title: GtrA Protein Rv3789 Is Required for Arabinosylation of Arabinogalactan in Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis possesses a thick and highly hydrophobic cell wall principally composed of a mycolyl-arabinogalactan-peptidoglycan complex, which is critical for survival and virulence. DprE1 is a well-characterized component of decaprenyl-phospho-ribose epimerase, which produces decaprenyl-phospho-arabinose (DPA) for the biosynthesis of mycobacterial arabinans. Upstream of dprE1 lies rv3789, which encodes a short transmembrane protein of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides. We demonstrate that rv3789 and dprE1 are cotranscribed from a common transcription start site situated 64 bp upstream of rv3789. Topology mapping revealed four transmembrane domains in Rv3789 and a cytoplasmic C terminus consistent with structural models built using analysis of sequence coevolution. To investigate its role, we generated an unmarked rv3789 deletion mutant in M. tuberculosis. The mutant was characterized by impaired growth and abnormal cell morphology, since the cells were shorter and more swollen than wild-type cells. This phenotype likely stems from the decreased incorporation of arabinan into arabinogalactan and was accompanied by an accumulation of DPA. A role for Rv3789 in arabinan biosynthesis was further supported by its interaction with the priming arabinosyltransferase AftA, as demonstrated by a two-hybrid approach. Taken together, the data suggest that Rv3789 does not act as a DPA flippase but, rather, recruits AftA for arabinogalactan biosynthesis.
  
  
  OBJECTIVE
  Upstream of the essential dprE1 gene, encoding a key enzyme of the decaprenyl phospho-arabinose (DPA) pathway, lies rv3789, coding for a short transmembrane protein of unknown function. In this study, we demonstrated that rv3789 and dprE1 are cotranscribed from a common transcription start site located 64 bp upstream of rv3789 in M. tuberculosis. Furthermore, the deletion of rv3789 led to a reduction in arabinan content and to an accumulation of DPA, confirming that Rv3789 plays a role in arabinan biosynthesis. Topology mapping, structural modeling, and protein interaction studies suggest that Rv3789 acts as an anchor protein recruiting AftA, the first arabinosyl transferase. This investigation provides deeper insight into the mechanism of arabinan biosynthesis in mycobacteria.
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland.
authors.forename: Gaëlle S
authors.initials: GS
authors.lastname: Kolly
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland.
authors.forename: Raju
authors.initials: R
authors.lastname: Mukherjee
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Comenius University in Bratislava, Faculty of Natural Sciences, Department of Biochemistry, Bratislava, Slovak Republic.
authors.forename: Emöke
authors.initials: E
authors.lastname: Kilacsková
authors.affiliation: Laboratory for Biomolecular Modeling, Ecole Polytechnique Fédérale de Lausanne and Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors.forename: Luciano A
authors.initials: LA
authors.lastname: Abriata
authors.affiliation: Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland.
authors.forename: Mahé
authors.initials: M
authors.lastname: Raccaud
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Comenius University in Bratislava, Faculty of Natural Sciences, Institute of Chemistry, Bratislava, Slovak Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Blaško
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Sala
authors.affiliation: Laboratory for Biomolecular Modeling, Ecole Polytechnique Fédérale de Lausanne and Swiss Institute of Bioinformatics, Lausanne, Switzerland.
authors.forename: Matteo
authors.initials: M
authors.lastname: Dal Peraro
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Comenius University in Bratislava, Faculty of Natural Sciences, Department of Biochemistry, Bratislava, Slovak Republic.
authors.forename: Katarína
authors.initials: K
authors.lastname: Mikušová
authors.affiliation: More Medicines for Tuberculosis (MM4TB) Consortium‡ Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland stewart.cole@epfl.ch.
authors.forename: Stewart T
authors.initials: ST
authors.lastname: Cole
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D005685:Galactans / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013729:Terpenes / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005685:Galactans; D013729:Terpenes; C089328:decaprenyl-P-arabinose; D001089:Arabinose; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; C005653:arabinogalactan