GplH might act as a critical activator of the amino acid adenylation activity of one or more of the four amino acid adenylation domains predicted by sequence analysis of the Mps1-Mps2 NRPS system [22, 23]. Biochemical studies will be required to investigate this possibility. MbtH-mediated cross-talk between GPL biosynthesis and mycobactin biosynthesis We noted that Ms has two potential mbtH-like genes located outside the GPL biosynthetic gene cluster. One of these genes is the mbtH orthologue in the mycobactin biosynthetic gene cluster of Ms

mentioned above [35]. The second gene, MSMEG_0016, is clustered with DMXAA manufacturer genes implicated in the production of the siderophore exochelin [48–50]. The protein products of these two Ms gplH paralogues have considerable amino acid sequence identity between themselves and with GplH and M. tuberculosis MbtH (this website Figure 3B). The GPL deficiency of Ms ΔgplH indicates that neither of these two Ms gplH paralogues can support the production of GPLs in Ms ΔgplH to a meaningful level under our culturing conditions. It is worth noting that Ms mbtH and MSMEG_0016 are associated with siderophore production pathways known to be repressed during growth under iron-rich

conditions [51, 52]. This fact raises the possibility that neither of these IWP-2 genes is expressed (or they are poorly expressed) in the iron-rich standard Middlebrook media used in our studies. With this consideration in mind, we explored whether an increase in expression of Ms mbtH (encoding the paralogue with the higher homology to GplH, Figure 3) could complement the GPL deficiency of Ms ΔgplH. To this end, we evaluated GPL production in Ms ΔgplH after transformation of the mutant with pCP0-mbtHMs

(expressing Ms mbtH). TLC analysis of lipid extracts from the transformant revealed the presence of GPLs, thus indicating that plasmid-directed constitutive expression of Ms mbtH complements the Amino acid GPL deficient phenotype of Ms ΔgplH (Figure 5). Thus, it appears that Ms MbtH has the potential to functionally replace GplH if present in sufficient quantities. This cross-complementation phenomenon is in line with recent cell-based studies demonstrating MbtH-like protein-mediated cross-talk between NRPS systems [41, 44]. Our finding is also consistent with reported in vitro enzymology indicating that, at least in some cases, the activity of amino acid adenylation domains of NRPSs can be stimulated not only by bona fide MbtH-like protein partners, but also by MbtH-like protein homologues from disparate natural product biosynthetic pathways [39, 40]. Deletion of gplH leads to a pleiotropic phenotype Colony morphotype, biofilm formation and sliding motility are properties that have been shown to be altered in GPL deficient mutants [18–20, 23]. Loss of GPL also perturbs bacterial surface properties [19, 32] and reduces the cell-wall permeability barrier to chenodeoxycholate uptake [19].