Cloning, sequencing and variability analysis of the gap gene from Mycoplasma hominis

The gap gene encodes the glycolytic enzyme glyceraldehyde 3-phosphate dehyd rogenase (GAPDH). The gene was cloned and sequenced from the Mycoplasma hom inis type strain PG21(T). The intraspecies variability was investigated by inspection of restriction fragment length polymorphism (RFLP) patterns afte r polymerase chain reaction (PCR) amplification of the gap gene from lj str ains and furthermore by sequencing of parr of the gene in eight strains. Th e M. hominis gap gene was found to vary more than the Escherichia coli coun terpart, but the variation at nucleotide level gave rise to only a few amin o acid substitutions. To verify that the gene was expressed in M. hominis, a polyclonal antibody was produced and rested against whole cell protein fr om 15 strains. The enzyme was expressed in all strains investigated as a 36 -kDa protein. All strains except type strain PG21(T) showed reaction to a 1 04-kDa band in addition to the expected 36-kDa band. The protein reacting a t 104 kDa is a M. hominis protein with either an epitope similar to one on GAPDH. or it is an immunoglobulin binding protein. (C) 2000 Federation of E uropean Microbiological Societies. Published by Elsevier Science B.V. All r ights reserved.