Abstract
Thin-layer chromatographic analysis of extracts of D-[1-14C]glucosamine-labelled rhizobia was used to analyze the effects of nodI, nodJ, and nodT on secretion of lipochitin oligosaccharide (LCO) signal molecules. Secretion was analyzed by comparing quantities of radiolabelled LCOs present in the cellular and spent growth medium fractions. A second rapid and sensitive method was introduced to estimate the secreted LCO fractions by using D-[1-14C]glucosamine-labelled cells grown in medium supplemented with chitinase. At various times after induction of LCO synthesis, the quantity of degradation products of LCOs was compared with the amount of nondegraded LCOs. In wild-type strains of Rhizobium leguminosarum biovars viciae and trifolii the nodI and nodJ genes (but not the nodT gene) strongly enhance the secretion of LCOs during the first 5 h after the induction of LCO synthesis. In LCO-overproducing strains the enhancement of secretion was observed only during the first 3 h after induction. At times later than 5 h after induction, a significant influence of the presence of the nodI and nodJ genes on LCO secretion was detectable neither in the wild type nor in LCO-overproducing strains. By using plasmids in which the nodI and nodJ genes are cloned separately under control of a flavonoid-inducible promoter, it was shown that both genes are needed for a wild-type level of LCO secretion. Therefore, these results demonstrate that nodI and nodJ play a role in determining the efficiency of LCO secretion.