Restoration of cytoadherence to an adherence-deficient mutant of Mycoplasma arthritidis by genetic complementation.
Mycoplasma arthritidis causes a severe septic arthritis in rats under natural and experimental conditions. An earlier study implicated a membrane lipoprotein designated MAA1 in cytadherence of M. arthritidis. In addition, a spontaneous adherence-deficient mutant was shown to contain a nonsense mutation in the gene encoding MAA1, resulting in production of a truncated product, MAA1Delta. In the present study, a wild-type maa1 gene carried on transposon Tn4001T was introduced into the low-adherence mutant by polyethylene glycol-mediated transformation. The presence of the tranposon and the wild-type maa1 gene in the chromosome of transformants was confirmed by PCR and Southern hybridization. The latter procedure also confirmed that each transformant contained a single copy of the transposon. Western immunoblotting showed that transformants produced both wild-type MAA1 and MAA1Delta, indicating that the introduced wild-type maa1 gene was functional. This phenotype was stably maintained after multiple subcultures even in the absence of antibiotic selection. Finally, transformants were shown to adhere to rat L-2 lung cells in culture at wild-type levels, providing confirmation for an important role for MAA1 in adherence.
Washburn
Leigh R
LR
Division of Basic Biomedical Sciences, University of South Dakota, Vermillion 57069, USA. lwashbur@usd.edu
Bird
Daniel W
DW
Dybvig
Kevin
K
D000818:Animals; D001422:Bacterial Adhesion / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics; D005816:Genetic Complementation Test; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009174:Mycoplasma / Q000235:genetics / Q000502:physiology*; D051381:Rats; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D008074:Lipoproteins; D008565:Membrane Proteins