@article{mbs:/content/journal/micro/10.1099/mic.0.26672-0, author = "Novotny, René and Schäffer, Christina and Strauss, Joseph and Messner, Paul", title = "S-layer glycan-specific loci on the chromosome of Geobacillus stearothermophilus NRS 2004/3a and dTDP-l-rhamnose biosynthesis potential of G. stearothermophilus strains", journal= "Microbiology", year = "2004", volume = "150", number = "4", pages = "953-965", doi = "https://doi.org/10.1099/mic.0.26672-0", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.26672-0", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", keywords = "TPR, tetratricopeptide repeat", keywords = "PAS, periodic acid-Schiff", abstract = "The ∼16·5 kb surface layer (S-layer) glycan biosynthesis (slg) gene cluster of the Gram-positive thermophile Geobacillus stearothermophilus NRS 2004/3a has been sequenced. The cluster is located immediately downstream of the S-layer structural gene sgsE and consists of 13 ORFs that have been identified by database sequence comparisons. The cluster encodes dTDP-l-rhamnose biosynthesis (rml operon), 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, whereas sgsE is 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 related G. stearothermophilus strains, of which only strain NRS 2004/3a possessed a glycosylated S-layer. Chromosomal DNA preparations of these strains were screened for the presence of the rml operon, because l-rhamnose is a frequent constituent of S-layer glycans. From rml-positive strains, flanking regions of the operon were sequenced. Comparison with the slg gene cluster of G. stearothermophilus NRS 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 the sgsE transcript 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 the rml locus is highly conserved among G. stearothermophilus strains, and that in the investigated rml-containing strains, dTDP-l-rhamnose is actively synthesized in vitro. However, in contrast to previous reports for G. stearothermophilus wild-type strains, an increase in growth temperature did not switch an S-layer protein phenotype to an S-layer glycoprotein phenotype, via the de novo generation of a new S-layer gene sequence.", }