An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.     

Plasmid ColE3 specifies a lysis protein.

Tn5 insertion mutations in plasmid ColE3 were isolated and characterized. Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium. Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells. All of these lysis mutants mapped near, but outside of, the structural genes for colicin E3 and immunity protein. Cells carrying the insertion mutations which did not release colicin E3 into the medium were not killed by UV exposure at levels that killed cells bearing wild-type plasmids. The protein specified by the lysis gene was identified in minicells and in mitomycin C-induced cells. A small protein, with a molecular weight between 6,000 and 7,000, was found in cells which released colicin into the medium, but not in mutant cells that did not release colicin. Two mutants with insertions within the structural gene for colicin E3 were also characterized. They produced no colicin activity, but both synthesized a peptide consistent with their map position near the middle of the colicin gene. These two insertion mutants were also phenotypically lysis mutants--they were not killed by UV doses lethal to wild-type colicinogenic cells and they did not synthesize the small putative lysis protein. Therefore, the lysis gene is probably in the same operon as the structural gene for colicin E3.     

Structural and functional organization of the colicin operon in the ColD-CA23 plasmid

The organization of the genes involved in colicin D synthesis was studied. These are colicin, immunity and lysis genes. The nucleotide sequence of the immunity gene, its structural and regulatory regions were determined. This gene was shown to be located next to the colicin gene on the same strand and followed by the lysis gene. When colicin synthesis is induced with mitomycin C the immunity gene is transcribed from the general SOS-dependent promotor as a part of the colicin operon. However it has its own SOS-independent promotor in normal growth conditions. A high homology in amino acid sequences of Co1D lysis protein and that of Co1E1, Co1E2, Co1E3, Co1DF13, Co1A was revealed. A detailed scheme of Co1D-CA23 colicin operon structural organization is suggested.     

Characterisation of the ColE8 plasmid, a new member of the group E colicin plasmids

We have determined the restriction map of the ColE8-J plasmid after cloning it into the pBR322 vector. By subcloning and transposon mutagenesis we have localized the colicin immunity gene, the colicin structural gene, and lys, the region that determines MC sensitivity. In contrast to the ColE3-CA38 plasmid, the genes coding for colicin E8 production and immunity cannot be cloned on a single EcoRI fragment. Insertion of Tn5 transposons into the colicin structural gene region of the recombinant plasmid inactivated colicin production and MC sensitivity. Insertion of transposons into the lys region reduced colicin E8 production and MC induced lysis, the extent of which was dependent upon the precise site of insertion. We propose that the colicin E8 structural gene and lys must be transcribed from a common promoter situated proximal to the structural gene, whilst the colicin E8 immunity gene is transcribed from a second promoter. The lys region is responsible both for cell lysis after MC induction and positive regulation of colicin E8 synthesis.     

Characteristics of replication of small colicinogenic plasmids

Specificity of small multicopy colicinogenic plasmids ColA, ColD, ColE2 and ColK replication has been compared with the one of ColE1 plasmid. Copy number for these plasmids per host cell has been estimated under the normal conditions of cellular growth and under the conditions of chloramphenicol-inhibited growth. DNA polymerase I and dnaB protein, an obligatory component for elongation step in replication, have been shown to be necessary for the plasmids replication. Initiation of plasmids replication has been demonstrated to be independent of dnaA and dnaC proteins. Replication of plasmid ColE2, being similar in its main features to replication of other plasmids from this group, has an important distinction. It requires de novo protein synthesis implying that ColE2 replicon may be different from ColA, ColD, ColK, ColE1 replicons. Thus study of the inducible A, D, K, El colicin synthesis coded by the corresponding plasmids has revealed the similarity regulation of genes, determining the synthesis of each of the mentioned colicins.     

beta-Galactosidase and alkaline phosphatase do not become extracellular when fused to the amino-terminal part of colicin N

A series of plasmids encoding hybrid proteins comprising various lengths of the NH2-terminal region of colicin N coupled to almost complete beta-galactosidase or alkaline phosphatase polypeptides was constructed by transposon mutagenesis of ColN plasmid derivatives. Synthesis of the hybrid proteins, like that of colicin N itself, was regulated by the SOS response. Large quantities of the hybrid proteins accumulated in the cytoplasm (beta-galactosidase) or particulate fractions (alkaline phosphatase). When the gene fusions were expressed in cells that were producing colicin E2 and expressing the ColE2 lysis gene, only very low levels of the hybrid proteins were found in the medium. The results suggest that the amino-terminal part of colicin N does not contain sufficient biochemical information to promote the release of the hybrid proteins into the medium.     

Cloning and characterization of the ColE7 plasmid

The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.     

Genetic analysis of the functional relationship between colicin E3 and its immunity protein.

