A rapid and simple method for inactivating chromosomal genes in<i>Yersinia</i>

Derbise, Anne; Lesic, Biliana; Dacheux, Denis; Ghigo, Jean Marc; Carniel, Élisabeth

doi:10.1016/s0928-8244(03)00181-0

Cited by 218 publications

(207 citation statements)

References 10 publications

(17 reference statements)

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“…A kanamycin cassette was obtained as a BamHI fragment from pGEMTFRTKM (15) and was cloned into BamHI-digested pGEMTΔlpxO to generate pGEMTΔlpxOKm. The ΔlpxO::Km allele was PCR amplified using primers LpxOUPF and LpxODOWNR and 1 μg of PCR product was electroporated into either Kp51245 or 52145-Δwca K2 harboring the lambda-red protein-encoding plasmid pKOBEG-sacB (47). Mutants were selected onto LB agar containing kanamycin and a recombinant in which the wild-type allele was replaced by the mutant one and was selected and named 52145-ΔlpxOKm and 52145-Δwca K2 -ΔlpxOKm.…”

Section: Methodsmentioning

confidence: 99%

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

Llobet

Martínez-Moliner

Moranta

et al. 2015

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

134

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The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

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Section: Methodsmentioning

confidence: 99%

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

Llobet

Martínez-Moliner

Moranta

et al. 2015

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

134

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“…The construction of an in-frame replacement of pmrL with a kanamycin resistance cassette (kan) was based on the method of Derbise et al (46). A pmrA c derivative of E. coli strain DY330 (47), designated MST100 (28), which contains a prophage harboring the recombination genes exo, bet, and gam under control of a temperature-sensitive cI-repressor, was utilized as the parental strain.…”

Section: Methodsmentioning

confidence: 99%

An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin Resistance in Escherichia coli

Yan¹,

Guan²,

Raetz

2007

Journal of Biological Chemistry

135

136

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Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coli, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A and renamed these genes arn-BCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrA c parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. The lipid A moiety of lipopolysaccharide (LPS) 4 in the outer membranes of Gram-negative bacteria typically consists of a ␤,1Ј-6-linked disaccharide of glucosamine, modified with phosphate groups at the 1-and 4Ј-positions, and with R-3-hydroxyacyl chains at the 2-, 3-, 2Ј-, and 3Ј-positions (Fig.

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“…A further approach to impede protein degradation in the absence of a protease inhibitor cocktail is the deletion of genes encoding proteases such as OmpT and Lon in EcN to construct a suitable strain for protein expression comparable to E. coli K-12 strains BL21 (DE3) and KRX (Derbise et al 2003). This will be part of the ongoing work to optimize defensin production by EcN.…”

Section: )mentioning

confidence: 99%