A new family of conditional replicating plasmids and their cognate Escherichia coli host strains

Matsumoto-Mashimo, Chiho; Guérout, Anne-Marie; Mazel, Didier

doi:10.1016/j.resmic.2004.03.001

Cited by 19 publications

(14 citation statements)

References 23 publications

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“…Briefly, the HK (PG0719) from strain W83 was cloned into shuttle/expression vector pT-COW (kindly provided by N. Shoemaker, University of Illinois) [4] by PCR using primers with AvaI and BamHI adapters (Table 1). The resulting plasmid (pT719) was first transformed into E.coli S17-1 [5] with selection for ampicillin resistant transconjugants on Luria-Bertani plates (100 µg/ml ampicillin). For filter mating, a culture of plasmid donor strain E.coli S17-pT719 was grown to OD 550 nm 0.25 and recipient strain ATCC 33277 was grown anaerobically for 48 h on BAP.…”

Section: Methodsmentioning

confidence: 99%

A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis

Scott¹,

Klein

Duran-Pinedo³

et al. 2013

PLoS ONE

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Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with infection of the periodontia. The organism has a small number of two-component signal transduction systems, and after comparing genome sequences of strains W83 and ATCC 33277 we discovered that the latter was mutant in histidine kinase (PGN_0752), while the cognate response regulator (PGN_0753) remained intact. Microarray-based transcriptional profiling and ChIP-seq assays were carried out with an ATCC 33277 transconjugant containing the functional histidine kinase from strain W83 (PG0719). The data showed that the regulon of this signal transduction system contained genes that were involved in hemin acquisition, including gingipains, at least three transport systems, as well as being self-regulated. Direct regulation by the response regulator was confirmed by electrophoretic mobility shift assays. In addition, the system appears to be activated by hemin and the regulator acts as both an activator and repressor.

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Section: Methodsmentioning

confidence: 99%

A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis

Scott¹,

Klein

Duran-Pinedo³

et al. 2013

PLoS ONE

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“…Alleles carrying an internal deletion were generated in vitro using a two-step PCR construction method (20). Using genomic DNA of the strain LGP32, independent PCR amplifications of the regions encompassing the VSA1062, VS2864, and VSA576 open reading frames (ORFs) were performed using two primer pairs per ORF: 1062-1 and 1062-2 (yielding a 457-bp product) and 1062-3 and 1062-4 (yielding a 447-bp product), 2864-1 and 2864-2 (generating a 341-bp product) and 2864-3 and 2864-4 (giving a 341-bp product), and 576-1 and 576-2 (generating a 500-bp product) and 576-3 and 576-4 (resulting in a 465-bp product).…”

Section: Methodsmentioning

confidence: 99%

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products ofVibrio splendidus

Binesse

Delsert

Saulnier

et al. 2008

Appl Environ Microbiol

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Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (⌬vsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the ⌬vsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the ⌬vsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.Vibrionaceae are a predominant family of gram-negative bacteria found in aquatic environments (30). Bacteria within this family demonstrate a high degree of genetic diversity and are able to colonize very different types of niches. They live freely as planktonic forms in the water column or are associated in biofilms or with host organisms as pathogenic, commensal, or mutualistic bacteria. To date, eight genome sequences from Vibrionaceae have been made available: those of Vibrio cholerae strains N16961 and 0395, V. parahaemolyticus RIMD2210633, V. vulnificus strains YJ016 and CMCP6, V. fischeri ES114, V. harveyi ATCC BAA-1116, and Photobacterium profundum SS9 (3,10,19,24,32). More recently, we have completed the sequencing of the genome of V. splendidus strain LGP32, an oyster (Crassostrea gigas) pathogen

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“…Matings between E. coli (β2163) and Vibrionaceae were performed at 30°C as described earlier (13). Nucleotide substitutions were introduced by PCR assembly as described earlier (14). RNA isolation and northern blotting were performed using standard laboratory protocols.…”

Section: Methodsmentioning

confidence: 99%

Conserved small RNAs govern replication and incompatibility of a diverse new plasmid family from marine bacteria

Roux

Davis

Waldor

2010

Nucleic Acids Research

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Plasmids are autonomously replicating extrachromosomal DNA molecules that often impart key phenotypes to their bacterial hosts. Plasmids are abundant in marine bacteria, but there is scant knowledge of the mechanisms that control their replication in these hosts. Here, we identified and characterized the factors governing replication of a new family of plasmids from marine bacteria, typified by the virulence-linked plasmid pB1067 of Vibrio nigripulchritudo. Members of this family are prevalent among, yet restricted to, the Vibrionaceae. Unlike almost all plasmid families characterized to date, the ori regions of these plasmids do not encode a Rep protein to initiate DNA replication; instead, the ori regions encode two partially complementary RNAs. The smaller, termed RNA I, is ∼68-nt long and functions as a negative regulator and the key determinant of plasmid incompatibility. This Marine RNA-based (MRB) plasmid family is the first characterized family of replicons derived from marine bacteria. Only one other plasmid family (the ColE1 family) has previously been reported to rely on RNA-mediated replication initiation. However, since the sequences and structures of MRB RNA I transcripts are not related to those of ColE1 replicons, these two families of RNA-dependent replicons likely arose by convergent evolution.

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A new family of conditional replicating plasmids and their cognate Escherichia coli host strains

Cited by 19 publications

References 23 publications

A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis

A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products ofVibrio splendidus

Conserved small RNAs govern replication and incompatibility of a diverse new plasmid family from marine bacteria

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