Regions of homology in small colicinogenic plasmids

Restriction maps have been constructed for the colicinogenic plasmids (ColA, ColD, and ColK. Their regions of homology with the ColE1 plasmid and its deletion derivative pAO3 carrying the region responsible for autonomous replication of ColE1 plasmid were determined by means of blotting hybridization and heteroduplex analysis. The plasmids ColA, ColD, and ColK were shown to contain DNA fragments homologous to the region of ColE1 involved in the regulation of replication.     

Identification of functional regions of the colicinogenic plasmid ColA

Summary ColA is a colicinogenic plasmid of 6.72 kb. It is compatible with ColE1 but not with ColK. Transposon insertion mutagenesis as well as complementation studies have been carried out to investigate the location of the various functional regions of this plasmid. Four independent ColA::Tn1 and one ColA::Tn3 plasmids were isolated and the locations of insertions were determined. From these plasmids, six different deletion mutants were constructed. In addition, various restriction fragments of ColA have been cloned into pUC8 to carry out complementation studies. We have thus confirmed the location of the DNA regions involved in colicin production, colicin release and immunity function. The DNA region involved in conjugal mobility promoted by R64 drd11 has been identified and we have demonstrated that the ColE1 mobility proteins can act in trans on the bom (basis of mobility) site of ColA. The location of this site, as well as the region involved in stable maintenance of ColA, have also been determined. These results are discussed with regard to the homology in nucleotide sequence between ColA and ColE1.     

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.     

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.     

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.     

A ColE1-encoded gene directs entry exclusion of the plasmid.

To detect entry exclusion of the ColE1 plasmid, we established an assay system that was not influenced by incompatibility of extant plasmids in the recipient cells or by the viability of the cells due to the killing action of colicin E1 protein. The assay revealed that exc1 and exc2, assigned as genes directing entry exclusion, had no exclusion activity. Instead, mbeD, which had been characterized as a gene for plasmid mobilization, directed the exclusion activity. MbeD was overexpressed and identified as a 35S-labeled protein, which was recovered in both the soluble and membrane fractions, particularly in the inner membrane fraction. An amphipathic helical structure was predicted in the N-terminal region of MbeD as well as in the corresponding homologous proteins of ColA and ColK. These proteins may bind to the inner membrane via the N-terminal amphipathic helix and function in entry exclusion.     

Structural and functional organization of ColE2 and ColE3 replicons

Summary The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined. They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation. An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication. The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%). The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33 bp for ColE3. They are the smallest of all the prokaryotic replication origins so far reported. They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins. Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3. These results indicate that these plasmids share a common IncA determinant. A possibility that a small antisense RNA is involved in copy number control and incompatibility (IncA function) was suggested.     

Nucleotide sequences from the colicin E5, E6 and E9 operons: Presence of a degenerate transposon-like structure in the ColE9-J plasmid

Summary The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined. These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, theircis- ortrans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys^*) present in the ColE9-J plasmid. The ColE6 gene organisation, in the ordercol-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer). The corresponding genes in the two plasmids are 87%–94% homologous. In ColE9-J, the genes are organised ascol-imm-lys ^*-E5imm-lys. The E9col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer). Downstream from E9imm is an E5imm (designated E5imm[E9]) which istrans-acting. Neither the predicted structures of E5Imm[E9] nor thecis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced. Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conservedbtuB-specified receptor-binding domain. A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid. This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101. One effect of ISE9 is the presence of the atypical lysis gene,lys ^*. The presence of a transposon-like element in the ColE9 plasmid exemplifies a new phenomenon relevant to the evolution of colicin E plasmids. Issued as NRCC publication no. 30065     

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70 kb, pRK10 is only 4241 bp long with 53% G + C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5α. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.     

pUB2380: characterization of a ColD-like resistance plasmid

A detailed analysis of the mobilizable, ColE1-like resistance plasmid, pUB2380, is reported. The 8.5-kb genome encodes six (possibly seven) major functions: (1) a ColD-like origin of replication, oriV, with associated replication functions, RNAI and RNAII; (2) a set of active mobilization functions highly homologous to that of ColE1, including the origin of transfer, oriT; (3) a ColE1-like multimer resolution site (cer); (4) a kanamycin-resistance determinant, aph, encoding an aminoglycoside-3'-phosphotransferase type 1; (5) an insertion sequence, IS1294; and (6) two genes, probably cotranscribed, of unknown function(s). The GC content of the various parts of the genome indicates that the plasmid is a hybrid structure assembled from DNA from at least three different sources, of which the replication region, the mobilization functions, and the resistance gene are likely to have originated in the enterobacteriaceae.     

The importance of homologous recombination in the generation of large deletions in hybrid plasmids in Amycolatopsis mediterranei

The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined. pT3.2I is 15,390 bp long with a 53.7% GC content. Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp). Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids. Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB. Based on sequence comparisons, pT3. 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group.     

Colicin E3 and its immunity genes

A DNA segment of plasmid ColE3-CA38 was cloned into pBR328 and its nucleotide sequence was determined. This segment contains the putative promoter-operator region, the structural genes of protein A (gene A) and protein B (gene B) of colicin E3, and a part of gene H. Just behind the promoter region, there is an inverted repeat structure of two 'SOS boxes', the specific binding site of the lexA protein. This suggests that the expression of colicin E3 is regulated directly by the lexA protein. Genes A and B face the same direction, with an intergenic space of nine nucleotides between them. ColE3-CA38 and ColE1-K30 are homologous in their promoter-operator regions, but hardly any homology was found in their structural genes. On the other hand, ColE3-CA38 is fairly homologous to CloDF13 throughout the regions sequenced, with some exceptions including putative receptor-binding regions. By deletion mapping of the immunity gene and recloning of gene B, it was shown genetically that protein B itself is the actual immunity substance of colicin E3. It was also found that the expression of E3 immunity partially depends on the recA function. Thus, we propose two modes of expression of E3 immunity: in the uninduced state, only a slight amount of protein B is produced constitutively to protect the cell from being attacked by the exogenous colicin; and in the SOS-induced state, a large amount of protein B is produced to protect the protein synthesis system of the host cell from ribosome inactivation by endogenously produced colicin E3.     

Sequence analysis and characterization of plasmid pSFD10 from Salmonella choleraesuis

The nucleotide sequence of a small plasmid, designated pSFD10, is isolated from the vaccine strain Salmonella choleraesuis C500 in China, has been determined. This plasmid is 4091 bp long with a total G+C content of 51.4%, which is in the range of Salmonella genomic DNA. Analysis of the complete nucleotide sequence reveals that pSFD10 has a high degree of similarity to ColE1-type plasmid, having the possible cer and rom genes, and a putative mobilization origin of ColE1-type. Plasmid pSFD10 possesses six main open reading frames (ORFs), five of which have a very high degree of amino acid identity to ColE1-type plasmid gene products involved in mobilization and copy number control. The other ORF (ORF6) encodes a putative protein, which has 49% homology to the invasion plasmid antigen J protein (IpaJ) secreted by the type III secretion apparatus of Shigella flexneri. In addition, pSFD10 belongs to a different incompatibility group than ColE1-type and pMB1-type to which it is related. Plasmid pSFD10 can be mobilized by the plasmid RP4 in E. coli.     

Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro.

Hybrid plasmids were constructed in vitro by linking the Inc P-1 broad host range plasmid RK2 to the colicinogenic plasmid ColE1 at their EcoRI endonuclease cleavage sites. These plasmids were found to be immune to colicin E1, non-colicin-producing, and to exhibit all the characteristics of RK2 including self-transmissibility. These joint replicons have a copy number of 5 to 7 per chromosome which is typical of RK2, but not ColE1. Unlike ColE1, the plasmids will not replicate in the presence of chloramphenicol and are maintained in DNA polymerase I mutants of Escherichia coli. In addition, only RK2 incompatibility is expressed, although functional ColE1 can be rescued from the hybrids by EcoRI cleavage. This suppression of ColE1 copy number and incompatibility was found to be a unique effect of plasmid size on ColE1 properties. However, the inhibition of ColE1 or ColE1-like plasmid replication in chloramphenicol-treated cells is a specific effect of RK2 or segments of RK2 (Cri⁺ phenotype). This phenomenon is not a function of plasmid size and requires covalent linkage of RK2 DNA to ColE1. A specific region of RK2 (50.4 to 56.4 × 10³ base-pairs) cloned in the ColE1-like plasmid pBR313 was shown to carry the genetic determinant(s) for expression of the Cri⁺ phenotype.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.