§ Nucleotide sequence accession numbers (GenBank) of analyzed sequences,

protein accession numbers (PAN), available genomic locus tags (GLT), and gene names are shown. Underlined gene names are proposed herein. ¥ M. smegmatis sequence submission AY439015.3 shows a single gene (mps1) where the annotated complete genome (GenBank: CP000480.1) shows two contiguous genes (MSMEG_0400 and MSMEG_0401). Our sequence comparison revealed that CP000480.1 has an insertion of a “C” and a deletion of an “A” relative to AY439015.3. The events (112-bp apart) create a transient frameshift that splits mps1 into MSMEG_0400 and MSMEG_0401. We resequenced the region containing the discrepancies and found that check details our sequence matched that of AY439015.3. Based on this and the conservation of mps1 across species, we conclude that the correct gene organization is as shown herein. †The open reading frame corresponding to this gene has not been previously annotated. ‡Our sequence analysis (not shown) indicates

that the pstA appears to have originated from an mps1 buy Lazertinib and mps2 deletion-fusion rearrangement relative to the canonical mps1 and mps2 seen in M. avium strains 104 and 2151 and other GPL-producing species. This rearrangement leads to a gene encoding a 4,027-amino acid protein that appears to have segments MK-8776 derived from both Mps1 and Mps2. This protein would not be competent for D-Phe-D-alloThr-D-Ala-L-alaninol synthesis, a defect that alone would explain the known GPL-deficiency of M. avium subsp. paratuberculosis K-10. Figure 3 Sequence Avelestat (AZD9668) relatedness of GplH orthologues and related homologues. (A) Protein alignment and (B) table of percentage of amino acid identity. Conserved amino acids that match consensus are highlighted in white font over black background. The three conserved tryptophan residues that are the hallmark of the MbtH-like protein family are marked (*). The protein alignment and identity determination were performed with ClustalW (Lasergene software, DNASTAR, Inc). Mab, M. abscessus; Ma, M. avium; Map, M. avium subsp. paratuberculosis; Mc, M. chelonae; Mi, M. intracellulare; Ms, M. smegmatis; Mt, M. tuberculosis. Deletion of gplH

in M. smegmatis Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a mbtH-like gene, gplH. The involvement of this conserved gene in GPL production remains unproven. Herein, we sought to conclusively establish whether gplH was required for GPL production. To this end, we engineered Ms ΔgplH, a mutant with an unmarked, in-frame deletion of gplH (Figure 4A), the Ms gene upstream of the NRPS-encoding gene mps1 (Figure 2), and assessed the ability of this mutant to produce GPLs as described below. Ms was selected as a representative prototype of GPL producers for the studies presented herein due to its superior experimental tractability compared with other GPL producers (e.g., MAC members). Figure 4 Construction of M. smegmatis Δ gplH.