S-layer glycan-specific loci on the chromosome of Geobacillus stearothermophilus NRS 2004/3a and dTDP-l-rhamnose biosynthesis potential of G. stearothermophilus strains

Abstract

The ∼16·5 kb surface layer (S-layer) glycan biosynthesis (slg) gene cluster of the Gram-positive thermophileGeobacillus stearothermophilusNRS 2004/3a has been sequenced. The cluster is located immediately downstream of the S-layer structural genesgsEand consists of 13 ORFs that have been identified by database sequence comparisons. The cluster encodes dTDP-l-rhamnose biosynthesis (rmloperon), required for building up the polyrhamnan S-layer glycan, as well as for assembly and export of the elongated glycan chain, and its transfer to the S-layer protein. This is the first report of a gene cluster likely to be involved in the glycosylation of an S-layer protein. There is evidence that this cluster is transcribed as a polycistronic unit, whereassgsEis transcribed monocistronically. To get insights into the regulatory mechanisms underlying glycosylation of the S-layer protein, the influence of growth temperature on the S-layer was investigated in seven closely relatedG. stearothermophilusstrains, of which only strain NRS 2004/3a possessed a glycosylated S-layer. Chromosomal DNA preparations of these strains were screened for the presence of thermloperon, becausel-rhamnose is a frequent constituent of S-layer glycans. Fromrml-positive strains, flanking regions of the operon were sequenced. Comparison with theslggene cluster ofG. stearothermophilusNRS 2004/3a revealed sequence homologies between adjacent genes. The temperature inducibility of S-layer protein glycosylation was investigated in those strains by raising the growth temperature from 55 °C to 67 °C; no change of either the protein banding pattern or the glycan staining behaviour was observed on SDS-PAGE gels, although thesgsEtranscript was several-fold more abundant at 67 °C. Cell-free extracts of the strains were capable of converting dTDP-d-glucose to dtdp-l-rhamnose. Taken together, the results indicate that thermllocus is highly conserved amongG. stearothermophilusstrains, and that in the investigatedrml-containing strains, dTDP-l-rhamnose is actively synthesizedin vitro. However, in contrast to previous reports forG. stearothermophiluswild-type strains, an increase in growth temperature did not switch an S-layer protein phenotype to an S-layer glycoprotein phenotype, via thede novogeneration of a new S-layer gene sequence.