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Mol Microbiol. 2015 Dec;98(5):946-62. doi: 10.1111/mmi.13169. Epub 2015 Oct 1.

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD).

Author information

1
University Medical Center Utrecht, Medical Microbiology, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
2
Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, 3124 Biosciences Research Building, College Park, MD 20742, USA.
3
Division of Molecular Microbiology, University of Dundee, School of Life Sciences, Dow Street, DD1 5EH, Dundee, UK.
4
Complex Carbohydrate Research Center, Department of Chemistry, The University of Georgia, 315 Riverbend Road, Athens, USA.
5
Division of Biological Chemistry and Drug Discovery, University of Dundee, School of Life Sciences, Dow Street, DD1 5EH, Dundee, UK.
6
Rutherford Appleton Laboratory, Research Complex at Harwell, OX11 0FA, Didcot, UK.

Abstract

The 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 Streptococcus (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 Salmonella 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 gacA in a S. mutans rmlD 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 gacA 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.

PMID:
26278404
PMCID:
PMC4832382
DOI:
10.1111/mmi.13169
[Indexed for MEDLINE]
Free PMC Article

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