Lateral transfer of rfb genes: a mobilizable ColE1-type plasmid carries the rfbO:54 (O:54 antigen biosynthesis) gene cluster from Salmonella enterica serovar Borreze.

Plasmid pWQ799 is a 6.9-kb plasmid isolated from Salmonella enterica serovar Borreze. Our previous studies have shown that the plasmid contains a functional biosynthetic gene cluster for the expression of the O:54 lipopolysaccharide O-antigen of this serovar. The minimal replicon functions of pWQ799 have been defined, and a comparison with nucleotide and protein databases revealed this replicon to be virtually identical to ColE1. This is the first report of involvement of ColE1-related plasmids in O-antigen expression. The replicon of pWQ799 is predicted to encode two RNA molecules, typical of other ColE1-type plasmids. RNAII, the putative replication primer from pWQ799, shares regions of homology with RNAII from ColE1. RNA1 is an antisense regulator of DNA replication in ColE1-related plasmids. The coding region for RNAI from pWQ799 shares no homology with any other known RNAI sequence but is predicted to adopt a secondary structure characteristic of RNAI molecules. pWQ799 may therefore represent a new incompatibility group within this family. pWQ799 also possesses cer, rom, and mob determinants, and these differ minimally from those of ColE1. The plasmid is mobilizable in the presence of either the broad-host-range helper plasmid pRK2013 or the IncI1 plasmid R64drd86. Mobilization and transfer of pWQ799 to other organisms provides the first defined mechanism for lateral transfer of O-antigen biosynthesis genes in S. enterica and explains both the distribution of related plasmids and coexpression of the O:54 factor with other O-factors in different Salmonella serovars. The base composition of the pWQ799 replicon sequences gives an average percent G+C value typical of Salmonella spp. In contrast, the percent G+C value is dramatically lower with rfb0:54, consistent with the possibility that the cluster was acquired from an organism with much lower G+C composition.     

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.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Replication of ColE2 and ColE3 plasmids In vitro replication dependent on plasmid-coded proteins

Summary We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.     

Characterization of a mini ColE1 cloning vector.

Plasmid pHA105 (formerly pAC105), a mini ColE1 plasmid containing one restriction endonuclease EcoRI site, was further characterized using restriction endonuclease analysis thereby revealing its relationship to ColE1. The polypeptides specified by plasmid pHA105 in minicells are of low molecular weight making it a useful plasmid to define cloned polypeptides larger than 16,000 daltons and its use for that purpose was demonstrated. pHA105 was used to clone two different sized fragments of DNA containing the gal operon. pHA105 was also used to reclone a 2 Mdal fragment of DNA that, when expressed, represses the synthesis of capsular polysaccharide. The repression of polysaccharide synthesis was expressed when a plasmid containing one molecule each of pHA105 and the 2 Mdal fragment was prepared (pFM100). In contrast, a plasmid containing two copies of pHA105 and one of the 2 Mdal fragment (pHA138) did not repress polysaccharide synthesis. The results demonstrate that expression of a cloned fragment gene may be prevented in certain arrangements of the vector and cloned fragment. Plasmid pHA105 fails to exhibit relaxation after treatment with sodium dodecyl sulfate in contrast to ColE1 treated in the same way. pHA105 replicates as a dimer form while ColE1 usually does not. A hypothesis that a function of a DNA-protein complex is required for monomeric DNA circle formation is discussed.     

Primary structures of the ColE2-P9 and ColE3-CA38 lysis genes

The lysis genes of plasmids ColE2-P9 and ColE3-CA38 were identified by DNA sequencing and electrophoretic analysis of the products of both wild type and artificially introduced ochre mutant genes. The E2 and E3 lysis genes had identical primary structures and were shown to encode 47 amino acids with a calculated molecular weight of 4,861, which is much smaller than that proposed previously for the ColE3-CA38 lysis protein. They are homologous with ColDF13 gene H, except in their 3'-portions. The nine C-terminal amino acids of the E2 and E3 lysis proteins proved to be non-essential for the lysis phenotype.     

Assessing genetic diversity in plasmids from Escherichia coli and Salmonella enterica using a mixed-plasmid microarray

AIMS: To compare genetic composition of plasmids using microarrays composed of randomly selected fragments of plasmid DNA. METHODS AND RESULTS: Separate shotgun libraries were constructed from plasmid DNA pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used as probes for microarrays. Plasmid targets were labelled, hybridized overnight, and bound targets were imaged after enzymatic signal amplification. Control hybridizations demonstrated significantly higher signal when probes and targets shared >95% sequence identity. Diagnostic sensitivity and specificity of the assay was 95 and 99%, respectively. Cluster analysis showed close matches for replicate experiments with a high correlation between replicates (r = 0.91) compared with the correlation for nonreplicates (r = 0.09). Analysis of hybridization data from 43 plasmids generated five distinct clusters, two for known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and two for plasmids harboring a recently disseminated antibiotic resistance gene (bla(CMY-2)). CONCLUSION: Mixed-plasmid microarrays are suitable for comparing genetic content of wild-type plasmids and hybridization results from this study suggest several novel hypotheses about plasmid gene exchange between E. coli and S. enterica. SIGNIFICANCE AND IMPACT OF STUDY: Mixed-plasmid microarrays permit rapid, low cost analysis and comparison of many plasmids. This ability is critical to understanding the source, fate, and transport of plasmids amongst commensal and pathogenic bacteria.     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

