Sinorhizobium fredii USDA257 Releases a 22-kDa Outer Membrane Protein (Omp22) to the Extracellular Milieu When Grown in Calcium-Limiting Conditions

Calcium, which regulates a wide variety of cellular functions, plays an important role in Rhizobium-legume interactions. We investigated the effect of calcium on surface appendages of Sinorhizobium fredii USDA257. Cold-field emission scanning electron microscopy observation of USDA257 grown in calcium-limiting conditions revealed cells with unusual shape and size. Transmission electron microscopy observation revealed intact flagella were present only when USDA257 cells were grown in calcium-sufficient conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of flagellar preparations from USDA257 cells grown in calcium-limiting conditions showed the presence of a 22-kDa protein that was absent from cells grown in calcium-sufficient conditions. We have cloned and determined the nucleotide sequence of the gene encoding the 22- kDa protein. After successful expression in Escherichia coli, polyclonal antibodies were raised against the recombinant 22-kDa protein (Omp22). Subcellular fractionation analysis demonstrated that Omp22 was predominantly present in the extracellular fraction. Western blot analysis revealed the presence of immunologically related proteins from diverse rhizobia. Immunocytochemical localization of thin sections of USDA257 cells showed specific labeling of protein A-gold particles on protein inclusions found proximal to the cells. Accumulation of Omp22 was greatly reduced when USDA257 cells were grown in the presence of increasing calcium. Northern blot analysis indicated that calcium was the only divalent cation among those tested that down-regulated omp22 expression. An omp22 mutant was able to grow in calcium-limiting conditions at a rate similar to that of wild-type USDA257. Significantly more nodules were initiated by the omp22 mutant than by the wild-type on soybean cultivar Peking grown in calciumlimiting conditions.