exhibited little, if any, uptake of phenylalanine. All of the azaserine-resistant organisms tested harboured a mutation in the aroP+ gene. However, resistance to the antibiotic was not due solely to this lesion.     

大肠杆菌酪氨酸转运系统基因敲除对酪氨酸生产的影响

酪氨酸是三大芳香族氨基酸之一,广泛用于食品、医药和化工等领域。转运系统工程为代谢工程改造大肠杆菌选育酪氨酸生产菌株提供了一种重要的研究策略。大肠杆菌中酪氨酸胞内转运主要通过aroP和tyrP基因编码的通透酶进行调控。以酪氨酸生产菌株HGXP为出发菌株,利用CRISPR-Cas9技术成功构建了aroP和tyrP基因敲除菌,并通过发酵试验考察了调节转运系统对酪氨酸生产的影响。发酵结果表明,aroP和tyrP基因敲除菌酪氨酸产量分别达到3.74 g/L和3.45 g/L,较出发菌株酪氨酸产量分别提高了19%和10%。对诱导温度进行了优化,结果表明38℃为最佳诱导温度。在3 L发酵罐上进行了补料分批发酵,aroP和tyrP基因敲除菌酪氨酸产量进一步提高至44.5 g/L和35.1 g/L,较出发菌株酪氨酸产量分别提高了57%和24%。研究结果对代谢工程强化大肠杆菌生产酪氨酸具有重要的参考价值。     

A Genetic Characterization of the Nadc Gene of Salmonella Typhimurium

The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.     

Azaserine resistance in Escherichia coli: chromosomal location of multiple genes.

Resistance to azaserine in Escherichia coli is the result of mutations in at least three different loci. All spontaneously arising azaserine-resistant mutants harbor a lesion in the aroP gene. However, a lesion in this gene is not solely responsible for resistance. All spontaneously arising intermediate-level azaserine-resistant mutants also harbor a lesion in a gene designated azaA, which lies near min 43 on the chromosome. High-level resistant mutants harbor lesions in the aroP and azaA genes and in a third gene designated azaB, which lies near min 69 on the chromosome. Transport studies demonstrate that mutants harboring lesions in the azaA gene are not defective in the transport of the aromatic amino acids, but that mutants which harbor lesions in the azaB gene are defective in phenylalanine transport but not in tyrosine or tryptophan transport.     

大肠杆菌色氨酸转运系统多基因敲除对色氨酸生产的影响

大肠杆菌中色氨酸向胞内的转运主要是由mtr、tnaB和aroP 3个基因编码的通透酶进行调控.利用Red重组技术,在mtr单基因敲除菌的基础上,成功构建了mtr.tnaB和mtr.aroP双基因敲除菌以及mtr.tnaB.aroP三基因敲除菌,并通过发酵实验首次考察了色氨酸转运系统多基因缺失对大肠杆菌合成色氨酸的影响.发酵结果表明,mtr.tnaB和mtr.aroP双基因缺失后,色氨酸产量分别达到1.38 g/L和1.27 g/L,与出发菌株相比分别提高了17％和9％,而mtr.tnaB.aroP三基因缺失后,菌体生长受到了明显抑制,发酵后色氨酸产量仅为0.63 g/L.在补料分批发酵实验中,mtr.tnaB双基因敲除菌的色氨酸产量进一步提高至12.2 g/L,与出发菌株相比色氨酸产量提高了27％.     

Histidine and Aromatic Permeases of Salmonella typhimurim

Mutants defective either in the histidine permease (hisP) or in the aromatic permease (aroP) were isolated in Salmonella typhimurium and were characterized. The hisP locus had a 49% linkage to purF by phage transduction. The aroP locus was close to proA. Merozygotes diploid for the hisP gene were constructed by episomal transfer, and hisP(+) was dominant over hisP. The properties of merozygotes are described and discussed. A method for the selection of revertants of hisP mutants was devised. By this method, one of the hisP mutants was characterized as an amber mutant. The specificity of the aromatic permease was investigated by using as substrates analogues of the aromatic amino acids and of histidine.     

Functional analysis of sequences adjacent to dapE of Corynebacterium glutamicum reveals the presence of aroP, which encodes the aromatic amino acid transporter.

An initially nonclonable DNA locus close to a gene of L-lysine biosynthesis in Corynebacterium glutamicum was analyzed in detail. Its stepwise cloning and its functional identification by monitoring the amino acid uptakes of defined mutants, together with mechanistic studies, identified the corresponding structure as aroP, the general aromatic amino acid uptake system.     

Location of the gene for the low-affinity tryptophan-specific permease of Escherichia coli.

L-Tryptophan uptake was assayed under conditions in which the aroT gene had been inactivated by deletion and the product of the aroP permease was competitively inhibition. A mutant carrying a deletion from bgl through tnaA showed negligible L-tryptophan uptake, in contrast to a strain possessing an intact tna region or to strains carrying point mutations in tna. The ability to take up L-tryptophan was not restored by lysogenizing the tna-deleted strain with lambda tna+.     

Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: genetic characterization and regulatory properties of deletion mutants.

