Identification of a maintenance system on rolling circle replicating plasmid pVT736‐1

Galli, Dominique M.; LeBlanc, Donald J.

doi:10.1046/j.1365-2958.1997.4991867.x

Cited by 9 publications

(7 citation statements)

References 47 publications

(85 reference statements)

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“…(1989). DNA–DNA hybridization conditions, and transformation by electroporation have been described previously (Galli and LeBlanc, 1997). Standard three‐step PCR experiments were performed with Taq polymerase from Promega (Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

2006

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SummaryNeutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans , a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibodyopsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.

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Section: Methodsmentioning

confidence: 99%

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

2006

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“…Standard recombinant DNA techniques were performed as described by Sambrook et al (47). DNA-DNA hybridization conditions and transformation by electroporation were as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

“…Previous work has focused mainly on the characterization of pVT736-1, one of the first rolling circle replicating (RCR) plasmids isolated from gram-negative bacteria (17,18). It was shown that pVT736-1 was cryptic, that it was not related to RCR plasmids found in gram-positive bacteria, and that it encoded a new type of partitioning system (20).…”

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confidence: 99%

Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

et al. 2001

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The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.The gram-negative bacterium Actinobacillus actinomycetemcomitans is a capnophilic coccobacillus. The organism has been associated with several forms of periodontal disease such as localized juvenile periodontitis and rapidly progressive periodontitis, as well as with soft tissue abscesses and endocarditis (58). In a previous study 39 isolates of this periodontal pathogen had been screened for the presence of indigenous plasmids in an effort to evaluate the role(s) of such genetic elements in oral bacteria (32). Three plasmids, pVT736-1 (2 kb), pVT736-2 (Ͼ30 kb), and pVT745 (25 kb), were identified in two strains, suggesting that the occurrence of plasmids in A. actinomycetemcomitans was rare. The ultimate goal was to determine the biological properties of these plasmids, to assess their potential contribution to the pathogenicity of A. actinomycetemcomitans, and to evaluate their usefulness as tools in recombinant DNA technology. Previous work has focused mainly on the characterization of pVT736-1, one of the first rolling circle replicating (RCR) plasmids isolated from gram-negative bacteria (17, 18). It was shown that pVT736-1 was cryptic, that it was not related to RCR plasmids found in gram-positive bacteria, and that it encoded a new type of partitioning system (20).Preliminary characterization suggested that there was no obvious phenotype associated with pVT745 (41). Its size was a strong indication that the plasmid replicated by a theta mechanism rather than by a rolling circle mode. Although pVT745 had been isolated from one strain of A. actinomycetemcomitans (VT745) only, it was demonstrated by Southern hybridization that this plasmid shared sequence homologies with chromos...

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“…Plasmid DNA was isolated from A. actinomycetemcomitans and E. coli as described previously (11). Restriction endonucleases, Klenow polymerase I, and T4 DNA ligase were used in accordance with the manufacturer's instructions (Gibco-BRL).…”

Section: Methodsmentioning

confidence: 99%

“…Standard re-combinant DNA techniques were performed as described by Sambrook et al (23). DNA-DNA hybridization conditions and transformation by electroporation have been described previously (11). Standard three-step PCR experiments were performed with Taq polymerase from Promega (Madison, Wis.).…”

Section: Methodsmentioning

confidence: 99%

DNA Inversion on Conjugative Plasmid pVT745

2002

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Plasmid pVT745 from Actinobacillus actinomycetemcomitans strain VT745 can be transferred to other A. actinomycetemcomitans strains at a frequency of 10 ؊6 . Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5 end of the invertase gene, inv. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all A. actinomycetemcomitans strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet.

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Identification of a maintenance system on rolling circle replicating plasmid pVT736‐1

Cited by 9 publications

References 47 publications

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

DNA Inversion on Conjugative Plasmid pVT745

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