Four plasmid genes are required for colicin V synthesis, export, and immunity.

The colicin V production and immunity genes were isolated from plasmid pColV-K30. A HindIII-to-SalI fragment of 9.4 kilobases was cloned into the compatible vectors pBR322 and pACYC184. Mutants defective in colicin production were generated by Tn5 insertions and by constructing deletions in vitro. Physical analysis of these mutations identified a 4.4-kilobase region of this DNA which contains all the plasmid genes (cva) needed for the production of colicin V. The colicin V immunity determinant (cvi) is in a 700-base-pair fragment located within one end of this region. Complementation tests identified three genes, called cvaA, cvaB, and cvaC, required for colicin production. Analysis of the proteins labeled in minicells harboring various Tn5 insertions allowed us to identify protein products for the cvaA and cvaC genes. Mutations in cvaA and cvaB eliminated colicin activity in culture supernatants, but not within the cells. Mutations in cvaC, however, eliminated all detectable activity. From these results we conclude that the cvaC gene codes for the structural gene for colicin V, while cvaA and cvaB are apparently needed for the normal export of the colicin.     

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.     

Cloning and overexpression of the colicin E1 immunity gene

A DNA fragment containing only the putative immunity gene-coding sequence was cloned under the control of the trp and lambda PL promoters, generating pRKA11 and pIPL, respectively. Escherichia coli hosts containing either construction were immune to colicin E1. Cells harboring both pIPL and pNT204, which encodes a temperature-sensitive cI repressor, were sensitive to colicin E1 at 30 degrees C, but became immune after 0.5 h of incubation at 42 degrees C. In addition, pRKA11 directed the synthesis of a 14.5-kDA protein in maxicells, identical to that found with the wild-type immunity gene. This evidence identifies unequivocally the coding sequence of the immunity gene as that extending from bases 1214 to 1552 [OKA, A., et al., Mol. Gen. Genet. 172, 151-159 (1979)]. The entire immunity gene operon was also cloned under the control of the tac promoter, generating pTCU2, which, upon induction with isopropyl beta-D-thiogalactopyranoside, produced the imm gene product in amounts sufficient to be visualized by autoradiography.     

Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.     

Localization of genes responsible for replication and immunity to colicin A on plasmid ColA-CA31

The region containing the origin and regulatory sites for replication as well as the immunity gene (iaa) have been localized on the plasmid ColA-CA31. The region involved in replication functions of ColA can be hybridized with that of ColE1. It is located between 1 and 1 kb on the plasmid map previously published (Morlon et al. 1982a). A 0.50 Kb HincII fragment of ColA can be weakly hybridized to the ColE1 immunity region. This fragment contains iaa since directed in vitro mutagenesis at an internal restriction site can abolish the immunity to colicin A; however, it does not contain the entire iaa. Knowing the localization of regions involved in autonomous DNA replication and immunity, a mini-ColA plasmid was constructed that contains these two regions. The mini-ColA of 2.8 Kb can be amplified in the presence of chloramphenicol and confers the immunity to transformants. It thus constitutes a useful cloning vector. Expression of ColA and of the various constructed plasmids in the maxicell system suggests that the immunity protein has a molecular weight of about 18-20 Kd.     

A DNA segment within the colicin E1 structural gene on ColE1 affecting immunity to colicin

Summary Insertion of DNA at the EcoRI site of ColE1 results in increase of immunity to colicin killing in E. coli harboring such recombinant ColE1 plasmid as compared to E. coli (ColE1). This effect is neither due to cis or trans interactions originating from the inserted foreign DNA fragment, nor to changes in plasmid copy number. This defect in the immunity mechanism is not trans complemented for by wild type ColE1. Increase in immunity can also be obtained by deleting a DNA segment from the ColE1 genome. This segment is 120 bp left to the EcoRI site within the colicin structural gene. It is concluded that the structure of DNA per se, around the EcoRI site, within colicin structural gene, is the structure which affects immunity expression.     

Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.     

Iron-regulated synthesis and uptake of colicin V

Abstract Virulent strains of Escherichia coli frequently contain ColV plasmids. It is shown that synthesis of the marker protein, colicin V, is regulated by iron via the iron repressor encoded by the fur gene. Mutants in the Cir outer membrane protein, in tonB , as well as in the exbB gene are resistant to colicin V suggesting all three functions to be required for colicin V uptake.     

Sequence, expression and localization of the immunity protein for colicin B

Summary Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20 185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.     

Binding of the immunity protein inactivates colicin M

Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma-producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N-terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.     

Is the CvaA* protein, encoded within the colicin V export gene cvaA, required for colicin V transport?

Abstract We investigated the expression of important Actinobacillus pleuropneumoniae surface polysaccharides, namely, capsular polysaccharides (CPS) and lipopolysaccharides (LPS), after growth under iron-restricted conditions. Iron restriction did not seem to affect the production of CPS, as determined by labelling with a monoclonal antibody (mAb) against the serotype l K-antigen and flow cytometry analysis, and also as determined by electron microscopy. SDS-PAGE revealed that the LPS profiles of these cells were also unaffected by iron restriction. Using flow cytometry analysis, however, we observed that binding of mAb against serotype 1 O-antigen was altered in cells of A. pleuropneumoniae serotype l reference strain (4074) grown under iron-restricted conditions. This strain exhibited two subpopulations with distinct patterns of reactivity with the mAb against the O-antigen. When strain 4074 was grown under iron-restricted conditions, a shift from one cell subpopulation (moderately fluorescent) to another cell subpopulation (highly fluorescent, thus binding more antibodies) was observed. Our results indicate that growth of A. pleuropneumoniae serotype l under iron-restricted conditions did not seem to affect CPS production, but might alter, at least for the reference strain, the expression of LPS.     

Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes

A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease.     

Isolation and some properties of colicin V preparations.

E. coli strain CLI(V) produces colicin V which can exist in two chemically different forms. A heat-stable, liposaccharide-protein complex is present as a main component of the cell wash. An intracellular colicin is a heat-labile and seems to be a simple protein. Preliminary experiments have shown that colicin V inhibits simultaneously synthesis of protein, RNA and DNA. Its mode of action is similar to colicins: E1, B, K and A.     

志贺毒素保护性抗原的基因克隆与序列分析

根据GenBank报道的志贺毒素A、B亚单位基因序列设计合成两对引物,以I型痢疾志贺茵的基因组为模板经PCR扩增获得3条DNA序列,将其分别插入pMD18—T载体,测序证明所获得的3条序列为志贺毒素A、B亚单位基因及其串联片段,与GenBank中相应序列比较,同源性分别为99．5％、100％和99．8％。通过计算机软件对所推测的氨基酸序列进行了蛋白质参数分析。志贺毒素保护性抗原基因的获得为志贺毒素的免疫学研究准备了必要的材料。