PMC

Kinetics of A168, A169, and A168–A169 (in 10 mM phosphate, pH 7.0). (a) Observed rate constants for A168 and A169 alone. A168 has two folding phases, one fast phase (red filled circles) and one slow, proline-isomerization-limited phase (red filled triangles). A169 has a single slow phase (blue filled circles). (b) Observed rate constants for A168–A169 compared with the single domains. Orange filled circles represent the fast phase (due to the folding of the A168 domain); orange filled triangles represent a slow phase associated with the A168 proline-isomerization-limited phase; cyan filled circles represent the slow phases associated with folding and unfolding of A169. The fits of the chevron plots for the isolated domains from (a) are shown as red broken lines for A168 and a blue broken line for A169.

Equilibrium data. (a) A164 (red filled squares), A165 (blue filled squares), A164–A165 (black open squares), and A164–Gly–Gly–A165 (green filled squares) in low-salt buffer (10 mM phosphate, pH 7.0, and 150 mM NaCl). The apparent m value of A164–A165 is approximately twice that of the isolated domains. Compare with the equilibrium curve for an equimolar mixture of A164 and A165 (black filled squares); note that these equilibrium data are shifted towards A165 due to the fluorescence signal of A165 being approximately three times that of A164. Also note that the apparent m value of the A164–Gly–Gly–A165 mutant is no longer twice that of the isolated domains, indicating a loss of cooperativity at equilibrium. (b) A168 (red filled circles), A169 (blue filled circles), and A168–A169 (black open circles) in low-salt buffer (10 mM phosphate, pH 7.0, and 0 M NaCl). The [urea]_50% of A169 increases to that of A168 in the tandem, suggesting that the domains are interacting in A168–A169. Compare with the equilibrium curve for an equimolar mixture of A168 and A169 (black filled circles). (c) A168, A169, and A168–A169 in high-salt buffer (10 mM phosphate, pH 7.0, and 500 mM NaCl). The [urea]_50% of A169 is slightly higher than that of A168. The [urea]_50% value of the tandem lies approximately midway between that of A168 and A169, as would be expected if the domains were not interacting.

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.

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.