Cloning and analysis of the gene encoding the cytadherence phase-variable protein HMW3 from Mycoplasma pneumoniae.

We have cloned the gene encoding the Mycoplasma pneumoniae cytadherence-accessory protein HMW3 into Escherichia coli to study its phase-variable expression. A truncated HMW3 protein (HMW3'; 113 kDa), identified using HMW3-specific, affinity-purified antibodies, was expressed under the control of the lacZ promoter in lambda gt11. The protein did not react with beta-galactosidase (beta Gal)-specific antibodies, however, indicating that HMW3' was not a beta Gal fusion protein. The direction of transcription was determined by examining gene expression from inserts in opposite orientations with respect to the lacZpo in pUC18 and pUC19, to generate pKV5 and pKV6. Amino acid sequence data were obtained from an enzymatically generated HMW3 peptide fragment and used to create a degenerate 17-mer probe. The degenerate 17-mer hybridized to the mycoplasma DNA insert in pKV6; both the 17-mer and the pKV6 insert hybridized to a 9.4-kb EcoRI fragment from wild-type (wt) M. pneumoniae chromosomal DNA. This EcoRI fragment was cloned from wt M. pneumoniae and an HMW3-deficient variant in both orientations into pUC18. The HMW3'-encoding region was localized to the center of the 9.4-kb EcoRI fragment, and no differences were observed in restriction patterns between the wt and variant. Although the 9.4-kb EcoRI fragment included the DNA segment encoding HMW3', neither this protein, nor derivatives thereof, were detected in IPTG-induced E. coli containing the EcoRI fragment from either wt or variant M. pneumoniae, in either orientation in pUC18.