Eliminatory Action of Glycine on Drug Resistance of Escherichia coli K12 Harboring an R Factor

Glycine, known to inhibit the synthesis of a peptidoglycan component of the bacterial cell wall, was effective in eliminating drug resistance of Escherichia coli K12 JE2100 strain harboring the R100–1 factor, although in lower frequencies than that of sodium dodecyl sulfate (SDS). The action of glycine was found to be less effective on the same R factor in JE177 strain, and not effective on the F factor in W6. Infection of R factors from R⁺ cells to R^– cells was found to take place in the glycine broth as efficiently as in broth without glycine. This might result in lowering the apparent efficiency of the action of glycine on those plasmids. The segregation patterns of drug-susceptible clones obtained by the glycine treatment were different from those obtained after the SDS treatment. These results coupled with other evidences suggest that the mode of action of glycine on R⁺ cells may be different from those of other curing agents and may involve mechanisms other than selection of R^– or drug-susceptible segregants that are present in R⁺ culture.     

Temperature-sensitive Chloramphenicol Acetyltransferase from Escherichia coli Carrying Mutant R Factors

Bacteria carrying temperature-sensitive mutant R factors for chloramphenicol resistance were isolated. In the presence of chloramphenicol, these bacteria grew at 34 C but not at 43 C. The mutations in the chloramphenicol resistance gene of the R factors affected neither the resistance of the bacteria to dihydrostreptomycin and tetracycline nor the stability of the R factors at 43 C. The chloramphenicol acetyltransferase obtained from Escherichia coli K-12 carrying the mutant R factors was heat-labile as compared with that from a strain carrying the wild-type R factor. We could not find chloramphenicol acetyltransferase activity in 17 chloramphenicol-sensitive and 5 -resistant strains (selected in vitro) of E. coli examined. The results strongly suggest that the chloramphenicol resistance gene of the R factors is the structural gene of the chloramphenicol acetyltransferase rather than the genome controlling the expression of a chromosomal determinant for the enzyme. Furthermore, the studies confirm that the existence of the chloramphenicol acetyltransferase is the primary cause of chloramphenicol resistance of bacteria carrying the R factor. Both the enzyme activity producing the monoacetyl derivative from chloramphenicol and the subsequent formation of the diacetate from the monoacetyl product were heat-labile to the same degree. The results suggest that only one enzyme participates in two steps of chloramphenicol acetylation.     

Variant of Penicillinase Mediated by an R Factor in Escherichia coli

The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.     

Studies on Resistance Transfer Factor Deoxyribonucleic Acid in Escherichia coli

A variant of the derepressed R factor, R1, which does not contain any of the drug resistance markers, and represents, in large part, the resistance transfer factor (RTF) was studied in Escherichia coli. RTF deoxyribonucleic acid (DNA) was specifically labeled in a female cell after conjugation. Physical characterization of the molecule showed that RTF possessed an average molecular weight of 50 x 10(6) daltons and a buoyant density of 1.709 g/cm(3). By comparison to R1, we calculate that the region of DNA carrying the drug resistance genes is therefore about 20% of the R1 molecule and has a buoyant density of approximately 1.716 g/cm(3). These results support the hypothesis that the single species of R-factor DNA observed in E. coli represents a composite of the 1.709 and 1.716 g/cm(3) replicons seen in Proteus.     

Drug Resistance of Enteric Bacteria VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

Genetic Mapping of Cold Resistance Gene of Escherichia coli

Using cold resistant mutants, MET1 and MET2, obtained from Escherichia coli K-12, genetic mapping of the cold resistance gene(s) of E. coli was performed by the conjugation and transduction techniques. The gene(s) was confirmed to be located close to trpB at 28 min (revised chromosome linkage map, 1983) on the E. coli chromosome.     

Chromosome replication in an Hfr strain of Escherichia coli

The pattern of chromosome replication in an exponentially growing culture of an Hfr strain of Escherichia coli has been compared to that obtained with the same Hfr following a procedure which synchronizes rounds of DNA replication. The results indicate that there is significant replication from the integrated plasmid following the synchronization procedure, whereas in the exponentially growing culture replication starts most frequently from the normal origin with little, if any, replication from the sex factor, F.     

R Factor Deoxyribonucleic Acid in Chromosomeless Progeny of Escherichia coli

The deoxyribonucleic acid (DNA) of resistance (R) factor 222 carried by Escherichia coli strain P678-54 was found in the normally chromosomeless progeny (minicells) of that strain. The entry of the R222 DNA into minicells appears to be via segregation at the time of their formation from normal cells. The R222 DNA can replicate in minicells although the extent of its replication appears to be limited. An analysis of the R222 DNA structure indicates that it exists in minicells as double-stranded linear, open circular, and twisted circular monomers (molecular weight, about 6.2 x 10(7) daltons). The monomers visualized by electron microscopy are 31.0 +/- 0.5 mum in length. An examination of the effect of acridine orange on the replication of R222 and colicin E1 DNA indicates the dye intereferes with plasmid DNA replication.     

Enzymatic Reduction of Mercurous Ions in Escherichia coli Bearing R Factor

Mercurous ion (Hg(+))-dependent reduced nicotinamide adenine dinucleotide phosphate oxidation was demonstrated in an extract from cells of Escherichia coli W2252 that bear R factor.     

