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1.
J Bacteriol ; 194(21): 5922-31, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22942245

RESUMO

Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimX(XAC2398) from Xanthomonas axonopodis pv. citri interacts with PilZ(XAC1133) directly through the nonenzymatic EAL domain of FimX(XAC2398). Here we present experimental data to reveal that the strong interaction between FimX(XAC2398) and PilZ(XAC1133) is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimX(XAC2398) was identified as the binding surface for PilZ(XAC1133) by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species.


Assuntos
Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Mapeamento de Interação de Proteínas , Pseudomonas aeruginosa/fisiologia , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície
2.
Chembiochem ; 12(18): 2753-8, 2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-22021215

RESUMO

Messenger bagged: The design of a fluorophore-labeled protein biosensor for the bacterial messenger cyclic di-GMP is described. The biosensor responds to c-di-GMP with sub-micromolar sensitivity in a real-time fashion. The biosensor can be used for enzyme assays for diguanylate cyclases and c-di-GMP phosphodiesterases as well as the high-throughput screening of inhibitors.


Assuntos
Técnicas Biossensoriais , GMP Cíclico/química , Corantes Fluorescentes/química , Concentração Inibidora 50 , Modelos Moleculares , Espectrometria de Fluorescência
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