Abstract
SummaryThe sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac A in a S. mutans rml D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.
Highlights
The cell wall of Gram-positive bacteria is an intricate network of peptidoglycan, proteins and secondary cell wall polymers (SCWPs) that are covalently linked to peptidoglycan
Bioinformatics analysis suggests that gacA encodes a dTDP-4-dehydrorhamnose reductase, an enzyme that catalyzes the final step in the production of dTDP-Lrhamnose (Giraud and Naismith, 2000)
A similar split genomic architecture of the rhamnose biosynthesis genes rmlA-C and rmlD was previously observed in Streptococcus mutans (S. mutans), a cariogenic Gram-positive bacterium (Tsukioka et al, 1997)
Summary
The cell wall of Gram-positive bacteria is an intricate network of peptidoglycan, proteins and secondary cell wall polymers (SCWPs) that are covalently linked to peptidoglycan. Many β-hemolytic streptococcal species appear to lack expression of typical teichoic or teichuronic acid structures (Sutcliffe et al, 2008; Caliot et al, 2012) and instead express a rhamnoserich polymer, which comprises approximately half of the cell wall mass (McCarty, 1952). Expression of these evolutionary conserved glycans underlies classification of β-hemolytic streptococci in Lancefield groups van Sorge et al (2014) identified the gene cluster responsible for GAC biosynthesis and demonstrated that the GAC GlcNAc side-chain contributes to GAS virulence
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