Indolmycin resistance of Streptomyces coelicolor A3(2) by induced expression of one of its two tryptophanyl-tRNA synthetases

Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials. Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain. Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes. We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive. The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant. Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo. Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme. Tryptophan recognition (assessed by k(cat)/K(M)) by TrpRS1 is 4-fold lower than that of TrpRS2. Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin.     

Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis.

The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.     

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding

The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.     

VI型分泌系统2核心组分VgrG对禽致病性大肠杆菌致病性的影响

【目的】构建禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)VI型分泌系统2(Type VI secretion system 2,T6SS2)结构基因vgrG缺失株,研究其对APEC生物学特性及致病性的影响。【方法】通过Red同源重组方法构建DE719菌株vgrG基因缺失株,并利用低拷贝质粒pSTV28构建互补株。比较分析野生株、缺失株与互补株的生长特性、运动性、生物被膜形成能力、黏附侵袭能力、动物致病力等差异。【结果】vgr G基因缺失不影响DE719的生长速度、运动能力及生物被膜形成能力。致病性试验表明缺失vgrG导致体内定殖能力及致病力显著下降,然而对DF-1细胞的黏附能力增强。【结论】T6SS2核心组分VgrG在APEC感染过程中发挥重要作用,为了解APEC的致病作用提供参考。     

High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.     

Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. II. Increased level of gene expression resulting from IS1 element integration

The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.     

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.     

Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells.

Exponentially growing Escherichia coli cells containing additional copies of the shape-determining gene mreB were found to be elongated, whereas mreB mutant cells were spherical and overproduced penicillin-binding protein 3, a septum peptidoglycan synthetase. The effect of the mreB gene on expression of ftsI, the structural gene for penicillin-binding protein 3, was examined by using an ftsI-lacZ fusion gene on a plasmid. Formation of beta-galactosidase from the fusion gene was significantly increased in mreB129 mutant cells, and its overproduction was suppressed to a normal level by the presence of a plasmid containing the mreB gene. These results indicate a negative mechanism of control of cell division by this morphology gene and suggest that the gene functions in determining whether division or elongation of the cells occurs.     

Repression of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (trp) and enzymes of the tryptophan pathway in Escherichia coli K-12

Mutant strains of Escherichia coli K-12 have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (trp) is partially constitutive. The mutation causing derepression is closely linked to aroH [the structural gene for DAHP synthetase (trp)] and occurs in a locus designated aroJ. The aroJ mutation is not recessive in an aroJ(+)/aroJ(-) diploid strain, as the synthesis of DAHP synthetase (trp) is still derepressed in this strain. On the basis of its close linkage to aroH and its continued expression in an aroJ(+)/aroJ(-) diploid, it is postulated that aroJ is an operator locus controlling the expression of the structural gene aroH. In support of this conclusion, the synthesis of anthranilate synthetase is still normally repressible in aroJ(-) strains, whereas, in trpR(-) strains, both DAHP synthetase (trp) and anthranilate synthetase are synthesized constitutively. The synthesis of DAHP synthetase (trp) remains repressible in an operator-constitutive mutant of the tryptophan operon. In two trpS mutants which possess defective tryptophanyl transfer ribonucleic acid synthetase enzymes, neither DAHP synthetase (trp) nor anthranilate synthetase derepress under conditions in which the defective synthetase causes a decrease in growth rate. On the other hand, an effect of the trpS mutant alleles on the level of anthranilate synthetase has been observed in strains which are derepressed for the synthesis of this enzyme, because of a mutation in the gene trpR. Possible explanations for this effect are presented.     

suhB基因对绿针假单胞菌HT66生防能力的影响

[背景]绿针假单胞菌(Pseudomonaschlororaphis)HT66是一株兼具生防安全性和吩嗪-1-甲酰胺(Phenazine-1-Carboxamide,PCN)高产的植物根际促生菌,在生物防治、生态农业及可持续发展农业领域具有广阔的应用前景.非编码RNA(ncRNA)SuhB参与了细胞中多个过程的代谢调控...     