Partial deletions in the immunity gene of the colicin E3 operon were used to study possible functions of the immunity protein besides protection against exogenous colicin. Nuclease BAL-31 was used to create a series of carboxyl-terminal deletions of the immunity gene. Mutants displaying lowered immunity against exogenous colicin were found, and six that had reduced but detectable levels of immunity were chosen for further analysis. DNA sequence analysis of the deletions showed that all six terminated within the last five codons of the immunity gene. The wild-type immunity gene was replaced by each of the six mutated immunity genes in a plasmid containing an otherwise functional colicin E3 operon. Transformants containing the resulting plasmids produced smaller colonies on solid medium and grew more slowly in liquid culture than transformants carrying the wild-type colicin and immunity genes. This result suggested that immunity protein was required to protect the cell against endogenous colicin E3. This idea was confirmed in experiments in which the colicin E3 and immunity genes were independently cloned on two compatible plasmid vectors.     

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.     

Structuro-functional organization of segments of Co1A plasmid DNA, determining its stable inheritance

Two par regions were localized within the structure of a small colicinogenic plasmid ColA. One of them functions at the expense of plasmid multimere resolution. Analysis of the nucleotide sequence of the region revealed the existence of essential homology with the par locus of plasmid ColE1. As compared to E. coli C600, the function of multimere forms' resolution of plasmid DNA in E. coli C is reduced or absent due to par regions of the ColE1 type. Par regions of various degrees of homology with the par locus of ColE1 were localized by Southern hybridization within the structure of colicinogenic plasmids ColN and ColD. The stabilization of the colicinogenic plasmids is believed to be also determined by the functioning of genes connected with the synthesis and action of colicin.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Molecular characterisation of the colicin E2 operon and identification of its products

Summary The DNA sequence of the entire colicin E2 operon was determined. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB. A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology. These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively. Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins. The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides. Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase. The mature ColE2 lysis protein was located in the cell envelope. The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.     

Nucleotide sequence of the immunity and lysis region of the ColE9-J plasmid

We have determined the nucleotide sequence of a 1500 bp fragment of the ColE9-J plasmid which encodes colicin E9 immunity and colicin E5 immunity and contains two lys genes. Open reading frames corresponding to the four genes have been located and their position confirmed by transposon mutagenesis of sub-clones of the ColE9-J plasmid. The E9imm gene shows 69% homology at both the nucleotide and the amino acid level to the previously sequenced E2imm gene. The E5imm gene shows little homology to any other E colicin immunity gene which has been sequenced. The lys gene distal to the 3' end of the E5imm gene shows considerable sequence homology to all other previously sequenced E colicin lys genes. The lys gene distal to the 3' end of the E9imm gene is identical to the pColE2 and pColE3 lys genes for the first 59 nucleotides but encodes a much smaller gene product than any other lys gene which has been sequenced. The two lys genes sequenced here are exceptions to Shepherd's rule concerning the number of RNY codons in the three possible reading frames.     

Expression of a gene in a 400-base-pair fragment of colicin plasmid ColE2-P9 is sufficient to cause host cell lysis.

The colicin E2 immunity (ceiB) and lysis (celB) genes of colicin plasmid ColE2-P9 were cloned as a 900-base-pair insert under the control of the lac promoter in high-copy-number plasmid pUR222. Hosts carrying this plasmid were immune to colicin E2, produced increased amounts of immunity protein (molecular weight, 9,000) and two smaller proteins (molecular weights, 5,000 and 3,000), and lysed when incubated in medium containing isopropyl-beta-D-thiogalactopyranoside (IPTG). A 400-base-pair lacp-distal fragment derived from the insert in this plasmid was recloned in the same orientation into pUR222. Although hosts carrying this plasmid also lysed when grown in the presence of IPTG, they were sensitive to colicin E2 and produced increased amounts of the 5,000- and 3,000-molecular-weight proteins (but not the full-length immunity protein) when treated with IPTG. The results were consistent with the idea that expression of celB (production of the 5,000- and 3,000-molecular-weight proteins) is sufficient to cause host cell lysis in the absence of colicin production and derepression of the host cell SOS system.     

Growth inhibition of Escherichia coli by E colicin plasmids

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.     

Characterization of the ColE9-J plasmid and analysis of its genetic organization

We have determined the restriction and functional map of the ColE9-J plasmid. By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments. Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter. Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation.     

Plasmid pColBM-Cl139 does not encode a colicin lysis protein but contains sequences highly homologous to the D protein (resolvase) and the oriV region of the miniF plasmid

Colicins are usually released from producing cells by so-called lysis proteins. No sequence homologous to the structurally very similar colicin lysis genes was found in the gene cluster cmi cma cbi cba, which determines the activity and immunity proteins of colicin B and M on pColBM-Cl139. Instead, the region upstream of cmi contained sequences that showed 91% homology to the structural gene of protein D (resolvase) and 75.5% homology to the rfsF sequence of the Escherichia coli miniF plasmid. It is concluded that colicins B and M are not released via the activity of lysis proteins and that the highly homologous regions encode a resolvase and its target respectively.     

Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm.

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.     

Degradation of substituted phenylurea herbicides by Arthrobacter globiformis strain D47 and characterization of a plasmid-associated hydrolase gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).