Cloning, nucleotide sequencing, and expression in Escherichia coli of a Rhizobium leguminosarum gene encoding a symbiotically repressed outer membrane protein

Abstract

We describe the cloning of a gene from Rhizobium leguminosarum biovar viciae strain 248 encoding protein IIIa, the 36-kDa outer membrane protein forming a part of the outer membrane protein antigen group III. The expression of this antigen group is repressed in the bacteroid form during symbiosis (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). A cosmid clone expressing the strain 248-specific MAb38 epitope of this antigen group in a nonrelated strain was selected by a colony blot assay. Sequencing revealed one large open reading frame encoding a 39-kDa protein. N-terminal amino acid sequencing of the purified 36-kDa outer membrane protein IIIa revealed that the isolated gene, now designated ropA, is the structural gene for this protein and that the mature protein was formed by processing of the 22-residue N-terminal signal sequence. The gene is preceded by a promoter that was active in R. leguminosarum but not in Escherichia coli. This promoter, which showed no homology to known promoter sequences, was located approximately by determination of the transcription start site. The region upstream of the putative promoter was shown to contain two potential binding sites for integration host factor protein. Expression of protein IIIa under control of the inducible lac promoter in E. coli shows that, of its earlier described properties, the peptidoglycan linkage of protein IIIa is specific for R. leguminosarum but that outer membrane localization and calcium-stabilized oligomer formation can to a large extent also occur in E. coli.