Purification and characterization of a novel esterase (?-hydroxypalmitate methyl ester hydrolase) and prevention of the expression of virulence by Ralstonia solanacearum

Abstract

Aims: To screen novel micro‐organisms and enzymes capable of degrading 3‐hydroxypalmitic acid methyl ester (3‐OH PAME), the quorum‐sensing signal molecule (quormone), which regulates the virulence of Ralstonia solanacearum. Methods and Results: Ideonella sp. 0‐0013, a betaproteobacterium isolated from soil using the selective‐enrichment culture method, was grown on plates containing 3‐OH PAME as its main carbon source. β‐Hydroxypalmitate methyl ester hydrolase (βHPMEH) purified from the supernatant of the Ideonella sp. 0‐0013 culture exhibited high hydrolysing activity towards the ester bond of 3‐OH PAME and eliminated the 3‐OH PAME activity, thereby reducing the virulence of R. solanacearum. An Escherichia coli transformant of the βhpmeh gene expression vector degraded 3‐OH PAME, and the crude enzyme from the transformant inhibited in vitro production of the R. solanacearum exopolysaccharide (EPS). Conclusions: The ability of βHPMEH to hydrolyse 3‐OH PAME inhibited the production of EPS by the R. solanacearum wild‐type strain, indicating that βHPMEH inhibits the effects of activation of virulence genes. This ability will be potentially useful for pest control of the wilt disease caused by this bacterium. Significance and Impact of the Study: This enzyme is the first protein that has been found to degrade a quormone other than N‐acyl homoserine lactone.

A Cell Wall–Degrading Esterase of Xanthomonas oryzae Requires a Unique Substrate Recognition Module for Pathogenesis on Rice

“…Xanthomonas campestris pv campestris (strain ATCC 33913) XCC2374 has Thr in place of Gly-231 of LipA, although the strain has an additional LipA-like protein XCC2957 that retains Gly-231. A LipA-like protein in the soil bacterium Ideonella, with 27% identity to LipA, has been functionally characterized recently (Shinohara et al, 2007). Interestingly, it has been shown to degrade b-hydroxy palmitic (C16) acid methyl ester.…”

Ideonella azotifigens sp. nov., an aerobic diazotroph of the Betaproteobacteria isolated from grass rhizosphere soil, and emended description of the genus Ideonella

“…Strain 0-0013 was isolated from soil on media containing the quorum-sensing signal molecule 3-hydroxypalmitic acid methyl ester as the sole carbon source and was shown to degrade this compound (Shinohara et al, 2007). Three strains, B508-1, B511 and B513, isolated from the stems of cultivated rice plants on an N-free medium containing mannitol, sucrose, lactate and malate, have been shown to reduce acetylene and have been proposed as possible endophytes (Elbeltagy et al, 2001).…”

Evaluación del gen que codifica la enzima βHPMEH para la inhibición de la marchitez bacteriana causada por Ralstonia solanacearum

“…FERM BP-08660 (accesión: BAF64544.1) esto debido a que nuestra secuencia tiene una sustitución de nucleó-tido que llevó a la codificación de una Prolina (P) a una Serina (S), sin embargo esta no se situó dentro del sitio activo y por lo tanto puede que no afecte su actividad enzimática (datos no mostrados). Además, se comprobó que la secuencia de la proteína βHPMEH conserva el motivo consenso G-X-S-X-G entre todas las serine esterasas (Brenner 1988, Shinohara et al 2007) y que el motivo conservado se encuentra ubicado cercano al centro de toda la secuencia proteíca. Además ésta es una característica importante en enzimas que poseen motivos conteniendo Serina (Akoh et al 2004).…”

“…Ralstonia solanacearum posee un sistema quorum sensing para la regulación de la expresión de estos genes de virulencia, siendo la molécula 3-hydroxy-palmitic acid methyl ester (3-OH-PAME) el auto regulador de esta red (Flavier et al 1997). Esta característica convierte a la molécula 3-OH-PAME, en un potencial blanco para el desarrollo de inhibidores de los factores de virulencia (Shinohara et al 2007). El sistema de quorum quenching (interceptación del quorum) actúa bloqueando distintos pasos implicados en quorum sensing, tales como la generación del agente autoinductor, la acumulación del agente señal, o bien la recepción de la señal (Dong et al 2007).…”

“…El sistema de quorum quenching (interceptación del quorum) actúa bloqueando distintos pasos implicados en quorum sensing, tales como la generación del agente autoinductor, la acumulación del agente señal, o bien la recepción de la señal (Dong et al 2007). Shinohara et al (2007) purificaron y caracterizaron la molécula β-Hydroxypalmitate methyl ester hydrolase (βHPMEH), aislada a partir de Ideonella sp. La enzima βHPMEH hidroliza a la molécula 3-OH-PAME en 3-OH ácido palmítico y metanol, y anula así la señal quorum de R. solanacearum.…”