肠炎沙门氏菌鸡源株ompR 基因缺失株的构建及生物学特性与亲本株的比较

【目的】为了探讨ompR基因在肠炎沙门氏菌生物被膜形成及毒力中的作用。【方法】以肠炎沙门氏菌作为母本,运用自杀性载体pGMB151构建了ompR基因缺失株,结晶紫染色法和扫描电镜观察测定缺失株的生物被膜形成能力,细胞的吸附和侵入及小鼠攻毒试验测定缺失株的毒力。【结果】RT-PCR和蛋白表达证明了ompR基因缺失株构建成功;该缺失株不表达纤维素和菌毛,不形成生物被膜;上皮细胞吸附和侵入试验表明缺失株与野生株具有相同的吸附和侵入率;BALB/c鼠腹腔感染性试验表明,缺失株的半数致死量为106.67CFU,而野生株的半数致死量小于2 CFU。【结论】ompR基因既是肠炎沙门氏菌生物膜形成的调控基因,又是重要的毒力基因。     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Transfer of mlt mutations into polyomavirus intronless genomes by intramolecular recombination in bacteria

We describe a modification of the procedure of Weber and Weissmann [Nucl. Acids Res. 11 (1983) 5661-5669] for the formation of hybrid genes by in vivo recombination to introduce two separate mutations into the same gene. The mutants of interest are inserted as head-to-tail tandems in a bacterial plasmid in such a way that the 5'-proximal mutation is located upstream from the mutant with the more distal mutation. Propagation of the plasmid in a rec+ strain of Escherichia coli allows recombination between homologous sequences in the insert. DNA with the size expected for the recombinant plasmid is isolated by agarose gel electrophoresis, cloned in a recA strain, and characterized by restriction endonuclease mapping. Using this procedure, we have transferred the deletion from polyomavirus mutant dl-8 into other mutant genomes lacking the intervening sequences for either middle T or large T.     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.     

Cloning and characterization of chromosomally encoded cephalosporinase gene of Enterobacter cloacae

The cephalosporinase gene, cpa, which codes for an inducible class I chromosomal beta-lactamase in Enterobacter cloacae was cloned on a fragment of 6.05 kilobase pairs inserted into plasmid pACYC184 and transferred into Escherichia coli HB101 recipient cells. The constructed hybrid plasmid, designated pGGQ101, carried a genomic fragment which retained its parental inducibility characteristics, although its expression level in transformed E. coli cells fell to 40-65% of its initial level in E. cloacae. The localization of the cpa gene on pGGQ101 plasmid was determined by Bal31 exonuclease deletion mapping and further confirmed by subcloning HindIII-AvaI restriction fragment on pMB9 plasmid vector. Labeling with [35S]methionine of pGGQ101 specified proteins in a minicell system showed that six or seven proteins are encoded by the insert. Two proteins with apparent molecular mass of 42 000 and 39 500 daltons, respectively, most probably represent the premature and mature cephalosporinase forms.     

鲍曼不动杆菌铁蛋白的抗氧化功能研究

【目的】克隆鲍曼不动杆菌铁蛋白(Abferritin)基因,并研究其抗氧化功能。【方法】荧光定量PCR检测氧化应激下Abferritin基因的表达量,并将其基因克隆到表达载体p ET28a以构建重组质粒p ET28a-Abferritin,转化大肠杆菌BL21(DE3)得到重组菌BL/p ET28aAbferritin,IPTG诱导目的蛋白表达并利用镍柱亲和层析纯化该蛋白。比色法测定Abferritin蛋白的Fe2+氧化酶活性,自由基清除实验测定其抗氧化功能。菌落计数法观察重组大肠杆菌在H2O2应激条件下的存活率。【结果】Abferritin基因在氧化应激下表达增高。重组质粒在大肠杆菌BL21(DE3)中高效表达,通过Ni2+亲和层析纯化获得了Abferritin蛋白。该蛋白具有Fe2+氧化酶活性,能有效减少氧自由基的形成及提高大肠杆菌抵抗氧化应激的能力。【结论】氧化应激能诱导Abferritin基因表达上调,且该蛋白具有亚铁氧化酶活性和抗氧化功能。     

Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in Escherichia coli hosts

In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene (tk), a well regulated viral gene has been chosen for this study. A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this cosmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then used to transform E. coli tdk- mutant strains, Ky895 or C600tdk- for the selection of the IBRV tk gene. The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning. The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene. The E. coli mutant strain C600tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of C600tdk- mutant.