A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola.

Chao Wei,Jun Deng,John Maxwell Dow,Mengran Zhao,Wenxian Sun,Xin Zeng,Wendi Jiang,Dongli Jin,Junjie Ling

doi:10.1038/srep23769

Abstract

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Highlights

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria
We further demonstrated that in vitro purified XOC_4190 and XOC_2102 were enzymatically inactive, but were able to bind to c-di-GMP with high affinity
The results indicate that the EAL domain, but not the GGDEF domain is required for binding of c-di-GMP by XOC_4190

Summary

Introduction

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed from two GTP molecules by diguanylate cyclases (DGCs) that have a GGDEF domain and is broken into pGpG or GMP by phosphodiesterases (PDEs) containing either an EAL or HD-GYP domain[4] These domains involved in c-di-GMP metabolism are widely present in Gram-negative bacterial proteins. A major sub-group of proteins involved in c-di-GMP signaling contain both GGDEF and EAL domains arranged in tandem[13] Several such proteins have been demonstrated to have both DGC and PDE enzymatic activities; for example MsDGC-1 in Mycobacterium smegmatis[14], Lpl0329 in Legionella pneumophila[15], and ScrC in Vibrio parahemeolyticus[16]. LapD from Pseudomonas fluorescens serves as a high affinity c-di-GMP receptor via a degenerate EAL domain and controls biofilm formation through regulating localization of the large cell surface adhesin LapA19,20 Another GGDEF-EAL protein Filp in Xanthomonas oryzae pv. No functional study on GGDEF and/or EAL domain-containing proteins in Xoc has been reported so far

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Conclusion