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Expression in Mycoplasma pneumoniae of the recombinant gene encoding the cytadherence‐associated protein HMW1 and identification of HMW4 as a product
Author(s) -
Hahn TaeWook,
Krebes Kathleen A.,
Krause Duncan C.
Publication year - 1996
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 - 10.1046/j.1365-2958.1996.455985.x
Subject(s) - biology , gene , recombinant dna , mycoplasma pneumoniae , gene product , identification (biology) , mycoplasma , genetics , gene expression , virology , botany , history , archaeology , pneumonia
Mycoplasma pneumoniae is a major cause of tracheobronchitis and pneumonia in older children and young adults. The lack of adequate tools for genetic analysis has hindered the elucidation of function and regulation of mycoplasma virulence determinants. We describe here the use of a transposon vector to deliver the cloned gene for the cytadherence‐associated protein HMW1 in M. pneumoniae . A 4.95 kbp Bam HI fragment encoding all but the C‐terminal end of HMW1 was cloned into a modified Tn 4001 and transformed into wild‐type M. pneumoniae and into a non‐cytadhering mutant lacking HMW1–HMW5. Southern blot hybridizations confirmed insertion of the transposon and the presence of both the resident and recombinant hmw1 alleles. Analysis by Western immunoblotting revealed a truncated HMW1 (HMW1′) in the transformants, the level of HMW1′ being dependent upon the orientation of the hmw1 gene in the transposon and the site of insertion. Similar expression patterns were noted in wild‐type and mutant backgrounds. However, expression of wild‐type levels of HMW1′ in the mutant did not restore adherence. Finally, HMW4 and HMW1 were shown to be products of the same gene, HMW4 being a heat‐modified derivative of HMW1.