Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155

Abstract

The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.

FIG. 1.

Synthesis of dTDP-l-rhamnose. Crude extracts from A. thermoaerophilus DSM 10155 (line A), E. coli B (line B), and A. thermoaerophilus L420-91^T (line C) were used to synthesize dTDP-l-rhamnose from dTDP-d-glucose in the presence of NADPH.

FIG. 2.

Precursor formation in A. thermoaerophilus DSM 10155. (A) Map of the genes involved in formation of GDP-d-glycero-α-d-manno-heptose and dTDP-β-l-rhamnose. (B) Scheme of the biosynthetic pathway of the nucleotide-activated precursors of the S-layer glycoprotein of A. thermoaerophilus DSM 10155.

FIG. 3.

Localization of RmlA in A. thermoaerophilus DSM 10155 cells: electron micrograph of an ultrathin section after immunolabeling with colloidal gold, showing the cytoplasmic localization of the enzyme. Bar = 50 nm.