The Effect of an Alternate Start Codon on Heterologous Expression of a PhoA Fusion Protein in Mycoplasma gallisepticum

Abstract

While the genomes of many Mycoplasma species have been sequenced, there are no collated data on translational start codon usage, and the effects of alternate start codons on gene expression have not been studied. Analysis of the annotated genomes found that ATG was the most prevalent translational start codon among Mycoplasma spp. However in Mycoplasma gallisepticum a GTG start codon is commonly used in the vlhA multigene family, which encodes a highly abundant, phase variable lipoprotein adhesin. Therefore, the effect of this alternate start codon on expression of a reporter PhoA lipoprotein was examined in M. gallisepticum. Mutation of the start codon from ATG to GTG resulted in a 2.5 fold reduction in the level of transcription of the phoA reporter, but the level of PhoA activity in the transformants containing phoA with a GTG start codon was only 63% of that of the transformants with a phoA with an ATG start codon, suggesting that GTG was a more efficient translational initiation codon. The effect of swapping the translational start codon in phoA reporter gene expression was less in M. gallisepticum than has been seen previously in Escherichia coli or Bacillus subtilis, suggesting the process of translational initiation in mycoplasmas may have some significant differences from those used in other bacteria. This is the first study of translational start codon usage in mycoplasmas and the impact of the use of an alternate start codon on expression in these bacteria.

Fig 1. Localisation of PhoA proteins in M. gallisepticum cells.

(A) Proteins of the pTGP3 transformant were separated into hydrophobic and aqueous fractions by Triton X-114 partitioning. Equivalent amounts of the fractions were separated by SDS-PAGE in 10% polyacrylamide gels, Western transferred and probed with a monoclonal antibody against alkaline phosphatase. Lanes: W, whole cells; H, hydrophobic fraction; A, aqueous fraction. The molecular weight (MW) markers were the biotinylated protein ladder from Cell Signaling Technology. (B) The membrane and cytosolic fractions of transformant pTGP3 were separated by SDS-PAGE in a 10% polyacrylamide gel and Western transferred. Immunostaining with a monoclonal antibody to alkaline phosphatase demonstrated the presence of PhoA in the whole cells (lane W) and the membrane fraction (lane M), but not in the cytosolic fraction (lane C). (C) Whole cells of the pTGP3 transformant were treated with increasing concentrations of trypsin. The cells in lane 0 were treated with TS buffer only. Proteins were separated by SDS-PAGE in 10% polyacrylamide gels and immunostained with a monoclonal antibody against alkaline phosphatase.