1887

Abstract

The ∼16·5 kb surface layer (S-layer) glycan biosynthesis () gene cluster of the Gram-positive thermophile NRS 2004/3a has been sequenced. The cluster is located immediately downstream of the S-layer structural gene and consists of 13 ORFs that have been identified by database sequence comparisons. The cluster encodes dTDP--rhamnose biosynthesis ( 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 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 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 operon, because -rhamnose is a frequent constituent of S-layer glycans. From -positive strains, flanking regions of the operon were sequenced. Comparison with the gene cluster of 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 transcript was several-fold more abundant at 67 °C. Cell-free extracts of the strains were capable of converting dTDP--glucose to dtdp--rhamnose. Taken together, the results indicate that the locus is highly conserved among strains, and that in the investigated containing strains, dTDP--rhamnose is actively synthesized . However, in contrast to previous reports for wild-type strains, an increase in growth temperature did not switch an S-layer protein phenotype to an S-layer glycoprotein phenotype, via the generation of a new S-layer gene sequence.

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2004-04-01
2020-12-01
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