Rolling-circle plasmids from Bacillus subtilis : complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from Gram-positive bacteria (original) (raw)

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus

Journal of Bacteriology, 1987

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolidelincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxy-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.

The replication origin of a repABC plasmid

BMC Microbiology, 2011

Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides within the repC gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in promoting this phenotype.

Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis

Nucleic Acids Research, 1995

In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtills plasmids which use the rolling-circle mechanism of replication. The plasmids used in this study involved pTA1015, pTA1020, pTAI 030, pTAI 040, pTA1 050 and pTA1060. The SSO of pTAI01 5 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own. Sequence analysis revealed that the SSO of pTAI01 5 is almost identical to formerly described palT type SSOs. Also pTA1020 and pTAI 060 were shown to contain SSOs highly homologous to palT. Using Southem hybridization with the palT of pTA1015 as a probe, the SSO of pTA1040 was cloned. Sequence analysis revealed a region of 200 bp which is 77% identical to the palT of pTA101 5. The plasmids pTA1030 and pTAI050 contain an SSO which is highly homologous to the SSO of pTA1040. The majority of the SSOs of rolling-circle plasmids from B.subtills seem to belong to two related families which we denote as palTi (present on pTAI 015, pTAI 020 and pTAI 060) and palT2 (present on pTA1030, pTA1040 and pTAI050). Both families of SSOs are highly efficient singlestrand-conversion signals in B.subtilis.

Effects of dnats Genes on the Replication of Plasmids in Bacillus subtilis

Microbiology, 1987

An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pKW 1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H ; pC194 requires only dnaG and H ; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW 1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW 1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.