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Cloning and analysis of the gene encoding the cytadherence phase-variable protein HMW3 from Mycoplasma pneumoniae
K F Ogle1, K K Lee, D C Krause
1Department of Microbiology, University of Georgia, Athens 30602.
Abstract:
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.
Insights
Researchers cloned the Mycoplasma pneumoniae HMW3 gene into E. coli to study its expression. Despite successful cloning and identification of a truncated HMW3 protein, it was not detected in induced E. coli, suggesting challenges in expressing this cytadherence protein.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mycoplasma pneumoniae is a pathogen that causes respiratory infections.
- The cytadherence-accessory protein HMW3 is crucial for M. pneumoniae attachment to host cells.
- Understanding the expression of HMW3 is important for developing strategies to combat M. pneumoniae infections.
Purpose of the Study:
- To clone the gene encoding Mycoplasma pneumoniae HMW3 into Escherichia coli.
- To investigate the phase-variable expression of HMW3.
- To characterize the expressed HMW3 protein and its encoding gene.
Main Methods:
- Cloning of the HMW3 gene into lambda gt11 and pUC vectors.
- Expression studies using lacZ promoter and IPTG induction.
- Protein identification using HMW3-specific antibodies.
- DNA hybridization and restriction fragment analysis.
- Sequencing of HMW3 peptide fragments.
Main Results:
- A truncated HMW3 protein (HMW3') was expressed in E. coli but was not a beta-galactosidase fusion.
- The HMW3-encoding region was localized to a 9.4-kb EcoRI fragment of M. pneumoniae DNA.
- No differences in restriction patterns were observed between wild-type and HMW3-deficient M. pneumoniae DNA.
- HMW3' or its derivatives were not detected in induced E. coli containing the cloned fragment.
Conclusions:
- The cloning and initial characterization of the HMW3 gene in E. coli were successful.
- Challenges exist in expressing functional HMW3 protein in E. coli, hindering further study of its phase-variable expression.
- Further investigation is needed to understand the reasons for the lack of detectable HMW3 protein expression in E. coli.