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

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

Degradation of Alkanes in Rhodococcus

Alkanes are widely distributed in the environment as they not only constitute the large fraction of crude oil but are also produced by many living organisms. They are saturated hydrocarbons of different sizes and structures, which pose a variety of challenges to degradative microorganisms due to their physicochemical properties, i.e., the extremely limited solubility and the high energy required for activation. The hydrophobic cell surface of Rhodococcus spp., the ability to produce biosurfactants, and the possession of a wide range of oxygenases allow coping with such challenges. In particular, monooxygenase enzymes are involved in the activation of alkanes by converting them into alcohols, which undergo a series of oxidation steps before being converted to fatty acids. Rhodococcus alkane monooxygenases belong to different families (i.e., AlkB-like monooxygenase, soluble di-iron monooxygenase, cytochrome P450), have different genetic organization, and are subject to different regulatory mechanisms, which are poorly known. Because of their long-term survival capacity, broad catabolic abilities, and effective contact mechanisms with hydrocarbon molecules, alkanotrophic Rhodococcus strains have biotechnology applications and potential in bioremediation and biotransformation reactions