Spiroplasmas: gene structure and expression

Upon sequencing of the SpV4 genome, eight putative open reading frames (ORFs) including that for the 65-kilodalton (kDa) capsid protein were detected. They involve all three reading frames. Three promoter sequences were found, as well as a transcription terminator and the initiation site for complementary strand synthesis. Ribosome binding sites and regulatory sequences are closely related to those of Eubacteria. Codon usage analysis showed that A and T terminated codons are preferably used. UAA is the major termination codon. Upon cloning of the full-size SpV4 replicative form, the capsid protein gene could not be expressed in Escherichia coli, whereas the spiralin gene cloned in the same bacterium is expressed. These results suggest that in spiroplasmas, as in Mycoplasma capricolum, UGA is not a termination codon, but very probably codes for tryptophan. Spiralin contains no tryptophan. Hence, its gene contains no UGA codons and can thus be expressed in E. coli. On the other hand, the gene for capsid protein has nine UGA codons and cannot be fully expressed in the bacterium. Our results fully support the bacterial origin of spiroplasmas.