Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

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.     

Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.     

Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 (A. thermophilum), growing optimally at 70 degrees C, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence revealed 10 open reading frames (ORFs). The two largest of these, namely Orf21 and Orf41, showed similarity to a Bacillus plasmid recombinase and a Pseudoalteromonas plasmid replication protein, respectively. A sequence with homology to double stranded replication origins from rolling circle plasmids was found, but no single stranded intermediates, characteristic of rolling circle replication, were found on Southern blots. The larger plasmid, pBAL, was found to be a 8294 bp plasmid with a GC content of 39%. It revealed 17 ORFs, of which three showed similarity at the amino acid (aa) level to known proteins. Orf22 showed the strongest similarity (33% aa) to replication proteins from large multiresistance Staphylococcal and Lactococcal plasmids, all of which are believed to replicate via a theta-like replication mechanism. Orf32 showed similarity to both DNA repair proteins and DNA polymerases with highest similarity to DNA repair protein from Campylobacter jejuni (25% aa). Orf34 showed similarity to sigma factors with highest similarity (28% aa) to the sporulation specific Sigma factor, Sigma 28(K) from Bacillus thuringiensis.     

A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.

Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.     

Complete Sequence of Bacillus subtilis Plasmid p1414 and Comparison with Seven Other Plasmid Types Found in Russian Soil Isolates of Bacillus subtilis

We determined the complete sequence of a cryptic 7949-bp plasmid isolated from naturally occurring Bacillus subtilis found in Russian soil from Moscow. We found 15 putative open reading frames (ORFs), all of which were preceded by a ribosome binding site. One encodes the gene (rep) which should be essential for vegetative rolling circle replication (RCR). The putative double-stranded origin as well as a palT1-like single-stranded origin was also identified. The predicted product of another ORF showed similarity to a moblization protein while a third showed similarity to a ubiquitous family of small proteins whose members have so far been associated with stress response. We used fragments with these latter ORFs to probe representatives of seven other groups of cryptic RCR plasmids from geographically related B. subtilis isolates. All plasmids carried the mob function, suggesting a common ancestor for the rep/mob region but the putative hsp was present only on some of the plasmids. This suggests that the putative hsp gene is not an essential plasmid component and may therefore be present as a phenotypic marker-perhaps providing response to stress. This adds weight to the growing evidence that these small Bacillus plasmids may not be cryptic but may provide an adaptive advantage for the host in its natural environment.     

Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni

A new cryptic plasmid, pRS1, from an Oenococcus oeni strain isolated from Spanish wines is reported. Nucleotide sequence analysis (2523 bp) revealed the presence of three major open reading frames (ORFs) whose nucleotide sequence and encoded proteins exhibit high homology with those of pOg32, a previously described plasmid of O. oeni. Common features in other plasmids from O. oeni (i.e., pLo13 and pOg32) have been found in pRS1. They have three major ORFs in the same strand; the putative encoded proteins by two of these ORFs exhibit homology with the replication (Rep) and the recombination (Pre) proteins, respectively, of the pT181 plasmid family and related gram-positive bacteria plasmids; these plasmids contain the DNA sequences required for plasmid replication by the rolling circle mechanism and for recombination (i.e., double-strand origin, DSO; single-strand origin, SSO; recombination-specific sites, RSA and RSB); and finally, all these plasmids have a third ORF of unknown function. These features suggest that pRS1 could constitute together with pLo13 and pOg32 a family of small cryptic plasmids of O. oeni.     

Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli

A new collection of shuttle cloning vectors has been constructed that can be used in a broad host range, because they carry replication origins which are functional in Escherichia coli (p15A, pWV01, ColE1), Lactococcus lactis, lactobacilli, and Bacillus subtilis (pAMbeta1, pWV01). These plasmids contain the lacZ-T1T2 cassette from pJDC9, which allows the X-gal selection and cloning of DNA fragments that could cause plasmid instability in E. coli. In addition, they have been proved to be structurally and segregationally stable in Lactobacillus casei, in which their copy number has been determined by real-time quantitative PCR. Furthermore, the antibiotic resistance markers (beta-lactamase, chloramphenicol acetyl transferase, and erythromycin transacetylase) and the theta and rolling circle replicating origins have been combined to obtain this set of compatible plasmids (pIA family) that can be cotransformed, both in lactic acid bacteria and in E. coli.     

