Characterization of Rhizobium leguminosarum exopolysaccharide glycanases that are secreted via a type I exporter and have a novel heptapeptide repeat motif

Abstract

The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum 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 (plyA and plyB) 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 prsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. A plyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyB 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 plyA plyB 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 Ca2+-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.

FIG. 1

(a) Map of the 9.6-kb EcoRI fragment carring the prsDE genes. Downstream from prsDE and ORF3 and in the opposite orientation are four genes (pssFCDE) homologous to glycosyl transferases in R. leguminosarum bv. trifolii (45) and R. meliloti (19). Upstream from prsDE is plyA, encoding a putative polysaccharide lyase. (b) The plyB gene is located in a 3.6-kb EcoRI fragment. Tn5 insertions identified in prsD, plyA, and plyB are indicated by solid circles. E, EcoRI; B, BamHI.

FIG. 2

Degradation of CMC by ply mutants. CMC degradation is estimated by the lack of staining (seen as cleared regions) by Congo Red in the agar directly below the colonies. (a) Comparison of the CMC degradation by 8401 (wild-type control), A632 (plyA), A616 (plyB), A408 (prsD), and A636 (plyA plyB) on CMC agar. (b) Introduction of pIJ7647 (carrying plyB) results in increased degradation of CMC by A616 (plyB) but not by A408 (prsD). (c) Introduction of pIJ7871 (carrying plyA) causes strong degradation by A616 (plyB) but not by A408 (prsD). (d) Egl from A. caulinodans, expressed from pRGC1, causes increased CMC degradation by 8401 and A616 (plyB) but not by A408 (prsD). (e) Similar results were obtained with Egl expressed from pGV910-C1, except that the CMC degradation extended beyond the diameter of the colony.

FIG. 3

Two Coomassie blue-stained SDS-PAGE gels showing secreted proteins from R. leguminosarum bv. viciae mutants. Culture supernatant proteins were precipitated from the prsD mutant A412 (lane b), wild-type (WT) 8401(pRL1JI) (lanes c and d), the plyA mutants A501 (lane e) and A503 (lane f), and the plyB mutant A617 (lane g). Molecular size markers (94, 67, 43, and 30 kDa) are shown in lanes a and h. The strains were grown in the absence of the nod gene inducer hesperetin. Proteins present in 8401(pRL1JI) but absent from the secretion mutant A412 are indicated in lanes c and d. One of these proteins (arrowhead) is apparently also absent from A617 (lane g). The gel containing lanes a, b, and c was run slightly differently from the other gel; the lines highlight the positions of the proteins that were absent from the supernatant of prsD mutants.

FIG. 4

Nodulation of vetch by 8401 pRLIJI (▪), the plyA mutant A638 (◊), the plyB mutant A617 (⧫), and the double mutant A640 (plyA plyB) (▵). Standard errors are shown.

FIG. 5

(a) Repeat motifs found in PlyA, PlyB, and SpsR. The position in the sequence of the first amino acid in each repeat is indicated. A rough consensus sequence is shown. PlyA and PlyB each contain 10 copies of the motif; SpsR contains 6 copies. (b) Sequence alignment of PelC from E. chrysanthemi with PlyA and PlyB from R. leguminosarum bv. viciae, showing conservation of secondary structure. The mature form of PelC (lacking the N-terminal signal peptide) was used for the alignment. Residues that are conserved in all three proteins (○) and between PelC and either PlyA or PlyB (·) are indicated beneath the alignment. Structural elements are indicated in boldface type, with α-helices in italics and β-strands underlined. The secondary structure of PelC is known from the crystal structure (49). The β-strands that form the PB2 β-sheet in PelC are indicated. The residues which form stacks stabilizing the parallel β-helix of PelC are indicated by asterisks above the sequence. The stacks are composed mainly of Asn residues, which occur as the second residue after the end of the PB2 β-strands. The secondary structure of PlyA and PlyB was predicted by PHDsec (34) and aligns well with the known structure of PelC, suggesting that these proteins may have a similar fold.