A bacteriocinogenic factor of Enterobacter cloacae

Summary Enterobacter cloacae strain DF13 produces a bacteriocin which is able to kill other strains of Enterobacter and Klebsiella. This property can be transmitted to Enterobacter cloacae strain O 2 (up to 90% of the acceptor population became bacteriocinogenic), to E. coli K12F^- and E. coli K 12 Hfr. Transfer of chromosome material was never observed, suggesting that the production of the bacteriocin is determined by a plasmid. However all attempts to eliminate this plasmid failed. The plasmid F trp cys Col B Col V could be transferred from E. coli into Ent. cloacae DF13 and subsequently it could be eliminated by acridine orange treatment. Ent. cloacae DF13 harbours in addition two independently transferable R-factors, one determining resistance against streptomycin and sulfanilamide and the other resistance against penicillin.Most but not all Ent. cloacae O2 recombinants which have received only the bacteriocinogenic factor upon conjugation with Ent. cloacae DF 13, can transfer this property to Ent. cloacae O2 but not to E. coli. E. coli F^- recombinants, which have received only the bacteriocinogenic factor cannot transfer this property. The results suggest that the bacteriocinogenic factor cannot mediate its own transfer, but can be transferred when another transmissible plasmid is present. This may be the R(str sul) factor, the F-factor in E. coli Hfr or a transfer factor () in Ent. cloacae O2.Closed circular DNA molecules were selectively isolated from these strains and investigated by electron microscopy and velocity sedimentation. Ent. cloacae DF13 harbours small closed circular DNA molecules ranging from 0.5 to 3.2 in contour length, 98% of which corresponds to a size class of 0.7±0.1 . Ent. cloacae O2 also harbours closed circular DNA ranging from 0.8 to 3.0 in contour length, with major size classes of 0.9 and 1.4 respectively. Circular DNA of a contour length of 3.0±0.2 (S_20,w=26 S) corresponding to a molecular weight of 6.0×10⁶ daltons was transferred to E. coli and Ent. cloacae O 2 concomitantly with the ability to produce the bacteriocin. A significant difference was observed in the number of copies of the plasmid between Ent. cloacae and E. coli.     

Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA

The avian retrovirus pp32 protein possesses DNA endonuclease activity and unique DNA binding properties. An improved purification procedure was developed for pp32, resulting in a severalfold increase in the yield of this virion protein. By use of the nitrocellulose filter binding assay, the protein retains approximately 2-fold more supercoiled (form I) DNA molecules than equivalent linear duplex DNA molecules. Single-stranded DNA is only slightly preferred over double-stranded DNA for pp32 binding. The pp32 DNA binding sites on form I pBR322 DNA which contained an insert of avian retrovirus long terminal repeat (LTR) DNA were determined. A preformed protein-DNA complex was digested with one of several different multicut restriction enzymes and filtered through nitrocellulose filters. Fragments containing viral LTR DNA sequences and plasmid DNA containing promoter sequences for the ampicillin and tetracycline genes, sequences for the "left-end" inverted repeat of transposon 3, and sequences encompassing the carboxyl terminus of the beta-lactamase gene were preferentially retained on the filter by pp32. Partial mapping of pp32 DNA binding sites on LTR DNA was accomplished by generation of deletions in LTR DNA sequences. The pp32 protein preferentially bound viral DNA fragments which contain the viral promoter (TATTTAA) and the adjacent "R" repeat sequences. Computer analysis revealed that three of the four plasmid DNA fragments retained by pp32 contained LTR DNA promoter-like sequences (one mismatch only) which were part of statistically significant and thermodynamically stable hairpin structures.     

DNA binding and antigenic specifications of DNA gyrase.

Complexes of DNA gyrase and minichromosomal DNA containing the origin of replication of Escherichia coli (oriC) can be formed without metabolic energy and visualised by electron microscopy. The A subunit, part of the A2B2-DNA gyrase complex is the binding protein. Various binding sites are scattered around the minichromosomal DNA including oriC. The minimal origin contains the only prominent and reproducible binding site. Binding to this site is suppressed by oxolinic acid and the ATP analogue beta-y-imido ATP. If gyrase isolated from the gram-positive bacterium Bacillus subtilis is used no binding to oriC is seen. This observation is consistent with antigenic differences between the A subunits of the two microorganisms. The binding to oriC might reflect a requirement for DNA gyrase during the initiation of DNA replication.     

从耐药产气肠杆菌中首次发现DNA旋转酶A亚单位基因gyrA新的变异型

目的 全面了解2株耐药产气肠杆菌对β-内酰胺类、氨基糖苷类、喹诺酮类的耐药机制.方法 2株耐药产气肠杆菌:zjm001株和zjm002株,分离自2012年3月一家三级甲等医院住院患者送检的血液标本,做gyrA和parC基因扩增、测序、GenBank比对确认菌种,再用PCR法分析39种β-内酰胺类药物获得性耐药基因、14种氨基糖苷类药物获得性耐药基因、7种喹诺酮类耐药相关基因、11种可移动遗传元件标记.结果 zjm001株的β-内酰胺类耐药基因检出CTX-M-3,氨基糖苷类耐药基因检出ant(3”)-Ⅰ,喹诺酮类耐药基因检出gyrA突变,可移动遗传元件检出int Ⅰ 1、ISEcp1、IS26、IS903;zjm002株的β-内酰胺类耐药基因检出ACT-1、CMY-2,喹诺酮类耐药基因检出gyrA突变.2株耐药产气肠杆菌的gyrA基因序列均为新变异型,GenBank登录号分别为:JX268598,JX273641.结论 2株耐药产气肠杆菌的耐药基因与耐药表型相符.在耐药产气肠杆菌中发现DNA旋转酶A亚单位基因gyrA新变异型是国内首次报道.     

The influence of DNA gyrase on the transcription of linear DNA in vitro

To determine the mode of disulphide bond formation in conotoxin GI, a tridecapeptide amide with 4 Cys residues, all 3 of its peptides having different modes of disulphide-bond formation were synthesized by solution procedure using selectively removable protective groups at the Cys residues. After deprotection with HF, one pair of acetamidomethyl groups was left unremoved, and then two sets of disulphide bonds were formed selectively. The toxic potency in mice of one product was comparable with that reported for native conotoxin GI and was almost 10-fold as high as that of the other two products. The toxicity of the native toxin reportedly is not regenerated upon reduction and reoxidation, but this study showed that the most toxic product was the most readily formed one.     

Nonspecific induction of beta-lactamase in Enterobacter cloacae

Induction of beta-lactamase was monitored in a strain of Enterobacter cloacae exhibiting high resistance to most beta-lactam antibiotics. Large amounts of the enzyme were induced not only in the presence of beta-lactams, but also in the presence of other bicyclic molecules such as folic acid, thiamin, tryptophan or haemin. Moreover, complex media (such as Trypticase soy broth and Schaedler's broth) and various body fluids (serum, pleural fluid and cerebrospinal fluid) also possessed considerable induction potency. Neither 'specific' induction (by beta-lactams) nor 'non-specific' induction (by other bicyclic compounds) could be augmented by addition of exogenous cAMP. These findings indicate that inducible beta-lactamases deserve more attention, above all with respect to the development of resistance against third-generation cephalosporins.