The smr gene resides on a novel plasmid pSP187 identified in a Staphylococcus pasteuri isolate recovered from unpasteurized milk

This work describes a novel plasmid pSP187 (5550 bp) carrying the small multidrug resistance determinant smr encoding resistance to quaternary ammonium compounds (QACs). pSP187 was identified in a Staphylococcus pasteuri isolate recovered from bulk milk in a dairy cattle herd in Norway. Sequence analysis revealed 6 putative ORFs in addition to the smr gene within a cassette with identical genetic organization to that found in the pSK41-like Staphylococcus aureus plasmid pTZ22. A protein homology search suggested the gene product of ORF7 to be a putative replication initiation protein, while ORF2 was predicted to encode a protein homologous to members of FtsK/SpoIIIE cell division-DNA segregation protein families. Sequence similarities to some initiator proteins of rolling circle replicons (RCR) indicated that pSP187 uses a RCR mode of replication, supported by the detection of intermediate ssDNA using S1 nuclease treatment and hybridization analysis. Interestingly, a 30-bp sequence found upstream from ORF7 showed high similarity to other dyad symmetry motifs proposed as putative double-strand origins of replication in the plasmids pGI3 (Bacillus thuringiensis), pSTK1 (Bacillus stearothermophilus), and pER1-2 (Streptococcus thermophilus). In conclusion: The novel smr-containing plasmid pSP187 is the first member of RCR group VI to be identified in a Staphylococcus sp.     

Processes at the nick region link conjugation, T-DNA transfer and rolling circle replication

Data from prokaryotic replicative and conjugative systems, which interrelate DNA processing events initiated by a site-specific nick, are reviewed. While the replicative systems have been established in accordance with the rolling circle replication model, the mechanism of conjugative replication has not been elucidated experimentally. We summarize data involving random point mutagenesis of the RK2 transfer origin (oriT), which yielded relaxation-deficient and transfer-deficient derivatives having mutations exclusively in a 10bp region defined as the nick region. Features of the RK2 (IncP) nick region, including the DNA sequence, nick site position, and 5′ covalent attachment of the nicking protein, have striking parallels in other systems involving nicking and mobilization of single-stranded DNA from a supercoiled substrate. These other systems include T-DNA transfer occurring in Agrobacterium tumefaciens Ti plasmid-mediated tumorigenesis in plants, and the rolling circle replication of plasmids of Gram-positive bacteria and of φX174-like bacteriophage. The structural and functional similarities suggest that IncP conjugative replication, originating at the oriT, and T-DNA transfer replication, originating at the T-DNA border, produce continuous strands via a rolling circle-type replication.     

Structurally stable Bacillus subtilis cloning vectors

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.     

Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family.

The replication of pT181 and related plasmids of Staphylococcus aureus proceeds by a rolling circle mechanisms. The initiator proteins encoded by the plasmids of the pT181 family have sequence-specific DNA binding and topoisomerase activities. These proteins nick one strand of the DNA at the origin of replication. The free 3'-hydroxyl end at the nick is then used as a primer for the replication of the leading strand of the DNA. Although these initiator proteins are highly homologous, they show specificity in DNA binding and replication for their cognate DNAs. In this study, we have generated hybrid initiator proteins and studied their various biochemical activities in vitro. Our results show that 6 amino acids are sufficient to determine the DNA binding and replication specificities of such initiator proteins.     

Plasmid rolling circle replication and its control

Abstract This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.     

Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1.

Numerous bacterial replicons remain poorly characterized due to difficulties in localization of the replication origin. We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision. Analysis of the replication intermediates of wild-type molecules by two-dimensional gel electrophoresis confirmed the location of the plasmid origin. Our results demonstrate that pAM beta 1 replication proceeds unidirectionally by a theta mechanism. This work confirms the use of termination signals to localize origins, suggests that termination in B. subtilis occurs by a mechanism similar to that of Escherichia coli and establishes that in addition to rolling circle replicating plasmids, Gram positive bacteria harbour plasmids which replicate by a theta mechanism.     

Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

Rolling circle replication of single-stranded DNA plasmid pC194.

A group of small Staphylococcus aureus/Bacillus subtilis plasmids was recently found to replicate via a circular single-stranded DNA intermediate (te Riele et al., 1986a). We show here that a 55 bp region of one such plasmid, pC194, has origin activity when complemented in trans by the plasmid replication protein. This region contains two palindromes, 5 and 14 bp long, and a site nicked by the replication protein. DNA synthesis presumably initiated at the nick in the replication origin can be terminated at an 18 bp sequence homologous to the site of initiation, deriving from another plasmid, pUB110, or synthesized in vitro. This result suggests that, similar to the Escherichia coli single-stranded DNA phages, pC194 replicates as a rolling circle. Interestingly, there is homology between replication origins and replication proteins of pC194 and the phage phi mX174.