Altered DNA-protein relaxation complex in a replication mutant of plasmid ColE1

Summary Approximately 200,000 clones of Escherichia coli carrying mutagen-treated colicinogenic plasmid E1 (ColE1) were examined for irreversible loss of the plasmid at 43°. Thirty of these clones that appeared to be most defective in plasmid DNA replication at the non-permissive temperature were selected for the study of: (a) the kinetics of plasmid and chromosomal DNA replication during a temperature shift in either the presence or absence of chloramphenicol; (b) the temperature stability of the plasmid DNA-protein relaxation complex; and (c) the temperature effect on F-promoted conjugal transfer. Two mutant plasmids, pJC307 and pJC301, showed defects in their relaxation complex. The relaxation complex of pJC307 exhibited an altered temperature stability in vitro. Reversion to temperature resistant replication resulted in four out of five cases in a concomitant change in the temperature stability of the relaxation complex. Conjugal mobility of this mutant was not markedly reduced at the permissive or non-permissive temperature. Plasmid pJC301 could not be isolated in the form of a relaxation complex and it was very poorly mobilized in an F-promoted conjugation. These results indicate that the ColE1 plasmid codes for at least one of the proteins of the relaxation complex and that the relaxation complex is involved in ColE1 DNA replication. In addition, the properties of the mutant plasmid pJC301 are consistent with a role for the complex in the mobilization of ColE1 during conjugation.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.     

Comparative analysis of the replicon regions of eleven ColE2-related plasmids.

The incA gene product of ColE2-P9 and ColE3-CA38 plasmids is an antisense RNA that regulates the production of the plasmid-coded Rep protein essential for replication. The Rep protein specifically binds to the origin and synthesizes a unique primer RNA at the origin. The IncB incompatibility is due to competition for the Rep protein among the origins of the same binding specificity. We localized the regions sufficient for autonomous replication of 15 ColE plasmids related to ColE2-P9 and ColE3-CA38 (ColE2-related plasmids), analyzed their incompatibility properties, and determined the nucleotide sequences of the replicon regions of 9 representative plasmids. The results suggest that all of these plasmids share common mechanisms for initiation of DNA replication and its control. Five IncA specificity types, 4 IncB specificity types, and 9 of the 20 possible combinations of the IncA and IncB types were found. The specificity of interaction of the Rep proteins and the origins might be determined by insertion or deletion of single nucleotides and substitution of several nucleotides at specific sites in the origins and by apparently corresponding insertion or deletion and substitution of amino acid sequences at specific regions in the C-terminal portions of the Rep proteins. For plasmids of four IncA specificity types, the nine-nucleotide sequences at the loop regions of the stem-loop structures of antisense RNAs are identical, suggesting an evolutionary significance of the sequence. The mosaic structures of the replicon regions with homologous and nonhomologous segments suggest that some of them were generated by exchanging functional parts through homologous recombination.     

Plasmid ColE1 conjugal mobility: the nature of bom, a region required in cis for transfer

Summary Conjugal mobility of ColE1 and related plasmids is promoted by a wide range of conjugative plasmids. ColE1 produces trans-acting products and has a region required in cis (bom; basis of mobility) for such mobility. Here we show that plasmid pBR322 contains a functional bom sequence located within a 141 bp HhaI fragment. This bom region is functional for conjugation promoted by several different conjugative plasmids and is highly conserved in ColE1 and contains nic the putative origin of transfer. The orientation and position of bom with respect to the ColE1 vegetative origin of replication can be changed without affecting the frequency of conjugal mobility promoted by R64drd11.     

Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site.

Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site. Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site. Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids. We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.     

Homology between Escherichia coli plasmids ColE1 and p15A.

The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented.     

Effect of altering GATC sequences in the plasmid ColE1 primer promoter.

Plasmid ColE1 has three recognition sites for the Escherichia coli DNA adenine methylase in the immediate upstream region of the primer promoter. Two of these sites are conserved among all plasmid relatives of ColE1 and constitute parts of an inverted repeat that can conceivably form a cruciform structure. Recent experiments have indicated that hemimethylated ColE1-type plasmids are inefficiently replicated after transformation (D. W. Russell and N. Zinder, Cell 50:1071-1079, 1987). By mutating the three methylation sites, we found that disruption of all three GATC sites was necessary for complete relief of the hemimethylation-mediated inhibition of replication in vivo. We also found that these three methylation sites acted in a position-specific manner. The putative cruciform, if present, did not play a regulatory role in the hemimethylation-mediated inhibition of replication.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Characterization and replication properties of the Zymomonas mobilis ATCC 10988 plasmids pZMO1 and pZMO2

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2.     

The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1

Summary The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48×10⁶). Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 andClo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.     

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.