The development of nitrogen-fixing nodules of the Rhizobium-legume symbiosis, especially the early stages of root hair deformation and curling, infection thread formation, and nodule initiation, has been well studied from a genetic standpoint. In contrast, the factors important for the colonization of surfaces by rhizobia, including roots-an important prerequisite for nodule formation-have not been as thoroughly investigated. We developed conditions for analyzing the ability of two fast-growing rhizobia, Sinorhizobium meliloti and Rhizobium leguminosarum bv. viciae, to produce biofilms on abiotic surfaces such as glass, plastic microtiter plates, sand and soil as a prelude to characterizing the genes important for aggregation and attachment. Factors involved in adherence to abiotic surfaces are likely to be used in rhizobial attachment to legume root cells. In this report, we show that S. meliloti exopolysaccharide-deficient mutants as well as exopolysaccharide overproducers exhibit reduced biofilm phenotypes that show parallels with their nodulation abilities. We also investigated two flagella-less S. meliloti mutants and found them to have reduced biofilming capabilities. To investigate whether there was a symbiotic phenotype, we tested one of the Fla(-) mutants on two different S. meliloti hosts, alfalfa and white sweetclover, and found that nodule formation was significantly delayed on the latter.

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC, whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti. Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC, whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti. Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.