Actinobacteria have a protein O-mannosylation system that is conserved across the prokaryotic and eukaryotic kingdoms . The membrane bound protein-O-mannosyl transferase (Pmt), transfers mannose from polyprenol phosphate mannose to a target protein. Whilst many Pmt targets in actinobacteria remain undiscovered, pmt null mutants in Mycobacterium tuberculosis exhibit attenuated pathogenicity in mice and a growth retarded phenotype . Streptomyces coelicolor pmt null mutants also show impaired growth and furthermore, are resistant to ϕC31 phage infection and have increased susceptibility to vancomycin and multiple β-lactams [3,4]. Pmt is therefore a possible new target for the isolation of novel antimicrobials to be used against M. tuberculosis. To further understand the structure and function of Pmt, we performed sequence alignments and secondary structure homology modelling to identify targets for site-directed mutagenesis of the S. coelicolor pmt gene. The pmt mutant alleles were introduced into a pmt - (DT2008) S. coelicolor strain using conjugative integrative plasmids and scored for their ability to complement the phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-three highly conserved residues were each changed to alanine and six mutants (R82A, D113A, H159A, D233A, K332A, R510A) failed to complement DT2008, indicating essentiality. Western blotting showed an absence of all six mutants in purified membrane fractions and whole cell lysates. Modelling these six critical residues showed that five are close to the predicted catalytic ‘DE’ motif, a hallmark of the GT-C fold glycosyl transferases . Crucially, four of these residues have not previously been proposed to have functional significance in the Pmt family, and therefore could be important for the M. tuberculosis homologue.
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