Plasmids Of Gram-positive Bacteria Research Articles

Extraordinary long-stem confers resistance of intrinsic terminators to processive antitermination.

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.

Characterization of two cryptic plasmids from Kocuria palustris IPUFS-1 and construction of novel Escherichia coli–Kocuria shuttle vector for biocatalysis

Two cryptic plasmids, designated pKPAL1 and pKPAL2, were identified from the gram-positive bacterium Kocuria palustris IPUFS-1, which was isolated from a fish source. The 2251-bp and 2488-bp circular genomes of pKPAL1 and pKPAL2, respectively, were sequenced. Subsequent open reading frame (ORF) and homology search analyses suggested that pKPAL1 and pKPAL2 possess two and three ORFs, respectively, and encode the putative replication proteins, RepA and RepB, like the genomes of several plasmids in gram-positive bacteria. Thus, pKPAL1 and pKPAL2 were inferred to belong to the ColE2 plasmid family. We constructed novel Escherichia coli-Kocuria shuttle vectors pKITE101-103 based on pKPAL1. The constructed shuttle vector was stably maintained in Kocuria transformant cells, and vector copy number was estimated to be about 60 per cell. Leifsonia sp. S749 alcohol dehydrogenase (LSADH) was efficiently expressed in Kocuria rhizophila DC2201 using the pKITE103P vector under the control of the promoter of glyceraldehyde 3-phosphate dehydrogenase (gapdh). Herein, we demonstrate that the novel shuttle vector is a useful tool for developing biocatalysts based on organic solvent-tolerant Kocuria cells.

Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.

Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

Plasmid Replication Control by Antisense RNAs.

Plasmids are selfish genetic elements that normally constitute a burden for the bacterial host cell. This burden is expected to favor plasmid loss. Therefore, plasmids have evolved mechanisms to control their replication and ensure their stable maintenance. Replication control can be either mediated by iterons or by antisense RNAs. Antisense RNAs work through a negative control circuit. They are constitutively synthesized and metabolically unstable. They act both as a measuring device and a regulator, and regulation occurs by inhibition. Increased plasmid copy numbers lead to increasing antisense-RNA concentrations, which, in turn, result in the inhibition of a function essential for replication. On the other hand, decreased plasmid copy numbers entail decreasing concentrations of the inhibiting antisense RNA, thereby increasing the replication frequency. Inhibition is achieved by a variety of mechanisms, which are discussed in detail. The most trivial case is the inhibition of translation of an essential replication initiator protein (Rep) by blockage of the rep-ribosome binding site. Alternatively, ribosome binding to a leader peptide mRNA whose translation is required for efficient Rep translation can be prevented by antisense-RNA binding. In 2004, translational attenuation was discovered. Antisense-RNA-mediated transcriptional attenuation is another mechanism that has, so far, only been detected in plasmids of Gram-positive bacteria. ColE1, a plasmid that does not need a plasmid-encoded replication initiator protein, uses the inhibition of primer formation. In other cases, antisense RNAs inhibit the formation of an activator pseudoknot that is required for efficient Rep translation.

Characterization of the transfer-related tra region of the conjugative plasmid p19 from a Bacillus subtilis soil strain

The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.

Geminiviruses: a tale of a plasmid becoming a virus

BackgroundGeminiviruses (family Geminiviridae) are small single-stranded (ss) DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps) of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons.ResultsPhylogenetic and clustering analyses of various RCR Reps suggest that Rep proteins of geminiviruses share a most recent common ancestor with Reps encoded on plasmids of phytoplasmas, parasitic wall-less bacteria replicating both in plant and insect cells and therefore occupying a common ecological niche with geminiviruses. Capsid protein of Satellite tobacco necrosis virus was found to be the best template for homology-based structural modeling of the geminiviral capsid protein. Good stereochemical quality of the generated models indicates that the geminiviral capsid protein shares the same structural fold, the viral jelly-roll, with the vast majority of icosahedral plant-infecting ssRNA viruses.ConclusionWe propose a plasmid-to-virus transition scenario, where a phytoplasmal plasmid acquired a capsid-coding gene from a plant RNA virus to give rise to the ancestor of geminiviruses.

Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

Conjugation is a major contributor to lateral gene transfer in bacteria, and pheromone-inducible conjugation systems in Enterococcus faecalis play an important role in the dissemination of antibiotic resistance and virulence in enterococci and related bacteria. We have genetically dissected the determinants of DNA processing of the enterococcal conjugative plasmid pCF10. Insertional inactivation of a predicted relaxase gene pcfG, via insertion of a splicing-deficient group II intron, severely reduced pCF10 transfer. Restoration of intron splicing ability by genetic complementation restored conjugation. The pCF10 origin of transfer ( oriT) was localized to a 40-nucleotide sequence within a non-coding region with sequence similarity to origins of transfer of several other plasmids in gram positive bacteria. Deletion of the oriT reduced pCF10 transfer by more than five orders of magnitude without affecting pCF10-dependent mobilization of co-resident oriT-containing plasmids. Although the host range for pCF10 replication is limited to enterococci, we found that the pCF10 conjugation system promotes mobilization of oriT-containing plasmids to multiple bacterial genera. Therefore, this transfer system may have applications for gene delivery to a variety of poorly-transformed bacteria.

Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector

Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

Characterization of the Replication Region of Plasmid pLS32 from the Natto Strain ofBacillus subtilis

Plasmid pL32 from the Natto strain of Bacillus subtilis belongs to a group of low-copy-number plasmids in gram-positive bacteria that replicate via a theta mechanism of replication. We studied the DNA region encoding the replication protein, RepN, of pLS32, and obtained the following results. Transcription of the repN gene starts 167 nucleotides upstream from the translational start site of repN. The copy number of repN-coding plasmid pHDCS2, in which the repN gene was placed downstream of the IPTG (isopropyl-1-thio-beta-D-galactopyranoside)-inducible Pspac promoter, was increased 100 fold by the addition of IPTG. Histidine-tagged RepN bound to a specific region in the repN gene containing five 22-bp tandem repeats (iterons) with partial mismatches, as shown by gel retardation and foot printing analyses. Sequence alterations in the first three iterons resulted in an increase in plasmid copy number, whereas those in either the forth or fifth iteron resulted in the failure of plasmid replication. The iterons expressed various degrees of incompatibility with an incoming repN-driven replicon pSEQ243, with the first three showing the strongest incompatibility. Finally, by using a plasmid, pHDMAEC21, carrying the sequence alterations in all the five iterons in repN and thus unable to replicate but encoding intact RepN, the region necessary for replication was confined to a 96-bp sequence spanning the 3'-terminal half of the fourth iteron to an A+T-rich region located downstream of the fifth iteron. From these results, we conclude that the iterons in repN are involved in both the control of plasmid copy number and incompatibility, and we suggest that the binding of RepN to the last two iterons triggers replication by melting the A+T-rich DNA sequence.

Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus.

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.

Molecular characterization of functional modules of plasmid pWKS1 of Paracoccus pantotrophus DSM 11072.

The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined. The G+C content of the sequence of this plasmid was 62 mol%. Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44.1 and 37.8 kDa, respectively. Mutational analysis showed that ORF2 was crucial for plasmid replication. The translational product of ORF2 shared local homologies with replication proteins of several theta-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts. An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV). ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria. A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli. MOB modules of pWKS1 and pBBR1 were functionally complementary to each other. Hybridization analysis revealed that only plasmid pSOV1 (6.5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1. Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E. coli.

An antisense RNA-mediated transcriptional attenuation mechanism functions in Escherichia coli.

Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

PS86, a new theta-replicating plasmid from Enterococcus faecalis.

The complete nucleotide sequence of the small (5149 bp) and cryptic plasmid pS86 from Enterococcus faecalis ssp. faecalis S-86 has been determined. Sequence analysis revealed six putative open reading frames (ORFs) encoding polypeptides of 28.3, 11.5, 8.4, 65.1, 7.3, and 11.96 kDa each. Based on sequence similarity, two cassettes have been identified in pS86: ORF1 codes for the replication initiation protein (Rep); ORF4 codes for a putative mobilization protein that shows similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. No function could be assigned to the other putative ORFs found. According to our results, pS86 plasmid could use a theta-mode of replication, similar to the recently described theta-type replicons from pUCL287 (Tetragenococcus halophila) and pLA1 or pLA105 (Lactobacillus acidophilus) plasmids.

Identification and Characterization of an Active Plasmid Partition Mechanism for the Novel Lactococcus lactis Plasmid pCI2000

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.

Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression.

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

Sequence requirements for the termination of rolling-circle replication of plasmid pT181.

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.

Fine Mapping and DNA Sequence of Replication Functions of Bacillus thuringiensis Plasmid pTX14-3

pTX14-3 is a 7.5-kb cryptic plasmid isolated from a Bacillus thuringiensis subspecies israelensis strain. Like many other small plasmids in gram-positive bacteria, pTX14-3 replicates via a single-stranded DNA intermediate. The nucleotide sequence of the replication region was determined and an open reading frame of 636 base pairs encoding a protein necessary for plasmid replication was identified by deletion analysis. No significant homology was found between this open reading frame and those encoding replication proteins identified on other plasmids isolated from gram-positive bacteria, nor could we find any homology to plus origins from other single-stranded DNA plasmids. Consequently, it seems that the replicon of pTX14-3 belongs to a new family of replicons in the group of single-stranded DNA plasmids. The sequence of the single-strand origin (i.e., the minus origin) responsible for the conversion of single-stranded plasmid DNA to double-stranded plasmid DNA was also determined. A partial homology between the minus origin of pTX14-3 and the Bacillus subtilis plasmid pBAA1 was identified. A previously identified locus that suppresses formation of high molecular weight multimers was also minimized and sequenced.

Biochemical and Genetic Analysis of the Unidirectional Theta Replication of the S. agalactiae Plasmid pIP501

pIP501 is a broad-host-range plasmid originating from Streptococcus agalactiae. In this report we show that (i) it replicates by a theta mechanism initiating at the 3′ end of the gene encoding the replication protein RepR and progressing in the direction of transcription of this gene; (ii) its replication origin lies within or a few nucleotides downstream from ORF R and not upstream from it as suggested in the literature (Brantl et al. (1990) Nucleic Acids Res. 18, 4783-4789); (iii) the RepR protein positively regulates pIP501 copy number; and (vi) the main function ensured by the sequences located upstream from ORF R is to express this ORF. Since the replication properties of pIP501 are indistinguishable from those of the highly related Enterococcus faecalis plasmid pAMβ1, we conclude that these elements form the first family of theta replicating plasmids in gram-positive bacteria. Based on sequence similarities, we extend this family to the S. pyogenes plasmid pSM19035 and to 12 other plasmids isolated from streptococci or enterococci.