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A Tn phoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM‐specific nodulins of infected soybean ( Glycine max ) cells
Author(s) -
Müller P.,
Ahrens K.,
Keller T.,
Klaucke A.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.18050831.x
Subject(s) - biology , bradyrhizobium japonicum , homologous chromosome , gene , signal peptide , gene expression , microbiology and biotechnology , genetics , bacteria , rhizobiaceae , peptide sequence , symbiosis
Bradyrhizobium japonicum mutant 132 was obtained by random Tn phoA mutagenesis of strain 110 spc 4. A 6.5 kb Bam HI 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 Bam HI— Cla I fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The Tn phoA insertion in mutant 132 was found to be in frame near the 3′ end of sipS . The resulting SipS—PhoA hybrid polypeptide was shown to be expressed in free‐living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody 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 specifically disturbed in the interaction with soybean ( Glycine max ) when the bacteria were released from the infection threads. The bacteroids were 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 peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by 4.4 kb Bam HI fragment including sipS . Subfragments thereof also complemented a temperature‐sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lep ts in E. coli . Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype.