ABSTRACTMycobacterium tuberculosispossesses 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 ofdprE1liesrv3789, which encodes a short transmembrane protein of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides. We demonstrate thatrv3789anddprE1are cotranscribed from a common transcription start site situated 64 bp upstream ofrv3789. 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 unmarkedrv3789deletion mutant inM. 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.IMPORTANCEUpstream of the essentialdprE1gene, encoding a key enzyme of the decaprenyl phospho-arabinose (DPA) pathway, liesrv3789, coding for a short transmembrane protein of unknown function. In this study, we demonstrated thatrv3789anddprE1are cotranscribed from a common transcription start site located 64 bp upstream ofrv3789inM. tuberculosis. Furthermore, the deletion ofrv3789led 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.