Searching for tubulin-like genes in free-living bacteria and symbionts of ciliates. Characterization of the genomic environment of tubulin genes from Prosthecobater

Tubulins are typical eucaryotic genes, which have recently been found in the free-living bacteria Prosthecobacter (Verrucomicrobiales). Nevertheless, microtubule-like structures were never observed in Prostecobacter, whereas they were reported in epixenosomes which are symbiotic Verrucomicrobiales of the ciliate Euplotidium. In the present work, an extensive search for tubulin like genes was performed with several combinations of PCR-primers, designed according to different criteria. Although some combinations of these primers are able to amplify simultaneously eukaryotic α- and β-tubulin as well as Prosthecobacter tubulin genes, no bacterial-like tubulin genes were identified in other Verrucomicrobiales species. Because of host derived contaminations, even a subtractive PCR approach, using microtiter-plate-subtractive-hybridization, was used with the epixenosomes. The characterization of the genomic environment of the Prosthecobacter tubulin genes was performed by gene walking. The sequences obtained from P. debontii revealed a so-far undiscovered gene duplication. The bacterial tubulin operon looks conserved among Prosthecobacter species and includes an α-tubulin, a β-tubulin and a kinesin like protein. Upstream of two operons a repetetive sequence could be found which could be a hint for horizontal gene transfer. The analysis of the genomic environments of the tubulin genes provided open reading frames, which have homology to Verrucomicrobium spinosum genes (ongoing genome project data), whereas up to now the blast search did not reveal tubulin-like genes in this organism. Present results suggest, that the Prosthecobacter tubulin genes are possibly of recent origin, maybe acquired by horizontal gene transfer, or lost by other so far studied Verrucomicrobiales. The origin and nature of microtubular genetic determinants from epixenosomes remains open.