A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains

Demarre, Gaëlle; Guérout, Anne-Marie; Matsumoto-Mashimo, Chiho; Rowe‐Magnus, Dean A.; Marlière, Philippe; Mazel, Didier

doi:10.1016/j.resmic.2004.09.007

Cited by 279 publications

(232 citation statements)

References 36 publications

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“…In brief, an internal 201 bp fragment of the rfaH gene was PCRamplified using Bam HI restriction site-containing primers rfaH-F and rfaH-R (Table S1). The Bam HI-digested PCR product was ligated to Bam HI-digested and shrimp alkaline phosphatase (SAP)-treated suicide vectors pSW23T and pSW25T (Demarre et al, 2005). The suicide constructs obtained, pSW23T-rfaH and pSW25T-rfaH, were subsequently introduced to strains 6471/76 and 6471/76-c by conjugation to obtain strains YeO3-DrfaH and YeO3-c-DrfaH, respectively.…”

Section: Methodsmentioning

confidence: 99%

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Leskinen

Varjosalo

et al. 2015

Microbiology

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The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA sequencing of the WT and DrfaH strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the DrfaH strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the DrfaH strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the DrfaH strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the DrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in Y. enterocolitica O:3. It may be speculated that RfaH might have an in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.

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Section: Methodsmentioning

confidence: 99%

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Leskinen

Varjosalo

et al. 2015

Microbiology

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“…The borders of the conserved region in the oriV in each plasmid were defined by the CLUSTAL W alignment (Larkin et al, 2007) of the sequences of the three plasmids. The PCR products were then digested with SphI and EcoRI, and cloned into the SphI-EcoRI sites of a promoterless vector pSW29T (Demarre et al, 2005). This gave rise to minireplicons pLR301 (minipKS208), pLR302 (mini-pMBUI1) and pLR303 (mini-pBP136).…”

Section: Methodsmentioning

confidence: 99%

Host range diversification within the IncP-1 plasmid group

et al. 2013

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Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other traits. In spite of increasing information about the genomic diversity of closely related plasmids, the relationship between sequence divergence and host range remains unclear. IncP-1 plasmids are currently classified into six subgroups based on the genetic distance of backbone genes. We investigated whether plasmids from two subgroups exhibit a different host range, using two IncP1c plasmids, an IncP-1b plasmid and their minireplicons. Efficiencies of plasmid establishment and maintenance were compared using five species that belong to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The IncP-1b plasmid replicated and persisted in all five hosts in the absence of selection. Of the two IncP-1c plasmids, both were unable to replicate in alphaproteobacterial host Sphingobium japonicum, and one established itself in Agrobacterium tumefaciens but was very unstable. In contrast, both IncP-1c minireplicons, which produced higher levels of replication initiation protein than the wild-type plasmids, replicated in all strains, suggesting that poor establishment of the native plasmids is in part due to suboptimal replication initiation gene regulation. The findings suggest that host ranges of distinct IncP-1 plasmids only partially overlap, which may limit plasmid recombination and thus result in further genome divergence.

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“…nation complex, but nevertheless in vitro recombination and in vivo integration were observed. To stabilize the exchange of strands, tyrosine recombinases normally require one WatsonCrick bp interaction on each side of the recombination complex immediately after the position of cleavage of the recombinases (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). The only notable exception to this rule is the integrase of some bacteroides conjugative transposons (32).…”

Section: Discussionmentioning

confidence: 99%

“…Classical and El Tor phage replication and integration machinery (RS elements) were amplified using genomic DNA of V. cholerae strains 569B or N16961, respectively, as templates. The amplicons were cloned into the suicide vector pSW23T (30). Plasmids carrying hybrid phage variants were engineered by inverse PCR.…”

Section: Methodsmentioning

confidence: 99%

Molecular keys of the tropism of integration of the cholera toxin phage

Das

Bischerour

Val

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

109

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International audienceCholera toxin is encoded in the genome of CTXvarphi, a lysogenic filamentous phage of Vibrio cholerae. CTXvarphi variants contribute to the genetic diversity of cholera epidemic strains. It has been shown that the El Tor variant of CTXvarphi hijacks XerC and XerD, two host-encoded tyrosine recombinases that normally function to resolve chromosome dimers, to integrate at dif1, the dimer resolution site of the larger of the two V. cholerae chromosomes. However, the exact mechanism of integration of CTXvarphi and the rules governing its integration remained puzzling, with phage variants integrated at either or both dimer resolution sites of the two V. cholerae chromosomes. We designed a genetic system to determine experimentally the tropism of integration of CTXvarphi and thus define rules of compatibility between phage variants and dimer resolution sites. We then showed in vitro how these rules are explained by the direct integration of the single-stranded phage genome into the double-stranded bacterial genome. Finally, we showed how the evolution of phage attachment and chromosome dimer resolution sites contributes to the generation of genetic diversity among cholera epidemic strains

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A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains

Cited by 279 publications

References 36 publications

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Host range diversification within the IncP-1 plasmid group

Molecular keys of the tropism of integration of the cholera toxin phage

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