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PubMed1993

The giant linear plasmid pHG207 from Rhodococcus sp. encoding hydrogen autotrophy: characterization of the plasmid and its termini.

As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MR11 and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids--on pHG201 (270 kb) in strain MR11 and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possible functions.

Bacterial Proteins↗

PubMed1998

Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73.

Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis, Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.

Bacillus↗

PubMed1976

The formation of a dissociable plasmid cointegrate from the Flac factor and the resident plasmid of Salmonella typhimurium LT2.

The Flac factor showed unstable maintenance in Salmonella typhimurium dnaC MP10LT2. The properties of a more stable lac+ derivate (SD-1) are described. SD-1 was ts and carried the fi+ property and the ability to transfer the lac+ character. It contained a large plasmid of molecular weight about 129 X 10(6) DALTONS. The properties of SD-1 and its derivatives suggested that the large plasmid was a cointegate of Flac and the MP10lt2 plasmid. Lac+ transfer was efficient from SD-1 to M799 MP10LT2 and one lac+ exconjugant contained the intact cointegrate. The cointegrate was not successfully transferred to strains lacking MP10LT2. It dissociated into apparently unaltered Flac and MP10LT2 plasmids, but the deletion of small parts of one or both plasmids during cointegrate formation could not be ruled out. Cointegrate dissociation was more marked in M799 than in SD-1 especially during growth in glucose-Casamino acids minimal medium. In the presence of R1drd19, the cointegrate (like the MP10LT2 plasmid) was stable maintained in the dnaC strain; maintenance of Flac was, however, unstable. It seems likely that replication of the cointegrate was controlled by the MP10LT2 plasmid constitutent.

Conjugation, Genetic↗

PubMed1988

Plasmid profiles and restriction enzyme fragmentation patterns of plasmids of methicillin-sensitive and methicillin-resistant isolates of Staphylococcus aureus from hospital and the community.

The number, frequency distribution and restriction enzyme fragmentation patterns of plasmids harboured by 163 methicillin-sensitive isolates of Staphylococcus aureus (MSSA) and 53 methicillin-resistant isolates (MRSA) were compared. Plasmids were demonstrated in less than half of the MSSA isolates; their frequency distribution did not differ from that predicted by a simple model of plasmid distributions. In contrast, all the MRSA isolates harboured plasmids, their distribution suggesting dissemination of a limited number of clones within the hospital. Among 72 MSSA isolates harbouring plasmids, 38 different restriction patterns were identified. There were fewer patterns among MRSA isolates; 11 were observed, and two predominant patterns accounted for 68% of those identified. These restriction patterns correlated with the presence or absence of aminoglycoside resistance. A multicopy plasmid of 2.6 kb was present in both MSSA and MRSA isolates that harboured more than one plasmid; it had the same restriction pattern irrespective of its source. The importance of these results in choosing a method of studying the spread of staphylococci is discussed.

Cross Infection↗

PubMed1990

The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101.

The components for the mobilization function of a plasmid DNA during conjugation include a cis-acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins. By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain. Within a 2797 bse-pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6). Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010. Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101. Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites. By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1. An identical nick site, which is adjacent to a characteristic 10 base-pair inverted repeat sequence, is also found for plasmid RSF1010. A recombinant plasmid containing a 42 base-pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT-active.

Amino Acid Sequence↗

PubMed1995

The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase-encoding genes: identification of a new structural module on cryptic plasmids.

Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis plasmids pTA1015 and pTA1040. It is composed of two genes: one specifies an unidentified protein with a putative signal peptide; and the other (sipP) specifies a functional type 1 signal peptidase (SPase). The homologous, but non-identical, sipP genes of the two plasmids are the first identified plasmid-specific SPase-encoding genes. With respect to structure and activity, the corresponding enzymes (denoted SipP) are highly similar to the chromosomally encoded SPase, SipP, of B. subtillis and several newly identified SPases of other bacilli. Our findings suggest that plasmid-encoded SPases have evolved because, of under certain conditions, SPase can be a limiting factor for protein secretion in B. subtilis.

Amino Acid Sequence↗

PubMed2005

RepB protein of an Agrobacterium tumefaciens Ti plasmid binds to two adjacent sites between repA and repB for plasmid partitioning and autorepression.

