Detection of Arcobacter spp. in the Coastal Environment of the Mediterranean Sea

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.     

Antimicrobial resistance and virulence genes profiles of Arcobacter butzleri strains isolated from back yard chickens and retail poultry meat in Chile

This research aims to investigate the presence and pathogenic potential of Arcobacter in poultry meat samples purchased in the retail market of Valdivia (South of Chile) as well as in faecal samples from backyard chickens from rural areas around this city. The isolates obtained were identified by molecular methods. Furthermore, putative virulence genes were assessed by PCR and the antimicrobial resistance was tested by phenotypic methods. Arcobacter was present in 41·6% of the samples, with the highest value in retail poultry meat (55·7%) followed by backyard production (28·0%). Arcobacter butzleri was the most prevalent species (75·6%) followed by Arcobacter skirrowii (14·8%) and Arcobacter cryaerophilus (9·6%). An 8·5% of A. butzleri strains from meat were resistant to both ciprofloxacin and tetracycline and 6·1% were resistant to erythromycin, while none was resistant to gentamycin, unlike strains from domestic chickens, which showed no resistance. Furthermore, A. butzleri strains from chicken meat presented a higher prevalence of virulence genes than strains from domestic chickens. In fact, in this last group, some genes (hecA, hecB and irgA) were completely absent. Therefore, this study provides insight on the epidemiology of Arcobacter in Chilean poultry and suggests that under traditional breeding conditions strains are, apparently, less pathogenic and drug resistant.     

Assessment of the Genetic Diversity among Arcobacters Isolated from Poultry Products by Using Two PCR-Based Typing Methods

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.     

The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri

Background

Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018.

Methodology/Principal Findings

Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains.

Conclusion/Significance

The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts.     

Adherence to and Invasion of Human Intestinal Cells by Arcobacter Species and Their Virulence Genotypes

The genus Arcobacter is composed of 17 species which have been isolated from various sources. Of particular interest are A. butzleri, A. cryaerophilus, and A. skirrowii, as these have been associated with human cases of diarrhea, the probable transmission routes being through the ingestion of contaminated drinking water and food. To date, only limited studies of virulence traits in this genus have been undertaken. The present study used 60 Arcobacter strains isolated from different sources, representing 16 of the 17 species of the genus, to investigate their ability to adhere to and invade the human intestinal cell line Caco-2. In addition, the presence of five putative virulence genes (ciaB, cadF, cj1349, hecA, and irgA) was screened for in these strains by PCR. All Arcobacter species except A. bivalviorum and Arcobacter sp. strain W63 adhered to Caco-2 cells, and most species (10/16) were invasive. The most invasive species were A. skirrowii, A. cryaerophilus, A. butzleri, and A. defluvii. All invasive strains were positive for ciaB (encoding a putative invasion protein). Other putative virulence genes were present in other species, i.e., A. butzleri (cadF, cj1349, irgA, and hecA), A. trophiarum (cj1349), A. ellisii (cj1349), and A. defluvii (irgA). No virulence genes were detected in strains which showed little or no invasion of Caco-2 cells. These results indicate that many Arcobacter species are potential pathogens of humans and animals.     

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322 bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

Inhibitory Effects of Some Spice and Herb Extracts Against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii

Seventeen spice and medicinal plant extracts (methanol and chloroform) were assayed for their antimicrobial activity against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii. In general, all of the tested extracts were able, to a different extent, to inhibit the growth of the selected Arcobacter species. Cinnamon, bearberry, chamomile, sage and rosemary extracts showed strong antimicrobial activity toward arcobacter strains tested. Overall, the methanol extracts showed better activity than the chloroform extracts (P < 0.05); however, enhanced antibacterial activity of chloroform extracts of cinnamon and rosemary has been observed in comparison with their methanol counterparts. The inhibitory dose of the most active extracts (the diameter of zone of inhibition ≥ 20 mm) was determined using the disc-diffusion method as well.     

Presence and quantification of pathogenic Arcobacter and Campylobacter species in retail meats available in Japan

We present estimations for the amounts of Arcobacter (A. butzleri, A. cryaerophilus and A. skirrowii) and Campylobacter (C. jejuni, C. coli and C. fetus) species in retail chicken, pork and beef meat using PCR-MPN. Arcobacter butzleri, A. cryaerophilus and C. jejuni were found in 100, 60 and 55% of chicken samples, respectively. No other Arcobacter or Campylobacter species were found in chicken. The MPNs of A. butzleri, A. cryaerophilus and C. jejuni were greater than 10³ per 100 g in 50, 0 and 5% of samples, respectively. The MPN of A. butzleri was higher than that of C. jejuni in 95% of samples. In pork, A. butzleri and A. cryaerophilus were detected in 10 and 11 (50 and 55%) of 20 samples, respectively. No other Arcobacter or Campylobacter species were found in pork. Only one pork sample had more than 10³ MPN per 100 g of A. cryaerophilus. For beef, only two samples tested positive for A. cryaerophilus, at 4600 and 92 MPN per 100 g. Overall, we found that the presence and MPNs of Arcobacter species are very high in chicken. In contrast, the positive ratios of Arcobacter in pork were high as chicken samples, but MPNs were lower than in chicken.     

Sequence Analysis of the 144-Kilobase Accessory Plasmid pSmeSM11a, Isolated from a Dominant Sinorhizobium meliloti Strain Identified during a Long-Term Field Release Experiment

The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base-pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules.     

Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids

Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.     

Identification of alkane monoxygenase genes inAcinetobacter venetianus VE-C3 and analysis of mutants impaired in diesel fuel degradation

Cells ofAcinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process inA. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis ofalkM andrubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk^? mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and energy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genetic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be similar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also.     

First multi-locus sequence typing scheme for Arcobacter spp.

Background  

Arcobacter spp. are a common contaminant of food and water, and some species, primarily A. butzleri and A. cryaerophilus, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first Arcobacter multilocus sequence typing (MLST) method for A. butzleri, A. cryaerophilus, A. skirrowii, A. cibarius and A. thereius.     

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This core homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.Abbreviations Sym symbiotic sequences/plasmids - nod genes required for nodulation - nod putative nod genes - nif genes required for the synthesis of the enzyme nitrogenase     

Genetic Diversity of <Emphasis Type="Italic">Arcobacter</Emphasis> Species of Animal and Human Origin in Andhra Pradesh,India

Arcobacter is an emerging foodborne pathogen having zoonotic significance. Enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive sequence-based PCR (rep-PCR) analysis of a total of 41 Arcobacter isolates revealed a greater degree of genetic diversity. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power for ERIC and rep-PCR was found to be 0.997 and 0.996, respectively. Close clustering between isolates of animal and human origin are indicative of probable zoonotic significance.     

Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ Factors of Arcobacter butzleri

The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintenance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor, indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Rp4 Promotion of Transfer of a Large Agrobacterium Plasmid Which Confers Virulence

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Replication regions of Sinorhizobium meliloti plasmids

The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA- strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75-79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O'Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232-237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.