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The replication origin region, ori, of the Lactococcus lactis subsp. lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (RepB) essential for plasmid replication. Gel retardation assays, using lactococcal crude cell extracts in which RepB was overproduced, were used to demonstrate that the replication protein interacts with DNA sequences within the origin region. IR1 was identified as a RepB binding site. The -35 region of the repB promoter is contained within the loop of the potential stem-loop structure of IR1, suggesting autoregulation of repB. The pCI305 RepB failed to interact with DNA sequences within the minimal replicons of nine other members of the pCI305 family of plasmids and it was concluded that this DNA-protein interaction was replicon specific. In vivo studies were performed to determine the role of the three-and-one-half copies of the 22-bp sequences. When this sequence was provided in trans on a compatible vector, it resulted in the loss of pCI305 from the cell population (incompatibility).

A system for the positive selection of structural plasmid rearrangements in Bacillus subtilis was developed. Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of the cat-86 gene, under control of a constitutive bacteriophage promoter. The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were shown, by restriction analysis, to contain initially both the intact parental plasmid and a deletion variant. Sequence analysis of deletion derivatives revealed a consensus target site (5'-A-T-T-A-A/T-3') at or near deletion termini, which resembles topoisomerase I target sites. Endpoints on one side of the deletions were found to be clustered in the promoter region of the tetracycline resistance gene present on pGP100, the gene product of which is an integral membrane protein. Furthermore, deletion of the genes encoding the ATP-dependent exonuclease, AddAB, severely reduced the structural stability of pGP100. The data indicate that similar mechanisms underlie deletion formation in pGP100, and a different plasmid-based system, pGP1, which we have analyzed previously.

The effects of the single-strand origins (SSOs) of plasmid pMV158 on (i) the conversion of its single-stranded (ss) replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. The rolling-circle plasmid pMV158, which replicates via ssDNA intermediates, contains two single-strand origins (SSOs) of replication, palA and palU. In this paper the results obtained with Bacillus subtilis are described; complementary studies with Lactococcus lactis are presented in the accompanying paper (Meijer et al., 1995). While in L. lactis both SSOs are functional as ssDNA conversion signal, only palU appeared to be active B. subtilis. Similar to the situation in L. lactis, the accumulation of large amounts of ssDNA resulted in a severe decrease in plasmid maintenance in B. subtilis. In the latter bacterium large amounts of ssDNA were only accumulated, however, when plasmids lacking a functional SSO were propagated in RecA mutant strains. In wild-type RecA strains these plasmids accumulated only modest amounts of ssDNA and they were maintained at fairly stable levels. The results suggest that in B. subtilis a RecA-mediated alternative pathway exists for the conversion of ssDNA which can improve plasmid maintenance. In addition to ssDNA accumulation and the antagonizing role of RecA therein, two other plasmid regions were shown to affect pMV158 maintenance in B. subtilis. One was the mob gene region, which had a negative effect on plasmid maintenance, and the other the palA type SSO. Although palA was not functional as an ssDNA conversion signal in B. subtilis, its presence had a positive effect on pMV158 maintenance.

The effects of the single-strand origins (SSOs) of the broad-host-range streptococcal plasmid pMV158 on (i) the conversion of its single-stranded (ss) DNA replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. pMV158 is distinguished from most other plasmids that replicate by the rolling-circle mechanism by the presence of two single-strand origins of replication, palA and palU. In this paper the results obtained with Lactococcus lactis are presented; complementary studies with Bacillus subtilis are presented in the accompanying paper (Meijer et al., 1995). In the presence of both SSOs, no ss plasmid DNA was observed in L. lactis. The removal of either palA or palU resulted in the appearance of low amounts of ssDNA. High amounts of ssDNA were detected, however, when both SSOs were deleted. The results indicated that both SSOs were active, albeit that palU was the most effective of the two. In the presence of both SSOs, the plasmid was stably maintained in L. lactis under nonselective growth conditions. Also, the derivatives containing only one of the two SSOs were maintained rather stably. In contrast, the derivative devoid of both SSOs was poorly maintained. It was concluded that, in the absence of a functional SSO, the generation of large amounts of ssDNA drastically reduces the maintenance of pMV158 in L. lactis. The results also showed that the presence of the plasmid-located mob gene, required for conjugative mobilization, was involved neither in the accumulation of ssDNA nor in the maintenance of pMV158.

