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GacA is essential for G roup A S treptococcus and defines a new class of monomeric d TDP ‐4‐dehydrorhamnose reductases ( RmlD )
Author(s) -
Beek Samantha L.,
Le Breton Yoann,
Ferenbach Andrew T.,
Chapman Robert N.,
Aalten Daan M. F.,
Navratilova Iva,
Boons GeertJan,
McIver Kevin S.,
Sorge Nina M.,
Dorfmueller Helge C.
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13169
Subject(s) - biology , biochemistry , mutant , bacteria , enzyme , biosynthesis , heterologous expression , gene , genetics , recombinant dna
Summary The sugar nucleotide d TDP ‐ L ‐rhamnose is critical for the biosynthesis of the G roup A Carbohydrate, the molecular signature and virulence determinant of the human pathogen G roup A S treptococcus ( GAS ). The final step of the four‐step d TDP ‐ L ‐rhamnose biosynthesis pathway is catalyzed by d TDP ‐4‐dehydrorhamnose reductases ( RmlD ). RmlD from the G ram‐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 G ram‐negative and G ram‐positive RmlD homologues predicts that enzymes from all G ram‐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 G ram‐positive bacteria and a subset of G ram‐negative bacteria. These results will help future screens for novel inhibitors of d TDP ‐ L ‐rhamnose biosynthesis.