Twenty-eight spontaneous auxotrophic aroP mutants with deletions in the azi--nadC--aroP--aceE--aceF--lpd region of the Escherichia coli K12 chromosome were characterized genetically with respect to various azi, nadC, ace and lpd markers by P1-mediated transduction. One mutant (Kdelta18; aroP--lpddelta) had a deletion which extended through the aceE and aceF genes to end within the lpd gene. The polarity of the ace operon (aceE to aceF) was confirmed. It was concluded that 10 out of 15 deletions generating a strict requirement for acetate terminated in the aceE gene. Of the ten, three mutants (Kdelta22, Cdelta41 and Cdelta41) synthesized detectable dihydrolipoamide acetyltransferase (the aceF gene product) and seven were assumed to possess deletions generating polar effects on aceF gene expression. Five deletions appeared to extend into the aceF gene. A further five deletions, which limited the expression of the ace operon without generating an Ace- phenotype or a complete Ace- phenotype, ended closest to the aroP-proximal aceE markers. The opposite ends of all these deletions appeared to terminate before (10), within (2) or extend beyond (9) the nadC gene. There was no obvious correlation between the deletion end-points and the corresponding lipoamide dehydrogenase activities, which ranged from 30 to 95% of parental levels in different deletion strains. The remaining seven deletions simply extended between the aroP and nadC genes (nad--aroPdelta) without affecting expression of the ace operon. Regulation of the synthesis of the pyruvate and alpha-ketoglutarate dehydrogenase complexes was investigated in some of the parental and deletion strains under different physiological conditions including thiamin-deprivation. The results indicate that the syntheses of the two dehydrogenase complexes are independently regulated. Expression of the lpd gene appears to be coupled to complex synthesis but can be dissociated under some conditions. Mechanisms for regulating lpd gene expression are discussed and an autogenous mechanism involving uncomplexed lipoamide dehydrogenase functioning as a negatively acting repressor at the operator site of an independent lpd gene is proposed as the simplest mechanism which is consistent with all available information.     

大肠杆菌tyrR基因剔除及其对苯丙氨酸生物合成的影响

TyrR是大肠杆菌芳香族氨基酸生物合成和运输途径中的一种全局性调控蛋白质。采用双交换同源重组的方法定位突变大肠杆菌染色体tyrR基因 ,在该基因中插入带有卡那霉素抗性基因的DNA片段 ,使之失活 ,实现基因剔除。经PCR、DNA测序、lacZ报告基因等多种方法证实了基因剔除的可靠性。tyrR基因剔除后 ,大肠杆菌芳香族氨基酸生物合成中受TyrR蛋白调控的关键酶的酶活力有所提高 :3 脱氧 2 阿拉伯庚酮糖 7 磷酸合成酶(DAHPS ,由aroG编码 )酶活力提高了 1.0 8倍 ,转氨酶 (AT ,由tyrB编码 )酶活力提高了 2 .70倍 ;突变菌株发酵生产苯丙氨酸的能力提高了 1.5 9倍 ;同时 ,与芳香族氨基酸运输相关的通透酶基因aroP(P)的阻遏被解除 ,细胞运输芳香族氨基酸的能力提高了 70 .2 %。     

The LIV-I/LS system as a determinant of azaserine sensitivity of Escherichia coli K-12

The growth of Escherichia coli is inhibited by an antibiotic compound, azaserine (O-diazoacetyl-L-serine). Previous studies revealed the biochemical properties of azaserine, which involves inhibition of various enzymatic reactions as well as introduction of DNA breakage. However, genetically, nothing has been elucidated except that all the azaserine-resistant strains isolated so far carry lesions in the aroP gene as a primary determinant. Here, we demonstrate that, in addition to AroP, the LIV-I/LS system, an ATP-binding cassette type transporter, is involved in azaserine sensitivity of E. coli, by genetic analysis and transport studies, in which Ki value for azaserine was determined to be approximately 10(-3) M.     

Repression of the aroP gene of Escherichia coli involves activation of a divergent promoter.

The repression of aroP expression which is mediated by the TyrR protein with phenylalanine, tyrosine, or tryptophan has been shown to be primarily a direct result of TyrR-mediated activation of a divergent promoter, P3, which directs the RNA polymerase away from promoter P1. Evidence which has been presented to support this conclusion is as follows. Repression of P1 does not occur either in vitro or in vivo if wild-type TyrR protein is substituted by the activation-negative mutant RQ10 (with an R-to-Q change at position 10). Repression of P1 is greatly diminished if the P3 promoter is inactivated or if a 5-bp insertion is made between the P3 promoter and the binding sites for TyrR. Repression is also abolished if the promoter strength of P1 is increased or a putative UP element associated with P3 is altered. Repression of the second promoter, P2, still occurs if the wild-type TyrR protein is substituted with RQ10 or EQ274. The tryptophan-mediated repression of aroP does not involve the TrpR protein.     

Hybrid plasmids containing the pyruvate dehydrogenase complex genes and gene-DNA relationships in the 2 to 3 minute region of the Escherichia coli chromosome

A sample of colonies from the Clarke-Carbon ColE1-Escherichia coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, delta (aroP aceE aceF lpd). Two ColE1-lpd+ hybrid plasmids were identified: pGS2 (ColE1-ace lpd+; 24 kb) and pGS5 (ColE1-lpd+; 14 kb). Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex, whereas pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter). These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24.2 kb segment of bacterial DNA in the nadC-lpd region. A further 13 sites (six enzymes) were defined in a 5.4 kb sub-segment containing the lpd gene. lambda phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7.78 kb sub-segment flanked by AccI and NruI sites.     

Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.     

The lysP gene encodes the lysine-specific permease.

Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease.     

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.