FIG 6.

Nitrate influences the quantity and viscosity of extracellular polysaccharide (EPS) via nasA. (A) Colony morphology of R. solanacearum wild-type UW551, nasA mutant, nasA mutant complemented with pNascomp, or epsB mutant, grown on either CPG medium plates (left) or on CPG plus 40 mM nitrate (right) and imaged after 4 days of growth at 28°C. (B) Effect of nitrate supplementation and nasA mutation on viscosity of a crude EPS suspension. Colonies grown for 4 days on CPG with (+) or without (−) 40 mM nitrate were suspended in water to 10⁹ CFU/ml and centrifuged, and the viscosity of the cell-free supernatant (from 10⁹ CFU) was measured with a Brookfield model DV-1 viscometer. The viscosity shown is normalized to that of water, arbitrarily set at 1. Bars: ■, WT UW551; □, nasA mutant; , epsB mutant. Bars indicate the standard errors. Asterisks indicate significant differences from wild-type cells growing without nitrate (P < 0.05, t test). (C) Immunological quantification of EPS in supernatants using ELISA with anti-R. solanacearum EPS antibodies (OD₄₀₅). Colonies grown for 4 days on CPG with (+) or without (−) 40 mM nitrate were suspended in water to 10⁹ CFU/ml. EPS was quantified in 10⁷ CFU (10 μl) by ELISA. Asterisks indicate significant differences in OD₄₀₅ values from wild-type cells growing without nitrate (P < 0.05, t test). (D) Biochemical quantification of EPS hexosamines. Purified EPS from colonies grown for 4 days on CPG with (+) or without (−) 40 mM nitrate was quantified by using the Elson-Morgan assay with known concentrations of N-acetylgalactosamine as a standard. The total hexosamine was quantified and normalized to the amount of CFU in the samples. An asterisk indicates a significant difference from wild-type cells growing without nitrate (P = 0.0014; t test).