| Capsular polysaccharide (CPS) production is critical for virulence and pathogenesis of
many bacterial pathogens, including Streptococcus pneumoniae. In S. pneumoniae, CPS
production is modulated by the metal-dependent phosphoglucomutase (Pgm), which generates
essential sugar precursors for CPS biosynthesis. Unlike other Pgm homologs, S. pneumoniae
Pgm binds two metal ions per protomer and is most active with manganese (Mn). This study
aims to identify the metal-binding determinants in S. pneumoniae Pgm. Utilizing previous data
obtained from multiple bioinformatic approaches, ten potential metal-binding residues D303,
R308, R48, N80, N134, E190, H238, R93, D91, and N146 in S. pneumoniae Pgm were selected
for analysis. Each residue was independently replaced with alanine using site-directed
mutagenesis and homologous recombination techniques yielding 10 new mutant Pgm S.
pneumoniae strains. All mutant Pgm strains, except those expressing Pgm alleles N80A, N134A,
E190A, exhibited impaired growth patterns like a pgm-null mutant strain and failed to grow to
wildtype stationary turbidity levels. Further analysis using western blots probing for Pgm protein
expression showed that all mutant Pgm strains expressed similar levels of Pgm and were equal to
that of the wildtype strain, thereby confirming the growth defect exhibited by Pgm mutants
(D303A, R308A, H238A, R93A, D91A, and N146A) did not result from poor Pgm protein
expression. In vitro analysis of Pgm mutant alleles D303A, R308A, and R48A using a mag-fura-
2 metal competition assay further revealed loss of metal binding, suggesting that D303, R308,
and R48 are likely involved in metal-associated mechanisms in Pgm. When combined with the
bioinformatic analysis of Pgm, these data indicate that the conserved D303 and R308 residues
likely do bind metal in Pgm, and this binding is required for Pgm activity in S. pneumoniae.
Further studies are ongoing to understand the role of R48 and other Pgm mutant alleles that
showed impaired growth.. These data along with future data acquisition will aid in understanding
how and why Pgm binds two metal ions and is most active with Mn2+.
Keywords : Streptococcus pneumoniae, capsular polysaccharide, phosphoglucomutase,
metal binding residues, site-directed mutagenesis |