To screen for a possible role for the 19 kDa lipoprotein in mycobacterial physiology, we therefore

generated a deletion mutant lacking the 19 kDa molecule and complemented this mutant with the wild type and site-mutagenised copies of the 19 kDa molecule. Figure 1 Sequence alignment of 27 open reading frames belonging to the 19 kDa family. Highly conserved cysteine, and phenylalanine residues are highlighted. “”*”" indicates fully conserved positions; “”:”" OSI-027 nmr indicates strong conservation; “”.”" Indicates weaker conservation. The 0-glycosylated threonine residues in the M. tuberculosis LpqH are boxed. Fully compliant Lipobox acylation motifs are underlined. Figure 2 A. Neighbour-joining tree of 19 kDa homologs. Family members are found in both slow-growing and fast-growing mycobacteria and in the closely related genera, Nocardia and Rhodococcus. The predicted

19 kDa proteins fall into three sub-families: LpqH, LppE and Lp3. B. Nucleotide sequence of the N-terminal coding sequence of the 19 kDa gene indicating the sequences that were modified in the Δ19 strains complemented by the non-acylated or non-O-glycosylated 19 kDa molecule. The disruption to sequence encoding the N-Acyl diglyceride motif is indicated by underlined text and the disruption of the 2 threonine clusters shown in bold. The protein sequence of the wild type and each variant is also shown. Amino acid numbering is based upon the mature protein after cleavage of the signal peptide. Generation and characterization click here of recombinant M. tuberculosis strains PCR analysis showed Rv3763

to be absent from Δ19 and that this sequence had been successfully reintroduced into strains Δ19::19,, Δ19::19NA, and Δ19::19NOG (Figure 3A). Western Blotting of cellular pellet indicated that the 19 kDa was not produced in Δ19 (Figure 3B, lane 2). Expression of native protein of the same MW was restored close to normal Protein kinase N1 levels by reintroduction of the 19 kDa gene in strain Δ19::19 (Figure 3B, lane 3). 19 kDa protein was only detected in the supernatant of cultures of the non-acylated (NA) and non-O-glycosylated complemented strains and was of slightly lower MW than the native 19 kDa. In Middlebrook 7H9 broth the growth rate of the Δ19, Δ19::19, Δ19::19NA, and Δ19::19NOG strains was identical (Figure 4). Figure 3 Characterization of mutant M. tuberculosis strains. A. PCR analysis showed Rv3763 to be absent from Δ19 and that this sequence had been successfully reintroduced into strains Δ19::19,, Δ19::19NA, and Δ19::19NOG. B. Western Blotting of cellular pellet indicating that the 19 kDa is not produced in Δ19 (lane 2). Expression of native protein of the same MW is restored close to normal levels by reintroduction of the 19 kDa gene in strain Δ19::19. C. Analysis of pellet and culture supernatant of complemented mutant strains.