Plasmids of Agrobacterium tumefaciens replicate using the products of the repABC operon, which are highly conserved among plasmids and some chromosomes of the alpha-Proteobacteria. The products of repA and repB direct plasmid partitioning, while the repC gene encodes a replication initiator protein. The transcription of the repABC operon of tumour inducing (Ti) plasmids is both negatively autoregulated by the RepA and RepB proteins, and positively regulated by TraR. In the present study, we have identified a fourth gene (repD) in the repABC operon of an octopine-type Ti plasmid. repD is 78 codons in length, and maps between repA and repB genes. A repD-lacZ protein fusion demonstrated that repD is strongly expressed. Two identical binding sites for the RepB protein were found within the repD coding sequence, and these sites are required for plasmid stability and for maximal repression of repABC transcription. RepA protein enhances the binding of RepB at these binding sites, just as RepB increases the affinity of RepA for binding sites at the repABC P4 promoter. We propose that RepA and RepB form complexes that bind both sites, possibly causing a loop that is important for repression of the repABC operon. Binding at one or both sites may also be required for accurate plasmid partitioning.

Agrobacterium tumefaciens↗

PubMed1995

A mutation that decreases the efficiency of plasmid R1 replication leads to the activation of parD, a killer stability system of the plasmid.

The silent parD (kis/kid) stability operon of plasmid R1 is normally repressed by the co-ordinated action of the Kis and Kid proteins. In this report it is shown that a mutation in repA, the gene of the plasmid replication protein, that reduces two-fold the copy number of the plasmid, leads to the derepression of the parD system. This derepression can be prevented by a suppressor mutation in copB, a copy number control gene of plasmid R1, that increases the efficiency of replication of the repA mutant. Derepression of the wild-type parD system leads to high plasmid stability. These data show the activation of a plasmid stability operon by a mutation that reduces the efficiency of wild-type plasmid replication.

Bacterial Proteins↗

PubMed1996

Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome.

Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS80d and RN4220 strains of Staphylococcus aureus. The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PS80d. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed.

Arsenates↗

PubMed1999

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Biological Evolution↗

PubMed1987

Structural variability of 40-50 Mdal virulence plasmids from Yersinia enterocolitica. Geographical and ecological distribution of plasmid variants.

Restriction endonuclease analysis (BamHI and EcoRI) was used to investigate the structural variability of 40-50 Mdal plasmids from 129 Yersinia enterocolitica strains isolated from human patients, animals, and food in 12 countries in Europe, North America, and Asia. A total of 12 restriction patterns was detected among plasmids from the six serogroups examined. The DNA fragment profiles were found to vary, not only between serogroups, but also among plasmids isolated from strains with the same serogroup affiliation. Despite widespread geographical and ecological origin, the plasmids isolated from the 0:3 and 0:9 strains examined revealed a surprising stability, whereas plasmids from 0:8 and 0:5,27 showed substantial diversity. The genes associated with autoagglutination, calcium dependency, and mouse virulence were conserved in all 12 plasmid variants detected. Our finding that porcine and human isolates harboured plasmids with identical restriction patterns, provides additional support for the importance of pigs in the epidemiology of human Y. enterocolitica infection.

Animal Diseases↗

PubMed1991

Nosocomial spread of an amikacin resistance gene on both a mobilized, nonconjugative plasmid and a conjugative plasmid.

Resistance to amikacin among members of the family Enterobacteriaceae at a hospital in Venezuela rose from 2% in 1979 to 5% in 1984 and 10% in 1985 as amikacin usage rose 20-fold to exceed gentamicin usage. Resistance to gentamicin remained at 25 to 27%. We examined the plasmids from 21 isolates obtained in 1984 and 1985. Nine of eleven in 1984 and three of ten in 1985 carried aacA and sul on a 3.8-kb BamHI fragment of pBWH300, a 10.4-kb nonconjugative plasmid that had been mobilized into strains of six species by at least two different coresident conjugative plasmids. Six 1985 isolates of two species carried these genes on a similar BamHI fragment of the 104-kb conjugative plasmid pBWH303. One isolate in 1984 and one in 1985 carried the 69-kb conjugative plasmid pBWH301, which had aacA as the promoter-proximal gene of an operon that also encompassed the cat and aadB resistance genes. Another conjugative plasmid, pBWH302, was found in a single isolate. It carried a different aacA allele on the functional transposon Tn654, which appeared to be closely related to Tn1331, a transposon previously isolated in Argentina and Chile. Increased selection may thus have led to dissemination of an endemic aacA allele on two endemic plasmids, one spread by mobilization, with occasional intrusion of additional aacA alleles from outside.

