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.
Highlights
The number of genes involved in cellular metabolism have been reduced in mycoplasmas, the number of genes involved in transcription and translation are comparable to those of other bacteria [1, 2], and the basic mechanisms of transcription and translation in mycoplasmas are thought to be similar to those of low G+C Gram-positive bacteria
In order to explore whether use of a GTG translational initiation codon was more common in mycoplasmas than in other bacterial species, we initially examined the annotated genomes of a number of Mycoplasma species to determine the prevalence of use of alternative translational start codons in predicted coding sequences in this family of bacteria
The minimal free energy for optimal secondary structure was higher with the ATG codon (-2.5 kCal mol-1), which could have reduced the stability of the mRNA secondary structure and increased transcriptional efficiency
Summary
The number of genes involved in cellular metabolism have been reduced in mycoplasmas, the number of genes involved in transcription and translation are comparable to those of other bacteria [1, 2], and the basic mechanisms of transcription and translation in mycoplasmas are thought to be similar to those of low G+C Gram-positive bacteria. In the E. coli K-12 genome, of the 4288 open reading frames annotated, 82% have an ATG start codon, 14.3% a GTG start codon and 3% a TTG start codon [8]. A recent annotation of ten different E. coli strains found that 82.5% of the start codons were ATG, 12.3% were GTG and 5% were TTG, with CTG, ATT and ATC used at lower frequencies [9]. Translational efficiency has been shown to decrease in E. coli when a start codon other than ATG is used, with an eightfold reduction in translation seen with GTG or TTG start codons [10]. In B. subtilis ATG, TTG and GTG start codons are used in 78%, 13% and 9% of CDSs, respectively [11], with GTG shown to be three- to five-fold less efficient than ATG in translational initiation [12]
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