Characterization of <i>Rhizobium leguminosarum</i> Exopolysaccharide Glycanases That Are Secreted via a Type I Exporter and Have a Novel Heptapeptide Repeat Motif

<jats:title>ABSTRACT</jats:title> <jats:p> The <jats:italic>prsDE</jats:italic> genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from <jats:italic>Rhizobium leguminosarum</jats:italic> bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes ( <jats:italic>plyA</jats:italic> and <jats:italic>plyB</jats:italic> ) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a <jats:italic>prsD</jats:italic> (protein secretion) mutant. The <jats:italic>plyA</jats:italic> gene is upstream of <jats:italic>prsD</jats:italic> but appears to be expressed at a very low level (if at all) in cultured bacteria. A <jats:italic>plyB</jats:italic> ::Tn <jats:italic>5</jats:italic> mutant has a very large reduction in degradation of EPS and CMC. Cultures of <jats:italic>plyB</jats:italic> mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a <jats:italic>plyA plyB</jats:italic> double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca <jats:sup>2+</jats:sup> -binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS. </jats:p>