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Susumu Akasaka Koichi Takimoto Kazuo Yamamoto 《Molecular & general genetics : MGG》1992,235(2-3):173-178
Summary
Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020). Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E. coli, to give pKY241, a derivative of pACYC184. When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF. It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype. Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA. If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies. By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 × 10–7 per replication. Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts. The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions). These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies. 相似文献
3.
Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates 总被引:17,自引:0,他引:17
Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the O-terminus of the predicted open reading frame. A series of ciprofloxacin-resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10000-fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high-level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are invoived in the establishment of extreme levels of ciprofloxacin resistance. 相似文献
4.
Minoru Yonezawa Masahiro Takahata Naoko Banzawa Nobuyuki Matsubara Yasuo Watanabe Hirokazu Narita 《Microbiology and immunology》1995,39(7):517-520
The functional contributions of amino acid residue Asp87 of Escherichia coli gyrase A protein (GyrA) was analyzed by site-directed mutagenesis. We generated a series of mutants, in which Asp87 of GyrA was changed to Ala, Val, Phe, Asn, Ser, and Lys. By genetic analysis of gyrA genes in a gyrA temperature-sensitive (Ts) background, it was shown that all these mutations caused the quinolone-resistance. These results indicate that the 87th amino acid of E. coli GyrA must have negative charge in expressing the phenotype of quinolone sensitivity. These findings also suggest that the carboxyl group of Asp87 may interact with quinolone drugs. 相似文献
5.
Summary The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated in the developing oocytes to provide a nutrient source for embryogenesis. We have described the phenotype of a temperaturesensitive female-sterile mutant, fs(1) K313, and characterised its yolk proteins. This mutation affects the secretion of YP2 and is the first mutation affecting YP2 to be described. Using genetic and molecular tests we argue that the female-sterile phenotype results, at least in part, from the abnormal secretion of YP2 perturbing the follicle cell secretory pathway in general and thus causing defects in chorion protein secretion. The gene coding for YP2 in fs (1) K313 has been cloned and sequenced. Two amino acid substitutions have been found which probably cause the abnormal secretion of YP2 and the resulting female-sterile phenotype. 相似文献
6.
Minoru Yonezawa Masahiro Takahata Naoko Banzawa Nobuyuki Matsubara Yasuo Watanabe Hirokazu Narita 《Microbiology and immunology》1995,39(4):243-247
Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH2-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues. 相似文献
7.
Kazuhiro Ikenaka Kangla Tsung Dorothy E. Comeau Masayori Inouye 《Molecular & general genetics : MGG》1988,211(3):538-540
Summary We have fortuitously created an in-frame insertion mutation in the cloned ompR gene of Escherichia coli in the course of an experiment involving linker insertion mutagenesis. According to the DNA sequence, the mutant protein has an insertion at the 53rd amino acid residue, which replaced the original valine, with the sequence Ala-Leu-Glu. The expression level of the mutant protein, OmpRX6, in a minicell system, is similar to that of the wild-type protein and the size of the mutant is slightly larger than the wild type by approxiately 300 daltons. This mutant was completely unable to activate porin expression as the wildtype does, and in addition, this phenotype was shown to be dominant over the wild type. Comparison of the amino acid sequence of OmpRX6 with those of a family of homologous bacterial regulatory proteins revealed that the mutation lies in a domain which is highly conserved among these proteins. 相似文献
8.
Tsutomu Fukuda Kohsai Fukuda Akihisa Takahashi Takeo Ohnishi Tamotsu Nakano Masaru Sato Norio Gunge 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2000,470(2):125
Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (SmS) to its mutant phenotype (SmR). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations. 相似文献
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Barbara L. Randolph-Anderson John E. Boynton Nicholas W. Gillham Changzhi Huang Xiang-Qin Liu 《Molecular & general genetics : MGG》1995,247(3):295-305
The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg68 (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln53 to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu68 mutations in rps4. Instead, six of 15 contained a Gln73 (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln73 to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence. 相似文献
11.
The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms. 相似文献
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13.
We have cloned and sequenced a gene encoding O-acetylserine lyase from Streptococcus suis. The gene encodes a protein of 309 amino acids with a calculated molecular mass of 32,038 Da. The deduced amino acid sequence
showed more extensive similarities to the CysK proteins than to the CysM proteins of other bacteria. The cloned gene was inserted
into a pTrcHisB histidine hexamer expression vector. A 38-kDa fusion protein was expressed in a cysMK auxotrophic mutant of Salmonella typhimurium and complemented the auxotrophic properties of the mutant. Furthermore, the transformants could grow in minimal defined media
supplemented with not only sulfide but also thiosulfate as a sole sulfur source. These data indicated that the cloned gene
encodes a protein that was a functional homolog of the CysM in S. typhimurium.
Received: 1 July 1999 / Accepted: 10 August 1999 相似文献
14.
Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori. 相似文献
15.
Quinolone-resistant mutations of the gyrA gene of Escherichia coli 总被引:35,自引:0,他引:35
Hiroaki Yoshida Tsuyoshi Kojima Jun-ichi Yamagishi Shinichi Nakamura 《Molecular & general genetics : MGG》1988,211(1):1-7
Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA. 相似文献
16.
A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome C550, but the membrane-bound holocytochromes c1 and C552 were present at 5-10% of the levels observed in wild-type ceils, DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild-type DNA library. A hybridizing 3.05 kb Bam HI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DPI08 and the B. japonicum cycH mutant C0X3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor. 相似文献
17.
Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor σs. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS ? derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations. 相似文献
18.
Thomas W. Gibbs Deborah R. Gill George P. C. Salmond 《Molecular & general genetics : MGG》1992,234(1):121-128
Summary After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein. 相似文献
19.
Shu-Yun Qi Soila Sukupolvi C. David O'Connor 《Molecular & general genetics : MGG》1991,229(3):421-427
Summary We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485–510 kb) whereas sipC and sipD both map to 82 min (3850–3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium. 相似文献
20.
Identification of a two-component regulatory system controlling methanol dehydrogenase synthesis in Paracoccus denitrificans 总被引:3,自引:0,他引:3
Nellie Harms Willem N.M. Reijnders Hideharu Anazawa Carol J. N. M. van der Palen Rob J. M. van Spanning L. Fred Oltmann Adriaan H. Stouthamer 《Molecular microbiology》1993,8(3):457-470