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

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.

Keywords: Streptomyces coelicolor; alanine-scanning mutagenesis; antibiotic susceptibility; homology model; protein mannosylation.

Fig. 1.

Model for protein O-mannosylation in S. coelicolor . GDP-mannose is synthesised from mannose in consecutive reactions catalysed by ManA, ManB and ManC before transfer of the sugar onto polyprenol phosphate by polyprenol phosphate mannose synthetase (Ppm1). The polyprenol phosphate mannose is flipped to the periplasmic face of the membrane where it is a substrate for Pmt catalysed mannosylation of secreted protein substrates.

Fig. 2.

Protein mannosyl transferase topology model. Predicted membrane topology model for Pmt. The limits of the 11 membrane spanning segments as predicted in TMHMM are specified along with the locations of the residues (red) mutated to alanine in this study. Italics denote mutated residues predicted to be within the transmembrane regions.

Fig. 3.

Antibiotic sensitivity assays for DT1025 transformants. DT1025 strains harbouring genes encoding Pmt variants were assayed for their sensitivity to cell wall/membrane targeting antibiotics, nitrofurantoin and rifampicin. Antibiotic sensitivity was quantified by measuring the zone of inhibition in disc diffusion assays. Shown are the averages of four biological replicates with SEM. The antibiotic paper discs have a diameter of 5 mm, which is shown as the dotted line threshold. The amount of antibiotic used in μg is shown in brackets. The asterisks indicate a P<0.05 that the observed differences occurred by chance.

Fig. 4.

ϕC31cΔ25 Phage infection assays. DT1025 strains harbouring genes encoding Pmt variants were assayed for their sensitivity to ϕC31cΔ25 phage infection. Phage were plated out on the right side of DN agar plates and spores were subsequently streaked from left to right (from the regions lacking phage to those containing phage). Plates were incubated at 30 °C for 48 h. Images are representative of four biological replicates.

Fig. 5.

Expression assays. C-terminal StrepII-tagged Pmt is absent (a) and present (b) in the membrane fractions of DT1025 strains harbouring non-complementing (alanine substitutions at positions 83, 113, 159, 233, 302 and 510) and complementing (alanine substitutions at positions 160, 164, 228, 231, 335 and 421) mutations respectively. Membrane and soluble fractions were loaded onto a 12 % SDS-PAGE gel, which was run and stained with Coomassie blue (upper images) to ensure that the protein concentrations in the samples were comparable. After transfer of proteins from an SDS-PAGE gel to a PVDF membrane and washing, the membrane was incubated with a StrepII-Tag Antibody HRP Conjugate, substrate was added and chemoluminescence was detected on an X-ray film (lower image). pIJ: pIJ10257 control, WT: Pmt WT complemented strain control.

Fig. 6.

Phyre2 homology model of S. coelicolor Pmt. (a) Worms/tubes rendering of the overall structural model in the programme CCP4mg [33]. The chain is colour ramped from the amino terminus (magenta) to the carboxyl terminus (red). The peptide (Pro-Tyr-Thr) and polyprenol phosphate ligands derived from the S. cerevisiae Pmt1-Pmt2 coordinate set (6p25) are displayed in the context of the model following the overlay of protein chains using the ‘Superpose models’ routine in CCP4mg. Transmembrane helices are numbered 1 to 11 and horizontal helices on the periplasmic face of the membrane are numbered HH1 to HH5. The Cα atoms of residues mutated in this study are displayed as spheres - white for mutations that were associated with complementation of pmt deficiency in the in vivo assays and black for those that were not. A selection of these residues is labelled. (b) Close-up view of the active site region of the model. The peptide and polyprenol phosphate are displayed in cylinder format with the Cα and side chains of five of the six residues whose mutation to alanine led to loss of complementation of pmt deficiency in the in vivo assays. Atoms are coloured as follows: nitrogens, blue; oxygens, red; phosphorus, magenta; carbons light green for ligands and grey for protein.