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Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

Bruno Dombrecht

Ruth Venmans

Christophe Heusdens

Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, B-3001 Leuven, Belgium

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.

Correspondence
Jan Michiels
jan.michiels{at}biw.kuleuven.be

Present address: Ablynx, Technologiepark 4, B-9052 Ghent, Belgium.

Present address: Group T – Leuven Engineering College (Association K.U.Leuven), Andreas Vesaliusstraat 13, B-3000 Leuven, Belgium.

Present address: Scientia Terrae, Fortsesteenweg 30A, B-2860 St-Katelijne-Waver, Belgium.