Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component

Abstract

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

FIG. 1

Activation of glycanase by cells of mutants defective for extracellular glycanase production. In panels a and b, colonies of strain 8401/pRL1JI were grown adjacent to the protein secretion mutant A412 (prsD) or the glycanase mutant A640 (plyA plyB); the cells were grown for 3 days on Y medium containing CMC. In panels c and d, these strains were grown on the same medium that had been seeded with a lawn of A412 and incubated for 4 days. After growth, the cells were washed off, and the plates were stained with Congo red; the unstained regions correspond to areas where the CMC has been degraded.

FIG. 2

EPS⁻ mutants are defective for CMC degradation. Growth and staining conditions were as in Fig. 1a and b. The EPS-defective mutants A168, A507, and A517 have much less CMC degradation than their parent 8401/pRL1JI and have similar or lower levels of CMC degradation than the glycanase secretion mutant A412.

FIG. 3

The EPS-deficient mutant A168 can induce CMC degradation below an adjacent (EPS⁺) colony. Growth and staining conditions were as in Fig. 1a and b. Strain A168 produces no EPS, and A550 is defective for both EPS production and protein secretion. A168 but not A550 induced CMC degradation below part of the adjacent colony of the secretion mutant A412.

FIG. 4

Cross-stimulation of glycanase activity among EPS⁻, glycanase secretion, and glycanase-defective mutants. Cells, as indicated, were grown on CMC-agar (a), CMC-agar seeded with a lawn of the secretion mutant A412 (prsD) (b), and CMC-agar seeded with the EPS⁻ mutant A168 (pssA) (c). Growth and staining was as described for Fig. 1.

FIG. 5

Assays of EPS degradation by glycanase, secretion, and EPS⁻ mutants. The assays were carried out as described for CMC degradation (Fig. 1), except that EPS replaced the CMC in the agar. In panel a the colonies were grown on EPS-agar. The degradation below 8401/pRL1JI did not extend beyond the colony; there was a low level of degradation seen with the glycanase mutant A640 (plyA plyB) and the secretion mutant A412 (prsD), but none was seen with the EPS⁻ mutant A168. In panel b the EPS-agar was seeded with a lawn of A412 (prsD), and in panel c there was a lawn of A640 (plyA plyB).

FIG. 6

plyB but not plyA encodes a diffusible glycanase. CMC degradation was assayed using CMC-agar (a and c) or CMC agar seeded with a lawn of the protein secretion mutant A412 (b). Growth and staining were as in Fig. 1. The strains used are the parental strain 8401/pRL1JI and its derivatives carrying mutations in plyA (A638), plyB (A600), or both plyA and plyB (A640) and A640 derivatives carrying cloned plyA (on pIJ7871) or plyB (on pIJ7709). In panel c, the A640 cells carrying cloned plyA or plyB were cultured at various distances from A412 to get a measure of the relative distance of cross activation by PlyB.

FIG. 7

plyB cloned in A. tumefaciens produces a glycanase that is not detected unless cells of R. leguminosarum are present. CMC degradation was assayed using CMC-agar (a) or CMC-agar seeded with a lawn of the protein secretion mutant A412 (b). In panel a, A. tumefaciens strain induces no CMC degradation even if it carries plyB cloned on pIJ7709, but plyB-dependent activity can be seen in panel b, where a lawn of A412 is present. Assay conditions are as described in Fig. 1.

FIG. 8

CMC degradation by various rhizobia. CMC degradation was assayed using CMC-agar (a), CMC-agar seeded with a lawn of the protein secretion mutant A412 (b), or CMC-agar seeded with a lawn of R. meliloti 1021 (c). The strains used are R. leguminosarum bv. viciae 8401/pRL1JI, R. etli CE3, R. leguminosarum bv. trifolii RCR5 and ANU843, R. leguminosarum bv. viciae 3855 and VF39, R. tropici CIAT899, S. fredii USDA193, R. meliloti 1021, Rhizobium sp. strain NGR234, and R. loti NZP2213.