In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described. The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemostat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched. We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAM beta 1 as selection vectors. The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties. The first class involved mutations in the pAM beta 1-derived selection plasmid. These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable. The other two classes were based on the insertion of additional, stability promoting, sequences in the selection plasmids. We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance. The site-specific resolution function of pAM beta 1 was used as an example of the latter functions. We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria.

We studied the effects of temperature on the segregational stability of derivatives of the rolling-circle-type plasmid pTB913 in Bacillus subtilis. This 4.5-kb plasmid is a deletion derivative of pTB19, which was originally isolated from a thermophilic Bacillus. pTB913 derivatives carrying large inserts or lacking the minus origin for complementary strand synthesis were segregationally unstable at 37 degrees C. In contrast, at 47 degrees C all pTB913 derivatives tested were stably maintained in B. subtilis. The increased stability at 47 degrees C was attributed, at least partly, to increased copy numbers at this temperature. Although considerable amounts of single-stranded and high-molecular-weight plasmid DNA were formed at 47 degrees C, these products did not reduce plasmid stability at this temperature. The increased stability and increased copy number of pTB913 at elevated temperatures extend the use of this plasmid as a cloning vector in B. subtilis and other bacilli.

Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Plasmid pGP1 carrying a penP-lacZ fusion was used to study structural plasmid instability in Bacillus subtilis. In only one of 28 sequenced deletion junction points in the penP-lacZ region short direct repeats (10 bp) and flanking imperfect inverted repeats (12 bp) were associated with endpoints. In 27 deletions no repeated sequences of more than 3 bp were present at the endpoints. In 15 of these the sequence 5'-T-G-T-A-3' was found within 10 bp from the left endpoint. At the left cleavage sites the sequence 5'-T-T-T-3', or the 5'-A-A-A-3' complement thereof, was frequently observed. Most of the left deletion endpoints were located in potential stem-loop structures in the penP transcription/translation regulatory region, which is very rich in hyphenated dyad symmetry. Near the right deletion endpoints a sequence consisting of four G/C residues, followed by three or four A/T residues, was found in 15 cases. It is speculated that DNA topoisomerase I is involved in the formation of the deletions studied.

Plasmid deletion formation was compared in wild-type, recE4, and addB72 strains of Bacillus subtilis. Deletion frequencies in plasmid pGP1, as monitored with a penP-lacZ fusion, were low in recE4 and high in addB72, in comparison to the wild-type strain. In the wild-type and recE4 strains, deletions between directly repeated sequences were rare. In contrast, about half of the deletions in the addB72 mutant resulted from recombination at direct repeats of 5 bp or more. The sequences at or near the left deletion endpoints showed striking similarities in the three strains. (1) 5'-T-T-T-3', or the complement 5'-A-A-A-3', was frequently located at these sites. (2) 5'-T-G-T-A-3' was found close to most of these termini. (3) Nearly all left termini occurred in a region rich in hyphenated dyad symmetry, which includes the penP transcription/translation regulatory sequences. It is assumed that DNA secondary structures, together with a sequence preference, specify the majority of the left deletion termini, which we speculate to be target sites for topoisomerase I. The right termini of deletions in the wild-type and addB72 mutant were frequently located close to a loose octanucleotide consensus sequence: 5'-G/C-G/C-G/C-G-A/T-A/T-A/T-A/G-3'. In contrast, in the recE4 mutant, the sequence 5'-C-A-G/C-G/C-G/C-G/C-T/G-3' was more frequently found at this position.

Plasmid pGP1, containing a fusion between the penicillinase gene of Bacillus licheniformis and the beta-galactosidase gene of Escherichia coli, was constructed. This plasmid enabled a study of structural plasmid instability in Bacillus subtilis wild-type cells and a variety of B. subtilis strains, defective in recombination- and DNA-repair functions. Large differences with respect to the level of stability of this plasmid were observed in the various genetic backgrounds.