Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60 232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large Gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.     

Complete nucleotide sequence of plasmid Rts1: implications for evolution of large plasmid genomes

Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence of tus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.     

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

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

Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis

The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.     

The organization of two rRNA (rrn) operons of the slow-growing pathogen Mycobacterium celatum provides key insights into mycobacterial evolution

The slow-growing Mycobacterium celatum is known to have two different 16S rRNA gene sequences. This study confirms the presence of two rrn operons and describes their organization. One operon (rrnA) was found to be located downstream from murA and the other (rrnB) was found downstream from tyrS. The promoter regions were sequenced, and also the intergenic transcribed spacer (ITS1 and ITS2) regions separating the 16S rRNA, 23S rRNA and 5S rRNA gene coding regions. Analysis of the RNA fraction revealed that rrnA is regulated by two (P1 and PCL1) promoters and rrnB is regulated by one (P1). These data show that the two rrn operons of M. celatum are organized in the same way as the two rrn operons of classical fast-growing mycobacteria. This information was incorporated into a phylogenetic analysis of the genus based on both 16S rRNA gene sequences and (where possible) the number of rrn operons per genome. The results suggest that the ancestral Mycobacterium possessed two (rrnA and rrnB) operons per genome and that subsequently, on two separate occasions, an operon (rrnB) was lost, leading to two clusters of species having a single operon (rrnA); one cluster includes the classical pathogens and the other includes Mycobacterium abscessus and Mycobacterium chelonae.     

Expression of the immunity protein of plantaricin 423, produced by Lactobacillus plantarum 423, and analysis of the plasmid encoding the bacteriocin

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.     

Primary Structure of NM.<Emphasis Type="Italic">Bst</Emphasis>SEI Operon from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>, the Producer of N.<Emphasis Type="Italic">Bst</Emphasis>SEI Site-Specific Nicking Endonuclease

The nucleotide sequence of Bacillus stearothermophilus SE-589 DNA fragment including an operon for the site-specific nicking-modification (NM) system with a gene for BstSEI nicking endonuclease (nickase) has been determined. An analysis of the regions adjacent to the nickase gene has revealed two genes encoding DNA methyltransferases belonging to different classes. Three genes that form the system operon are separated by short open reading frames (ORFs). An analysis of these ORFs has shown that the polypeptides they encode are homologous to different parts of BstSEI nickase, NatB protein, and arginase. A difference in the GC content of the beginning and ending regions of the cloned DNA fragment and the presence of short ORFs similar to genes for known proteins indicate that the NM.BstSEI system operon has probably evolved by horizontal DNA transfer.     

Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.     

Complete nucleotide sequence of pGS18, a 62.8-kb plasmid from <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> strain 18

The complete nucleotide sequence (62.8 kb) of pGS18, the largest sequenced plasmid to date from the species Geobacillus stearothermophilus, was determined. Computational analysis of sequence data revealed 65 putative open reading frames (ORFs); 38 were carried on one strand and 27 were carried on the other. These ORFs comprised 84.1% of the pGS18 sequence. Twenty-five ORFs (38.4%) were assigned to putative functions; four ORFs (6.2%) were annotated as pseudogenes. The amino acid sequences obtained from 29 ORFs (44.6%) had the highest similarity to hypothetical proteins of the other microorganisms, and seven (10.8%) had no significant similarity to any genes present in the current open databases. Plasmid replication region, strongly resembling that of the theta-type replicon, and genes encoding three different plasmid maintenance systems were identified, and a putative discontinuous transfer region was localized. In addition, we also found several mobile genetic elements and genes, responsible for DNA repair, distributed along the whole sequence of pGS18. The alignment of pGS18 with two other large indigenous plasmids of the genus Geobacillus highlighted the presence of well-conserved segments and has provided a framework that can be exploited to formulate hypotheses concerning the molecular evolution of these three plasmids.     

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.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli

The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.     

The complete sequence and analysis of the large virulence plasmid pSS of Shigella sonnei

The complete sequence of pSS, which is the large virulence plasmid of Shigella sonnei, was determined. The 214-kb plasmid is composed of segments of virulence-associated genes, the O-antigen gene clusters, a range of replication and maintenance genes, and large numbers of insertion sequence (IS) elements. Two hundred and forty-one open reading frames (ORFs) were identified, of which 117 are highly homologous to IS elements or transposases, 57 are homologous to known pathogenesis-associated proteins, and 30 are related to replication, plasmid maintenance, or other metabolic functions. Thirty-seven ORFs have no similarity to proteins with a known function, including two with no significant similarity to any hypothetical proteins. Interestingly, 10 ORFs encoding O-antigen gene clusters were identified on the plasmid and this is markedly different from most other Shigella spp. virulent plasmids. A novel toxin-antitoxin system, a series of stbDE homologs, was found on the plasmid immediately downstream of the replication region; the sole segregation stability system may be responsible for the instability of pSS. The pSS plasmid is a mixture of genes with different origins and functions. The sequence suggests a remarkable history of IS-mediated recombination and acquisition of DNA across a range of bacterial species.     

Complete DNA sequence of a ColBM plasmid from avian pathogenic Escherichia coli suggests that it evolved from closely related ColV virulence plasmids

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5' end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the "conserved" portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its "variable" portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this "conserved" cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied.     

pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni

The complete nucleotide sequence of pEOC01, a plasmid (11,661 bp) from Pediococcus acidilactici NCIMB 6990 encoding resistance to clindamycin, erythromycin, and streptomycin was determined. The plasmid, which also replicates in Lactococcus and Lactobacillus species contains 16 putative open reading frames (ORFs), including regions annotated to encode replication, plasmid maintenance and multidrug resistance functions. Based on an analysis the plasmid replicates via a theta replicating mechanism closely related to those of many larger Streptococcus and Enterococcus plasmids. Interestingly, genes homologous to a toxin/antitoxin plasmid maintenance system are present and are highly similar to the omega-epsilon-zeta operon of Streptococcus plasmids. The plasmid contains two putative antibiotic resistance homologs, an ermB gene encoding erythromycin and clindamycin resistance, and a streptomycin resistance gene, aadE. Of particular note is the aadE gene which holds 100% identity to an aadE gene found in Campylobacter jejuni plasmid but which probably originated from a Gram-positive source. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut.