pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria

Abstract A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315. It had a theta-type mechanism of replication in its natural host. Its minimal replicon, Rep 281, was isolated on a 1.6-kb Eco RI fragment. The Rep 287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus . Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria. As assessed by hybridization, Rep2Sl is dissimilar to pAMβ1, pIP50l and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria. Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria.     

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.     

Identification of the replication region of Lactobacillus acidophilus plasmid pLA103

Abstract The structure of the region necessary for replication of the plasmid pLA103 from Lactobacillus acidophilus TK8912 has been characterized. Sequence analysis revealed that the replication region contained an open reading frame (OrfA) encoding a 282-amino acid peptide preceded by a 22-bp tandem repeat sequence region. The predicted OrfA protein showed homology to the replication protein of a plasmid from Pediococcus halophilus . The plasmid containing the repeat sequence region preceding OrfA was able to replicate in the Lactobacillus host when provided with OrfA in trans , suggesting that the repeat sequence region contains the origin sequence essential for the pLA103 replication.     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

Comparative and evolutionary analysis of plasmid pREN isolated from Lactobacillus rennini, a novel member of the theta-replicating pUCL287 family

Here, we describe plasmid pREN of Lactobacillus rennini ACA-DC 1534, isolated from traditional Kopanisti cheese. pREN is a circular molecule of 4371 bp. Orf calling revealed a novel repA-orf2 operon with the deduced product of orf2 showing no similarity to other known proteins. Downstream of this operon, a gene cluster encoding different mobilization proteins, namely mobC, mobA1, mobA2 and mobB, was detected. Based on the sequence of the origin of replication (ori) and the similarity pattern of RepA, pREN was placed in the pUCL287 family of theta-replicating plasmids. Multiple sequence alignment demonstrated for the first time the degree of conservation in the pUCL287 oris. Our analysis supported that the identified conserved repeats could drive similar secondary structures in the oris of all plasmids. Furthermore, comparative mapping of pREN with its related plasmids (i.e. pLB925A03 and pLJ42) showed that they retain a unique combination in the architecture of their replication and mobilization elements within the pUCL287 family. Phylogenetic analysis also established that these plasmids have undergone a modular evolutionary process in order to acquire their mob genes. Research on plasmids from uncommon lactic acid bacteria will expand our appreciation for their divergence and will aid their rational selection for biotechnological applications.     

Characterization, sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

 The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites. Received: 25 April 1996 / Accepted: 20 June 1996     

Molecular characterisation of a 5.75-kb cryptic plasmid from Bifidobacterium breve NCFB 2258 and determination of mode of replication

A small cryptic plasmid originating from Bifidobacterium breve NCFB 2258 was cloned and its complete nucleotide sequence determined. pCIBb1 is a circular DNA molecule, 5750 bp in size with a GC composition of 57%. Computer-assisted analysis identified 10 possible open reading frames (ORFs), seven of which could be assigned no function from homology searches. One ORF, rep (380 amino acids), was postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. Demonstration of single-stranded forms of the plasmid in cell lysates that could be specifically degraded by S1 nuclease provided experimental evidence to substantiate a replication mechanism via single-stranded intermediates. Two other ORFs, par (199 amino acids) and an ftsK-like gene (286 amino acids), were assigned putative functions based on the presence of conserved motifs in their deduced proteins.     

Identification and sequence analysis of the replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503

Abstract The replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503 was localised to within a 10-kb Hin dIII restriction fragment. A 6.3-kb Bgl II- Hin dIII subclone of this fragment, cloned into a replication probe vector, allowed replication in Lactococcus but not in Bacillus or Lactobacillus . Sequence analysis revealed an ORF of 1152 bp preceded by a putative ori region containing a 22-bp sequence tandemly repeated three and three-quarter times, a second smaller direct repeat and two inverted repeats. Extensive homology was observed with the well characterised replication region of the small cryptic plasmid pCI305 (Hayes, F., Vos, P., Fitzgerald, G.F., de Vos, W. and Daly, C. Plasmid 25, 16–26).     

The effect of the location of the proteic post-segregational stability system within the replicon of plasmid pTF-FC2 on the fine regulation of plasmid replication

The broad host-range IncQ-2 family plasmid, pTF-FC2, is a mobilizable, medium copy number plasmid that lacks an active partitioning system. Plasmid stability is enhanced by a toxin–antitoxin (TA) system known as pas (plasmid addiction system) that is located within the replicon between the repB (primase) and the repA (helicase) and repC (DNA-binding) genes. The discovery of a closely related IncQ-2 plasmid, pRAS3, with a completely different TA system located between the repB and repAC genes raised the question of whether the location of pas within the replicon had an effect on the plasmid in addition to its ability to act as a TA system. In this work we demonstrate that the presence of the strongly expressed, autoregulated pas operon within the replicon resulted in an increase in the expression of the downstream repAC genes when autoregulation was relieved. While deletion of the pas module did not affect the average plasmid copy number, a pas-containing plasmid exhibited increased stability compared with a pas deletion plasmid even when the TA system was neutralized. It is proposed that the location of a strongly expressed, autoregulated operon within the replicon results in a rapid, but transient, expression of the repAC genes that enables the plasmid to rapidly restore its normal copy number should it fall below a threshold.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

Analysis of replication region of the cryptic plasmid pAG20 from Acetobacter aceti 3620

The DNA sequence of small cryptic plasmid pAG20 in Acetobacter aceti was determined at 3064 bp with 51.6% GC pairs. The plasmid encoded a 186 amino acid protein which is important for plasmid replication in Gram-negative bacteria except Escherichia coli. Two 21 bp large direct repeat sequence 1 and two 13 bp direct repeat sequence 2 were determined in the regulation region upstream from gene encoded Rep protein. Vector pAG24 with kanamycin gene and two deletion derivatives pAG25 and pAG26 without rep gene from plasmid pAG20 were constructed. Plasmid pAG24 was replicated in a broad host range like E. coli, Acetobacter pasteurianus, A. aceti, Comanomonas spp., Serratia marcescens, and Shigella spp.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides

Abstract The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the Bgl II restriction enzyme, ligated to the 2.7 Bgl II fragment of transposon Tn 10 , which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides .     

recA Gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites

Summary Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli. Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected. By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42° C was scored. Strains that failed to grow at 42° C depended upon the recA gene for replication. Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42° C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks. Two classes of hypotheses concerning the function of the recA gene are considered.     

The replicator region of the Rhizobium leguminosarum cryptic plasmid pRL8JI

Abstract The replicator region of the cryptic plasmid pRL8JI from Rhizobium leguminosarum strain 3841 was cloned and sequenced. The recombinant plasmid (pYK3) was selected by function from a partial Eco RI library of total DNA cloned in pSUP202 and shows incompatibility with plasmid pRL8JI when conjugated into R. leguminosarum strains 3841 and its derivative 1062. The cloned insert (∼ 10.5 kb) comprises five Eco RI fragments none of which confers replicative stability when cloned individually. A single 5.0-kb Bam HI fragment, that spans all five Eco RI fragments and confers replicative stability on pSUP202 in R. leguminosarum , has been sequenced. This replicator region shows organisational and sequence similarity to the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b. It has three open reading frames ( repA, repB, repC ) and a conserved intergenic sequence.