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Alanine-scanning mutagenesis of protein mannosyl-transferase from Streptomyces coelicolor reveals strong activity-stability correlation

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JournalMicrobiology (Reading, England)
DateAccepted/In press - 13 Sep 2021
DatePublished (current) - 22 Oct 2021
Issue number10
Volume167
Original languageEnglish

Abstract

In Actinobacteria, protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and β-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.

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© 2021 The Authors

    Research areas

  • alanine-scanning mutagenesis, antibiotic susceptibility, homology model, protein mannosylation, Streptomyces coelicolor

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