Signature Tagged Mutagenesis of &lt;I&gt;Pseudomonas aeruginosa&lt;/I&gt;

Wiehlmann, Lutz; Salunkhe, Prabhakar; Larbig, Karen; Ritzka, Margit; Tümmler, Burkhard

doi:10.1166/gl.2002.014

Cited by 11 publications

(8 citation statements)

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“…Winson et al (1998) Generation and complementation of the P. aeruginosa vqsR mutant. The P. aeruginosa vqsR mutant was generated by Tn5 transposon mutagenesis as described previously (Wiehlmann et al, 2002). Plasmid pME6010vqsR, used for complementation in trans, was constructed by cloning the 1254 bp of the PA2591 (vqsR) gene generated by PCR with primers 59-CTTGAACAAGCTTTCGTCCT-GCGCGTA-39 and 59-GATTATAGATCTGTGGATATCGCATTGC-AC-39 into the BglII/EcoRI-restricted shuttle vector pME6010.…”

Section: Methodsmentioning

confidence: 99%

Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa

2004

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Pathogenesis of Pseudomonas aeruginosa is controlled to a major extent by the two quorum-sensing systems las and rhl. The previously uncharacterized gene PA2591 was identified as a major virulence regulator, vqsR, in the quorum-sensing hierarchy. vqsR is a member of the LuxR family and possesses a las box in its upstream region. Transposon inactivation of vqsR abrogated the production of N-acylhomoserine lactones and the secretion of exoproducts and diminished bacterial virulence for Caenorhabditis elegans. Cytotoxicity towards macrophages was not affected. vqsR mRNA was expressed more strongly in the presence of human serum and oxidative stress than under standard growth conditions. High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type strain and a vqsR mutant. One-hundred-and-fifty-one and 113 genes were significantly differentially expressed in the presence of H 2 O 2 and human serum, respectively. The disruption of vqsR repressed the expression of genes that are known to be promoted by quorum sensing and activated the expression of genes that are known to be repressed by quorum sensing. Moreover, the vqsR mutant harboured less mRNA transcript for the production of siderophores and membrane-bound elements of antibiotic resistance. The protein encoded by PA2591 regulates several traits of pathogenicity; hence, the name vqsR ('virulence and quorum-sensing regulator') was assigned to PA2591. INTRODUCTIONThe opportunistic human pathogen Pseudomonas aeruginosa is the leading source of Gram-negative nosocomial infections (Van Delden & Iglewski, 1998) and causes chronic lung infections in individuals suffering from cystic fibrosis (Tümmler & Kiewitz, 1999). The analysis of the fully sequenced P. aeruginosa genome revealed that more than 9 % of the assigned open reading frames (ORFs) encode known or putative transcriptional regulators and twocomponent systems (Stover et al., 2000). It is thought that the large amount of regulatory factors allows the organism to adapt to various environments and thus represents the key for the understanding of its enormous metabolic versatility.Evidence has accumulated over the past few years that for the expression of pathogenic traits a quorum-sensing circuitry that is operating in P. aeruginosa is of central importance (for recent reviews, see de Kievit & Iglewski, 2000;Williams et al., 2000;Camara et al., 2002). This form of gene regulation ensures that virulence factors are expressed in a population-density-dependent manner. Two quorumsensing systems that rely on diffusible N-acylhomoserine lactone (AHL) signal molecules have been identified in P. aeruginosa and were shown to orchestrate expression of virulence factors and participate in the development of biofilms: the las system consisting of the transcriptional activator LasR and the AHL synthase LasI which directs the synthesis of N-3-oxo-dodecanoyl-homoserine lactone Abbreviations: AHL, N-acylhomoserine lactone; C 4 -HSL, N-butanoylhomoserine lactone; 3-oxo-C 12 -HSL, N-3-...

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Section: Methodsmentioning

confidence: 99%

Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa

2004

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“…Pseudomonas aeruginosa strain TB was used as a wild‐type in this study [10]. The P. aeruginosa VqsR mutant was generated by Tn5 transposon mutagenesis as described previously [11]. Bacterial cultures were grown in 15 ml of ABC minimal medium (0.15 M (NH 4 ) 2 SO 4 , 0.33 M Na 2 HPO 4 × 2H 2 O, 0.22 M KH 2 PO 4 , 0.5 M NaCl, 2 mM MgCl 2 × 6H 2 O, 0.1 mM CaCl 2 × 2H 2 O, 0.003 mM FeCl 3 × 6H 2 O, 10 mM Sodium citrate) (AB medium [12] supplemented with 10 mM citrate) in 125 ml Capsenberg flasks at 250 rpm on a rotatory shaker at 30 °C up to an optical density of 1.0 at 600 nm.…”

Section: Methodsmentioning

confidence: 99%

GeneChip expression analysis of the VqsR regulon ofPseudomonas aeruginosaTB

Juhas

Wiehlmann

Salunkhe

et al. 2005

FEMS Microbiology Letters

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Two interlinked quorum sensing circuits, las and rhl, which control pathogenesis of Pseudomonas aeruginosa are further modulated by numerous regulators, including VqsR (virulence and quorum sensing regulator). High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type and VqsR mutant in ABC minimal medium. The expression of a large group of metabolic genes, ECF sigma factors as well as of many quorum-sensing genes previously not assigned to VqsR-regulon was found to be affected by the disruption of vqsR.

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“…Moreover no more than 5% of the colonyforming units (CFU) per conjugation experiment were included in the library in order to avoid redundant clones. Pulsed-field gel electrophoresis and Southern hybridization showed that the redundancy was lower than 10% (Wiehlmann et al, 2002).…”

Section: Article In Pressmentioning

confidence: 97%

“…The flow chart provides an overview of the experimental approach. A transposon library was constructed in P. aeruginosa TBCF10839 with the plasposon pTnModOGm (Dennis and Zylstra, 1998) carrying variable signature tags (Wiehlmann et al, 2002). The mucoid strain TBCF10839 (right upper corner) had caused severe chronic lung infection in an individual with CF (see chest roentgenogram, left upper corner).…”

Section: Introductionmentioning

confidence: 99%