Gene recombination and segregation of resistance factor R in Escherichia coli

Hashimoto, Hajime (Osaka University, Osaka, Japan), and Yukinori Hirota. Gene recombination and segregation of resistance factor R in Escherichia coli. J. Bacteriol. 91:51-62. 1966.-Independent chloramphenicol-sensitive (CM(s)) mutants of the drug-resistance factor R were isolated. Introduction of two different R factor CM(s) mutants into a single bacterium, by conjugation or transduction, gave chloramphenicol-resistant (CM(r)) colonies when such strains were plated on a medium containing chloramphenicol (Cm). These CM(r) colonies resulted from recombination between two R factors contained within the same cell. Most of the CM(r) colonies were heterogeneous, and segregation of drug-resistance markers was observed among the progeny. Segregated bacteria which still carried the recombinant R factor were stable for resistance to Cm as well as for other markers of R. All the markers of recombinant R factors were cotransducible with high coincidence and at the same frequency as wild-type R. Sensitive mutants of R which had lost all the resistance markers of the R factor were found also. A mutation of R, referred to as SMA, which was sensitive to streptomycin and sulfanilamide, was capable of reverting to resistance to both of these drugs simultaneously. The sensitive alleles for SMA, CM, and TC were shown to be recessive to the resistance alleles. Mutants of R having multisite mutations or deletions in the CM gene were isolated and used to analyze the pattern of linked segregation of unselected markers of the recombinant R factor. The drug resistance factor R was shown to have two linkage groups, CM-SMA and TC-m.     

Integration of Gene Maps: Chromosome X

Omitting 1137 loci that are included in the location database but have only cytogenetic assignment, there are 605 loci in the integrated map that synthesizes physical and genetic data and subsumes a composite physical location, cytogenetic and regional assignments, mouse homology, rank, and references. With error filtration and allowance far interference the genetic length is 211 cM, to which the p arm contributes 100 cM. The physical length is 164 Mb, with 62 Mb in the p arm. Current problems in map integration are discussed and some solutions proposed.     

PQR转运体基因赋予大肠杆菌BL21百草枯抗性

前期研究中成功地分离了2个对百草枯具有高度抗性的土壤细菌SPQ03和SPQ14.本研究从这两个菌分别克隆了基因PnPQR和OaPQR,二者ORF全长均为1 233 bp,编码410个氨基酸残基,含有11个跨膜区(TMS),属于非典型的主要易化超家族(major facilitator superfamily, MFS).立体结构分析表明,蛋白的N端和C端分别由5个和6个由α-螺旋组成的跨膜区.只有P151L和P154V两个氨基酸不同.将两个基因在大肠杆菌BL21菌株中异源表达,能提高大肠杆菌对百草枯的抗性,但不能提高其对过氧化氢的抗性.     

The Structure of the Chloramphenicol Resistance Gene on a Transferable R Plasmid from the Fish Pathogen,Pasteurella piscicida

The chloramphenicol resistance gene (pp-cat) was cloned from a transferable R plasmid of Pasteurella piscicida, pSP9351, and the sequence of the gene was determined. Subcloning and deletion analysis localized the resistance gene, pp-cat, to within a 2.3 kb HincII-BamHI fragment. The fragment as a probe hybridized with the type I chloramphenicol acetyltransferase (CAT) gene and did not hybridize to CAT types II, III, and CAT-VA. The fragment hybridized to transferable R plasmids encoded with resistance to chloramphenicol, which were detected from P. piscicida isolated in different years. Nucleotide sequences of the coding and flanking regions of pp-cat (2031 bp) identified an open reading frame coding type I CAT of a molecular mass of about 25,000 Da. Comparison analysis of the sequences outside the cat open reading frame showed also that pp-cat has homology, in part, with the gene that coding for the endonuclease EcoRII and those that flank the cat gene derived from the Acinetobacter baumannii chromosome.     

Effect of p-Fluorophenylalanine on Chromosome Replication in Escherichia coli

The effect of p-fluorophenylalanine (FPA) on deoxyribonucleic acid (DNA) synthesis and chromosome replication was studied in a thymine-requiring mutant of Escherichia coli. The rate and extent of chromosome replication were followed by labeling the DNA with isotopic thymine and a density marker, bromouracil. The DNA was extracted and analyzed by CsCl gradient centrifugation. The block in chromosome replication caused by high concentrations of FPA occurred at the same point on the chromosome as that caused by amino acid starvation. In a random culture, DNA in cells treated with FPA replicated only slightly slower than the DNA from cells that were not exposed to the analogue. In cultures which had been previously starved for thymine, however, the DNA from the cells treated with FPA showed a marked decrease in the rate and extent of replication. It was concluded that the E. coli cell is most sensitive to FPA when a new cycle of chromosome replication is being initiated at the beginning of the chromosome.     

Bacteriophage Transcription Factor Cro Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli

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拟南芥AtTERT对大肠杆菌非生物胁迫抗性的影响

端粒酶是真核生物中维持染色体末端DNA完整性的一类特殊逆转录酶,研究拟南芥AtTERT对大肠杆菌生长及非生物胁迫的影响,为深入研究TERT蛋白非端粒功能奠定基础.将拟南芥AtTERT转入大肠杆菌,成功构建pET32a-AtTERT原核表达载体,优化诱导条件,纯化并鉴定GST-AtTERT融合蛋白,运用Western b...