Screening of plasmids in non-pathogenic corynebacteria

Abstract A screening of plasmids in 25 nonpathogenic coryneform bacteria was carried out. 11 Strains showed at least one plasmid, ranging in size from 4.2 to 55 kb. These plasmids did not encode bacteriocin production or resistance to a number of antibiotics or to ions such as arsenite, mercury(II) and cobalt(II). A detailed study of plasmid pBL100 from Brevibacterium linens is presented. pBL100 has a size of 7.75 kb, and contains single sites for the endonucleases: Hin dIII; Pst I, Bgl II, Eco RI and Bam HI. B. linens is easily and efficiently transformed with vectors derived from pBL1 isolated from Brevibacterium lactofermentum .     

Diversity and evolution of repABC type plasmids in Rhodobacterales

The repABC operon is the prevalent replication unit of alphaproteobacterial plasmids. Their semi-autonomy is ensured by the essential replicase gene repC as well as the repAB partitioning cassette. While conserved repAB modules are widespread among bacterial plasmids and homologues are even responsible for chromosome partitioning, repC genes are exclusively present in Alphaproteobacteria . RepABC operons contain two strong incompatibility regions, namely a small regulative antisense RNA gene ( incα ) and a palindromic centromere region ( incβ ), which were previously used to classify these replicons. The present survey pursued a complementary strategy essentially following the rationale that all plasmids identified from a single bacterium are per se compatible. We established a novel classification scheme for plasmids based on comprehensive phylogenetic analyses of repC , repA and repB genes. Our case study is focused on the Roseobacter clade ( Rhodobacterales ), one of the most successful lineages of the marine bacterioplankton. Its global significance was shown in several studies and the interest in these organisms is reflected by more than 40 upcoming genome projects. Based on phylogenetic RepC analyses we identified nine compatibility groups that are expected to stably coexist within the same cell. This prediction is supported by RepA and RepB phylogenies, moreover independent evidence is delivered by the group specificity of highly conserved palindromes ( incβ ).     

Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids.

Minireplicons from the I-complex plasmids R387 (IncK) and pIE545 (IncZ) were constructed, and the nucleotide sequences of their replication regions were compared with that of the B plasmid, pMU720. The coding sequence of the putative replication protein, RepA, of each plasmid was located. RepA of K and B plasmids were homologous, whereas RepA of Z resembled RepA1 of FII plasmid. Sequences upstream of RepA were conserved in the three I-complex plasmids. Group B and Z plasmids were incompatible.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.     

Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli

Four antibiotic-resistance plasmids isolated from thermophilic bacilli were characterized in detail. Three tetracycline-resistance (Tc1) plasmids were designated as pTHT9 (7.7 kilobases (kb], pTHT15 (4.5 kb) and pTHT22 (8.4 kb). From the results of restriction endonuclease analysis and the subsequent Southern hybridization, these were found to possess extensive genetic homology in the regions that include the replication origin and the Tcr gene. Detailed restriction maps of the smallest Tcr plasmid pTHT15 and a kanamycin-resistance (Kmr) plasmid pTHN1 (4.8 kb) were constructed. The positions of antibiotic-resistance loci and regions essential for plasmid replication were determined by cloning plasmid fragments in Bacillus subtilis. These four plasmids were found to replicate and express the resistance genes stably in both B. subtilis and B. stearothermophilus.     

Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.     

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.     

Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus

Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis, whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1.0 MDal EcoRI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1.0 MDal EcoRI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 10(3) to 10(4) times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.     

Molecular characterization of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> biovar <Emphasis Type="Italic">longum</Emphasis> NAL8 plasmids and construction of a novel replicon screening system

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users only.     

A Monte Carlo simulation of plasmid replication during the bacterial division cycle

Plasmids have cell cycle replication patterns that need to be considered in models of their replication dynamics. To compare current theories for control of plasmid replication with experimental data for timing of plasmid replication with the cell cycle, a Monte Carlo simulation of plasmid replication and partition was developed. High-copy plasmid replication was simulated by incorporating equations previously developed from the known molecular biology of ColE1-type plasmids into the cell-cycle simulation. Two types of molecular mechanisms for low-copy plasmid replication were tested: accumulation of an initiator protein in proportion to cell mass and binding of the plasmid origin to the cell membrane. The low-copy plasmids were partitioned actively, with a specific mechanism to mediate the transfer from mother to daughter cells, whereas the high-copy plasmids were partitioned passively with cell mass.The simulation results and experimental data demonstrate cell-cycle-specific replication for the low-copy F plasmid and cell-cycle-independent replication for the high-copy pBR322, ColBM, and R6K plasmids. The simulation results indicate that synchronous replication at multiple plasmid origins is critical for the cell-cycle-specific pattern observed in rapidly growing cells. Variability in the synchrony of initiation of multiple plasmid origins give rise to a cell-cycle-independent pattern and is offered as a plausible explanation for the controversy surrounding the replication pattern of the low-copy plasmids. A comparison of experimental data and simulation results for the low-copy F plasmid at several growth rates indicates that either initiation mechanism would be sufficient to explain the timing of replication with the cell cycle. The simulation results also demonstrate that, although cell-cycle-specific and cell-cycle independent replication patterns give rise to very different gene-expression patterns during short induction periods in age-selected populations, long-term expression of genes encoded on low-copy and high-copy plasmids in exponentially growing cells have nearly the same patterns. These results may be important for the future use of low-copy plasmids as expression vectors and validate the use of simpler models for high-copy plasmids that do not consider cell-cycle phenomena. (c) 1996 John Wiley & Sons, Inc.     

Effects of dnats genes on the replication of plasmids in Bacillus subtilis

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, pKW1, 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, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.     

Recombination-dependent replication of plasmids during bacteriophage T4 infection

The replication of plasmids containing fragments of the T4 genome, but no phage replication origins, was analyzed as a possible model for phage secondary (recombination-dependent) replication initiation. The replication of such plasmids after T4 infection was reduced or eliminated by mutations in several phage genes (uvsY, uvsX, 46, 59, 39, and 52) that have previously been shown to be involved in secondary initiation. A series of plasmids that collectively contain about 60 kilobase pairs of the T4 genome were tested for replication after T4 infection. With the exception of those known to contain tertiary origins, every plasmid replicated in a uvsY-dependent fashion. Thus, there is no apparent requirement for an extensive nucleotide sequence in the uvsY-dependent plasmid replication. However, homology with the phage genome is required since the plasmid vector alone did not replicate after phage infection. The products of plasmid replication included long concatemeric molecules with as many as 35 tandem copies of plasmid sequence. The production of concatemers indicates that plasmid replication is an active process and not simply the result of passive replication after the integration of plasmids into the phage genome. We conclude that plasmids with homology to the T4 genome utilize the secondary initiation mechanism of the phage. This simple model system should be useful in elucidating the molecular mechanism of recombination-dependent DNA synthesis in phage T4.     

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.     

Initiation of DNA replication at cloned origins of bacteriophage T7

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.