Genetic characterization of a Sinorhizobium meliloti chromosomal region involved in lipopolysaccharide biosynthesis

The genetic characterization of a 5.5-kb chromosomal region of Sinorhizobiu m meliloti 2011 that contains lpsB, a gene required for the normal developm ent of symbiosis with Medicago spp., is presented. The nucleotide sequence of this DNA fragment revealed the presence of six genes: greA and lpsB, tra nscribed in the forward direction; and lpsE, lpsD, lpsC, and lrp, transcrib ed in the reverse direction. Except for lpsB, none of the lps genes were re levant for nodulation and nitrogen fixation. Analysis of the transcriptiona l organization of lpsB showed that greA and lpsB are part of separate trans criptional units, which is in agreement with the finding of a DNA stretch h omologous to a "nonnitrogen" promoter consensus sequence between greA and l psB. The opposite orientation of lpsB with respect to its first downstream coding sequence, lpsE, indicated that the altered LPS and the defective sym biosis of lpsB mutants are both consequences of a primary nonpolar defect i n a single gene. Global sequence comparisons revealed that the greA-lpsB an d lrp genes of S. meliloti have a genetic organization similar to that of t heir homologous loci in R. leguminosarum by. viciae. In particular, high se quence similarity was found between the translation product of lpsB and a c ore related biosynthetic mannosyltransferase of R. leguminosarum by. viciae encoded by the lpcC gene. The functional relationship between these two ge nes was demonstrated in genetic complementation experiments in which the S. meliloti lpsB gene restored the wild-type LPS phenotype when introduced in to lpcC mutants of R. leguminosarum. These results support the view that S. meliloti lpsB also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core. Evidence is provided for the presence of othe r lpsB-homologous sequences in several members of the family Rhizobiaceae.