Downloaded from www.microbiologyresearch.org by IP: 54.144.54.146 On: Fri, 29 Jan 2016 16:33:47 Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues Maarten Fauvart, Natalie Verstraeten, Bruno Dombrecht,3 Ruth Venmans,4 Serge Beullens, Christophe Heusdens1 and Jan Michiels Correspondence Jan Michiels jan.michiels@biw.kuleuven.be Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, B-3001 Leuven, Belgium Received 16 January 2009 Revised 25 May 2009 Accepted 27 May 2009 While establishing a nitrogen-fixing symbiosis with leguminous plants, rhizobia are faced with the problem of penetrating the plant cell wall at several stages of the infection process. One of the major components of this barrier is pectin, a heteropolysaccharide composed mainly of galacturonic acid subunits. So far, no enzymes capable of degrading pectin have been isolated from rhizobia. Here, we make an inventory of rhizobial candidate pectinolytic enzymes based on available genome sequence data and present an initial biochemical and functional characterization of a protein selected from this list. Rhizobium etli hrpW is associated with genes encoding a type III secretion system, a macromolecular structure that allows bacteria to directly inject so-called effector proteins into a eukaryotic host’s cell cytosol and an essential virulence determinant of many Gram-negative pathogenic bacteria. In contrast to harpin HrpW from phytopathogens, R. etli HrpW possesses pectate lyase activity and is most active on highly methylated substrates. Through comparative sequence analysis, three amino acid residues crucial for the observed enzymic activity were identified: Trp192, Gly212 and Gly213. Their importance was confirmed by site-directed mutagenesis and biochemical characterization of the resulting proteins, with the tryptophan mutant showing no detectable pectate lyase activity and the double-glycine mutant’s activity reduced by about 80 %. Surprisingly, despite hrpW expression being induced specifically on the plant root surface, a knockout mutation of the gene does not appear to affect symbiosis with the common bean Phaseolus vulgaris. INTRODUCTION Rhizobia are a group of soil-dwelling, Gram-negative bacteria that can induce and infect specialized organs on the roots of leguminous host plants. Inside these so-called root nodules, the bacteria can fix atmospheric nitrogen for the host’s benefit in exchange for plant-derived carbon sources (Gage, 2004). Upon detection of flavon- oids indicative of a compatible host, rhizobia chemotac- tically migrate towards the plant roots. During root surface colonization, the bacteria secrete Nod factors. These lipochitooligosaccharides cause the root cortex cells to reinitiate cell division, giving rise to nodule primordia, while simultaneously eliciting early Nod factor responses such as calcium spiking and root hair cytoskeleton modification. Root hair curling allows the attached bacteria to settle within the deformation and enter an invagination in the cell membrane, called the infection thread, that grows inwardly toward the base of the root hair and nodule primordium. Once they reach the dividing nodule cells, the bacteria exit from the infection thread by a process resembling endocytosis. They subsequently undergo physiological, structural and mor- phological changes and differentiate into nitrogen-fixing bacteroids. During root infection and invasion, there are at least two stages at which the plant cell wall must be breached (Brewin, 2004; Gage, 2004). First, during infection thread initiation. This happens when bacteria become entrapped between two root hair cell walls. Infection thread ingrowth is believed to start after localized cell wall degradation at the site of infection. Secondly, during infection thread exit. As infection threads are bounded by plant cell wall material such as cellulose and pectin, this barrier has to be passed prior to rhizobial endocytosis and symbiosome Abbreviations: PGA, polygalacturonic acid; T3SS, type III secretion system. 3Present address: Ablynx, Technologiepark 4, B-9052 Ghent, Belgium. 4Present address: Group T – Leuven Engineering College (Association K.U.Leuven), Andreas Vesaliusstraat 13, B-3000 Leuven, Belgium. 1Present address: Scientia Terrae, Fortsesteenweg 30A, B-2860 St- Katelijne-Waver, Belgium. Microbiology (2009), 155, 3045–3054 DOI 10.1099/mic.0.027599-0 027599 G 2009 SGM Printed in Great Britain 3045