A TNPHOA INSERTION WITHIN THE BRADYRHIZOBIUM-JAPONICUM SIPS GENE, HOMOLOGOUS TO PROKARYOTIC SIGNAL PEPTIDASES, RESULTS IN EXTENSIVE CHANGESIN THE EXPRESSION OF PBM-SPECIFIC NODULINS OF INFECTED SOYBEAN (GLYCINE-MAX) CELLS

Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA muta genesis of strain 110spc4. A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment, DNA sequence analysis of a 3213 bp BamHI-Clal fragment revealed that three open reading frames (ORFs) were encoded in the same orientation, Based on sequence similarities t o other proteins in the database, the second ORF was called sipS (sign al peptidase). The TnphoA insertion in mutant 132 was found to be in f rame near the 3 ' end of sipS. The resulting SipS-PhoA hybrid polypept ide was shown to be expressed in free-living B. japonicum and in Esche richia coli cultures. An immunoblot analysis with a polyclonal antibod y directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide, A much stronger 52 kDa band was also detected. Mutant 132 was specifi cally disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids wer e not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroi d membrane indicated a lower expression of these proteins, The mutant phenotype was genetically complemented by a 4.4 kb BamHI fragment incl uding sipS. Subfragments thereof also complemented a temperature-sensi tive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lep(ts) in E. coli. Genetic studies sugges ted that the three genes are organized in one common operon which is e xpressed from a promoter upstream of the sequenced region, Inactivatio n of the gene downstream of sipS did not result in a detectable phenot ype.