Characterization of sulfhydryl sites within bacterial cell envelopes using selective site-blocking and potentiometric titrations

In this study, a novel approach was developed to estimate the concentration and acidity constants of sulfhydryl sites within bacterial cell envelopes, and we apply the approach to compare sulfhydryl site concentrations of Bacillus licheniformis, Bacillus subtilis, Bacillus cereus, Shewanella oneidensis and Pseudomonas fluorescens. The experiments involved the selective blocking of sulfhydryl sites using a thiol-specific molecule, coupled with total site concentration comparisons of blocked and un-blocked bacterial samples by potentiometric titration measurements to determine sulfhydryl concentrations. All five species studied contained measureable concentrations of sulfhydryl sites, ranging from 16.6 ± 3.3. μmol/g for B. cereus to 33.1 ± 7.6. μmol/g for S. oneidensis. No significant difference was found between sulfhydryl site concentrations on Gram-positive species relative to those on Gram-negative bacteria. However, the proportion of sulfhydryl sites relative to the total sites on each species was the highest for the thermophilic bacterium B. licheniformis with 14 ± 3%, and the four mesophilic species exhibited an average of 8 ± 2%. All species contained sulfhydryl sites with a p. K_a of 9.2-9.4, but B. subtilis and P. fluorescens exhibited significant concentrations of sulfhydryl sites with much lower p. K_a values as well. Our results suggest that sulfhydryl sites are present in relatively low concentrations over a wide range of bacterial diversity, but that their concentrations are high enough to control the binding of metals onto bacteria under low metal-loading conditions.