Insights into the genetic organization of the Bacillus mycoides cryptic plasmids pDx14.2 and pSin9.7 deduced from their complete nucleotide sequence

Bacillus mycoides, a member of the Bacillus cereus group of bacteria, can be easily distinguished from close species because of colony shape, made by filaments of cells, resembling fungal hyphae, curving clock- or counterclockwise depending on the strain. Two plasmids, one from a strain curving to the right (pDx14.2), the other from a strain curving to the left (pSin9.7), were sequenced and analyzed for gene content and replication mode. Rolling-circle replication modules and mobilization proteins were found, very similar to those of other plasmids of the B. cereus group bacilli, mostly Bacillus thuringiensis living in the same ecosystem, suggesting active plasmid exchange in nature.     

pSRD191, a new member of RepL replicating plasmid family from Selenomonas ruminantium

A numerous plasmid population was detected in strain 19 of Selenomonas ruminantium. The population was found to consist of six plasmids in size ranging from 1.4 to more than 20kb. The smallest 1.4kb cryptic plasmid pSRD191 was further characterized. Sequence analysis identified a single ORF encoding the 177-residue putative replication protein (Rep191) which shared significant homology with RepL family of replication protein from Firmicutes (staphylococci and bacilli). PCR analysis and Southern hybridisation showed that pSRD191 related plasmids are frequently encountered in rumen selenomonads.     

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.     

spbA locus ensures the segregational stability of pTH1030, a novel type of Gram-positive replicon

The replication region of the plasmid pHT1030 of Bacillus thuringiensis was previously mapped to a 2.9 kb DNA fragment. The DNA sequence was analysed and it was shown that the minimal replicon resides within a 1 kb fragment of DNA carrying no potential protein coding sequence. Moreover, no production of single-stranded DNA intermediates was detected in the plasmid-containing cells. pHT1030 therefore belongs to a class of replicons not previously described in Gram-positive bacteria. Examination of the segregational stability of deletion derivatives of pHT1030 in bacilli defined two stability regions. One is located within the minimal replicon of pHT1030, whereas the second (spbA) is not required for replication. spbA encodes a 15 kDa protein and ensures the segregational stability of the plasmid. This effect of spbA is particularly highlighted in sporulation. The absence of the spbA locus gives rise to plasmid-free spores at high frequency, whereas the spbA+ plasmids are stably maintained. The stability of the plasmids during sporulation seems to be correlated with an unequal division of the cell by the sporulation septum.     

A new method for integration and stable DNA amplification in poorly transformable bacilli

Abstract We have developed a strategy for the integration and stable amplification of DNA sequences in the chromosome of poorly transformable bacilli, which avoids the presence of a functional plasmid replication system in the integrated DNA. The parental vector for integration contains two plus origins of replication from pUB110 in the same orientation on a single plasmid. Due to the direct repeats, such plasmids produce two individual progeny vectors, one of which is dependent on the other for replication, as it lacks a functional rep gene. We have used such a progeny vector system to integrate and amplify DNA on the chromosome of Bacillus licheniformis , and show that the structure is stable in the absence of selective pressure.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

The localization of replication origins on ARS plasmids in S. cerevisiae

Replication intermediates from the yeast 2 microns plasmid and a recombinant plasmid containing the yeast autonomous replication sequence ARS1 have been analyzed by two-dimensional agarose gel electrophoresis. Plasmid replication proceeds through theta-shaped (Cairns) intermediates, terminating in multiply interlocked catenanes that are resolved during S phase to monomer plasmids. Restriction fragments derived from the Cairns forms contain replication forks and bubbles that behave differently from one another when subjected to high voltage and agarose concentrations. The two-dimensional gel patterns observed for different restriction fragments from these two plasmids indicate that in each plasmid there is a single, specific origin of replication that maps, within the limits of our resolution, to the ARS element. Our results strongly support the long-standing assumption that in Saccharomyces cerevisiae an ARS is an origin of replication.     

Comparative analysis of five related staphylococcal plasmids

The genomic organization of five small multicopy staphylococcal plasmids comprising the pT181 family has been analyzed. In addition to pT181, the family presently includes the streptomycin resistance plasmid pS194 and the chloramphenicol resistance plasmids pC221, pC223, and pUB112. Although they belong to five different incompatibility groups, the five plasmids have similar basic replicons, use the same basic copy control mechanism, and have a common structural organization. It has been demonstrated previously that pT181 and pC221 encode trans-active replication proteins (RepC and RepD, respectively) which specifically recognize the respective plasmid's origin of replication in both cases is initiated by site-specific nicking and 3' extension. The other three plasmids in this family encode similar replication proteins; 63% of the predicted amino acid residues are identical for all five and the least similar pair shows 75% identity at the amino acid level. However, despite this homology, the replication proteins and origins of replication of different members in this family did not show cross complementation in vivo. Outside of the basic replicon, which comprises about one-third of each plasmid's genome, functional organization is also conserved. The resistance determinants are all located in the same position, immediately downstream of the replication protein coding sequence, and all are transcribed in the same direction. The three chloramphenicol resistance determinants encode highly homologous chloramphenicol transacetylases which are unrelated to the tet and str gene products. Three of the five plasmids form relaxation complexes and the involved genome segments are closely related. The other two are not homologous to these three in the corresponding region, but are homologous to each other and encode a site-specific recombinase, Pre. It is suggested that the replication, resistance, and relaxation complex regions of these plasmids can be regarded as conserved segments ("cassettes") assembled in various combinations, but always with the same spatial arrangement.     

Evolution of the Replication Regions of IncI{alpha} and IncFII Plasmids by Exchanging Their Replication Control Systems

The basic replicons of bacterial plasmids consist of two setsof genetic systems, the replication-structural system and thereplication control system. Comparison of nucleotide sequencessuggested that the basic replicons of plasmids P307 (IncFI)and pMU2200 (IncZ) were generated by reciprocal recombinationbetween ancestors of R100 (IncFII) and ColIb-P9 (IncI), or viceversa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9,are structurally unrelated to each other. Based on this information,we constructed in vitro and analyzed P307-like chimeric repliconsfrom ColIb-P9 and R100. When the replication-structural regionof ColIb-P9 was combined with the whole replication controlregion of R100, the resultant replicon replicated stably asR100 did. These results revealed that the basic replicons ofthe plasmids diverged by exchanging their replication controlsystems. Thus, we propose that the replication control systemsof plasmids, in some cases, evolved independently of their structuralsystems, although these two systems work together to maintainthe replication functions. We also showed that the reciprocalrecombination was specified by the unique secondary structuresof RNA involved in the control of expression of the genes encodingthe replication initiator proteins.     

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.     

Rolling-circle replication of bacterial plasmids.

Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.     

Conjugation in bacilli

The review considers experimental data on the conjugal transfer of plasmids in the Bacillus cereus and Bacillus subtilis groups (the transfer of large self-transmissible plasmids and the mobilization of small plasmids). Conjugation in bacilli is compared with conjugation in E. coli dependent on the F factor. Conjugation of bacilli in their natural habitats is also discussed.     

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.     

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.     

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.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.