In this study, we provide evidence unequivocally establishing that the conserved mbtH-like gene (herein referred to as gplH) located in the GPL biosynthetic gene locus of Ms is essential for GPL production. This finding presents the first case of a mbtH-like gene Momelotinib cost required for biosynthesis of a cell wall component and provides the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Moreover, we show that loss of gplH leads to a mutant with atypical NVP-BGJ398 cell line colony morphology, lack of sliding motility, reduced biofilm formation capacity, and increased

antimicrobial drug susceptibility. Altogether, this study demonstrates a critical role for gplH in mycobacterial biology and advances our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the unique mycobacterial outer membrane. Results and discussion Conservation of a MbtH homologue in the GPL biosynthetic pathway

MbtH is a protein encoded in the mycobactin siderophore biosynthetic gene cluster of M. tuberculosis and the founding member of the MbtH-like protein family (NCBI CDD pfam 03621) [33]. Our analysis of available genome sequences of GPL producers revealed that every GPL biosynthetic LY2874455 gene cluster known to date contains a mbtH-like gene located upstream of NRPS-encoding genes required for D-Phe-D-alloThr-D-Ala-L-alaninol assembly

(Figure 2). The MbtH-like protein orthologues encoded by these mbtH-like Aurora Kinase genes are comprised of 69–93 amino acids and have remarkable sequence identity (80-100%) (Figure 3). This sequence identity extends to the three fully conserved tryptophan residues that are a hallmark of the protein family (NCBI CDD pfam 03621) [33] (Figure 3A). The open reading frame corresponding to the mbtH-like gene of M. avium 2151 (Figure 2) has not been previously annotated; however, our genome sequence analysis revealed its presence. The MbtH-like protein encoded by this gene is shown in the protein alignment (Figure 3A). The orthologous mbtH-like genes or MbtH-like proteins in the other species shown in Figure 2 have been annotated each as mbtH or MbtH, respectively [24, 46], presumably due to their sequence relatedness with M. tuberculosis MbtH. This name assignment is misleading as these genes are not orthologues of mbtH, the gene of the mycobactin biosynthetic pathway present in many mycobacteria, including M. smegmatis, M. abscessus, and M. avium[33, 35]. This name assignment leads to gene nomenclature confusion by resulting in more than one gene named mbtH in the same species. We proposed herein to name all the orthologous mbtH-like genes associated with GPL production as gplH, a name derived from glycopeptidolipid and mbt H and not previously assigned to any mycobacterial gene.