Alleles↗

PubMed1986

Location of plasmid-mediated citrate utilization determinant in R27 and incidence in other H incompatibility group plasmids.

Citrate utilization (Cit+) is encoded by a specific subgroup of incompatibility HI plasmids, viz., IncHI1 plasmids. Only one IncHI1 plasmid, pRG1271, which originated in a Mexican typhoid outbreak in 1972, did not specify Cit+. All other Cit+ plasmids hybridized to a Cit+ probe, a 2-kilobase BglII fragment derived from the Cit+ transposon Tn3411. The position of the Cit+ determinant was mapped to a 13.5-kilobase ApaI fragment within the prototype IncHI1 plasmid R27. No other functions have been mapped within this region. Citrate utilization mediated by IncHI1 was observed only after a prolonged lag period of approximately 150 h, and certain Escherichia coli strains, e.g., E. coli K-12 J53-1, were not able to utilize citrate specified by the H plasmids. Most E. coli strains harboring the multicopy Cit+ plasmid pOH2, a derivative of pBR322, required only 18 to 24 h to express the Cit+ phenotype, but E. coli J53-1 (pOH2) required at least 72 h for expression.

Citrates↗

PubMed1992

Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.

Bacteria↗

PubMed1985

Integration of the Vwa plasmid into the chromosome of Yersinia pestis strains harboring F' plasmids of Escherichia coli.

Conditional virulent strains of Yersinia pestis were used as recipients of an F-lac plasmid from either Escherichia coli 23.10S or E. coli CSH23. The transconjugants of Y. pestis were calcium independent; however, calcium dependence was restored after the loss of the E. coli plasmid. The plasmid contents of several Y. pestis F-lac clones were compared with those of the parent strains and several lac segregants. Although the 6- and 61-megadalton plasmids were detected in all strains tested, the Vwa plasmid was absent in the F-containing strains. The plasmid was evident, however, in the lac segregants derived from the F-lac clones. Results of Southern transfers indicated that the Vwa plasmid had integrated into the chromosome of Y. pestis strains harboring F-lac.

Calcium↗

PubMed2006

Complete DNA sequence of a ColBM plasmid from avian pathogenic Escherichia coli suggests that it evolved from closely related ColV virulence plasmids.

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5' end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the "conserved" portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its "variable" portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this "conserved" cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied.

Animals↗

PubMed1975

Chromosome-plasmid interaction in Escherichia coli K-12 carrying a thermosensitive plasmid, Rts1, in autonomous and in integrated states.

An Hfr strain of Escherichia coli K-12 was obtained by integrative suppression with a thermosensitive plasmid, Rts1. The R plasmid was integrated into the chromosome between rif and thr, and transfer of the chromosome occurred counterclockwise. The thermosensitivity of host cell growth due to the dnaA mutation was markedly but not completely reduced in this integratively suppressed Hfr strain. When the dnaA mutation was removed by transducing the dnaA+ genome to this Hfr, the thermosensitivity of cell growth due to existence of Rts1 was suppressed in contrast to strains carrying it autonomously. Thermosensitivity of cell growth appeared again when the plasmid was detached from the chromosome to exist autonomously. Contrary to the effect on cell growth, the transfer of the chromosome and the plasmid itself and the ability to "restrict" T-even phages were still thermosensitive in all of these strains carrying Rts1, irrespective of its state of existence. The detached plasmid as well as the original Rts1 were segregated upon growth at 42 C. These data are discussed in relation to chromosome-plasmid interaction. One of the most important conculusions is that some plasmid genes, related to their replication, are phenotypically suppressed by the chromosome when it is integrated.

Chromosomes, Bacterial↗

PubMed1977

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.

Colicins↗