A highly efficient electroporation system for transformation of Yersinia

Conchas, Ramón F.; Carniel, Élisabeth

doi:10.1016/0378-1119(90)90505-l

Cited by 104 publications

(66 citation statements)

References 21 publications

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“…Large-scale plasmid DNA preparations were purified on Qiagen columns. Recombinant plasmid DNA was introduced into Y. pseudotuberculosis by mating or electroporation (Conchas & Carniel, 1990 Synthetic oligonucleotides and PCR. Oligonucleotide primers (Table 2) were custom-synthesized (Sigma and Genset) for PCR generation of DNA fragments used for cloning or probing.…”

Section: Methodsmentioning

confidence: 99%

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence

et al. 2004

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The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

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Section: Methodsmentioning

confidence: 99%

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence

et al. 2004

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“…DNA fragments were purified from agarose gels using Geneclean (Bio101) or GenElute (Supelco). Plasmids were introduced into Yersinia by electroporation as described by Conchas and Carniel (1990).…”

Section: Dna Methodsmentioning

confidence: 99%

Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III‐dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments

Frithz‐Lindsten

Rosqvist

et al. 1997

Molecular Microbiology

198

262

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SummaryExoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. The amino-terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Recently, YopE was found to be translocated into the host cell by a bacteria-cell contact-dependent mechanism involving the ysc-encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti-phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an anti-phagocytic strategy.

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“…Isolation of plasmids and transformation were carried out by standard methods. Electroporation of Y. enterocolitica cells was performed as previously described (Conchas & Carniel, 1990). …”

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confidence: 99%