A new Bradyrhizobium japonicum gene required for free-living growth and bacteroid development is conserved in other bacteria and in plants.

Abstract

In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, a new DNA region, orf74, was discovered which is required for optimal free-living growth and, by consequence, also necessary for the formation of an effective symbiosis. A Tn5-233 insertion of orf14 resulted in a mutant, strain 74, that has a reduced growth rate in free-living cultures under all conditions tested and less than 1% residual symbiotic nitrogen fixation activity as compared with the wild type. Nodule distribution and nodule morphology are severely affected similarly as in a previously characterized B. japonicum nifA mutant. Protein databank searches revealed that the 142-amino-acid protein encoded by orf74 is homologous to a 161-amino-acid protein encoded by orf17 of Bacillus subtilis (approximately 46% identity; J. C. R. Struck, R. Kretschmer-Kazemi Far, W. Schröder, F. Hucho, H. Y. Toschka, and V. A. Erdmann, Biochim. Biophys. Acta, 1050:80-83, 1990) as well as to a 178-amino-acid protein encoded by orf178 of Escherichia coli (approximately 45% identity; K. L. Poulsen, N. W. Larsen, S. Molin, and P. Andersson, Mol. Microbiol., 6:895-905, 1992). Significant similarity was also found with unknown ORFs of two plant species. When expressed from a strong constitutive promoter, orf17 of B. subtilis could partially complement B. japonicum mutant 74. By contrast, orf74 of B. japonicum was unable to functionally complement an E. coli orf178 mutant. The conservation of this DNA region in gram-negative and gram-positive bacteria suggests that the gene is essential for a fundamental cellular process which is required in B. japonicum for both free-living and symbiotic growth.