Cell surface electrochemical heterogeneity of the Fe(III)-reducing bacteria Shewanella putrefaciens

Acid-base titration experiments and electrostatic force microscopy (EFM) we re used to investigate the cell surface electrochemical heterogeneity of th e Fe(III)-reducing bacteria, Shewanella putrefaciens. The acid-base titrati ons extended from pH 4 to 10; and the titration data were fit using a linea r programming pK(a) spectrum approach. Overall, a five-site model ac-counte d for the observed titration behavior with the most acidic sites correspond ing to carboxylic groups and phosphodiester groups, intermediate: sites pho sphoryl groups, and two basic sites equivalent to amine or hydroxyl groups. The pH for the point of zero charge on the bacteria was 5.4. In EFM images bf cells rinsed in solutions at pH 4.0, 7.0, and 8.0, a pronounced increas e in small (less than or equal to 100 nm diameter) high contrast patches wa s observed on the cells with increasing pH. The pH dependence of EFM image contrast paralleled the pattern of cell surface charge development inferred from the titration experiments; however, quantitative; analysis of high Co ntrast regions in the EFM images yielded lower surface charge values than t hose anticipated from the titration data. For example at pH 7, the calculat ed surface; charge of high contrast regions in EFM images of the bacterial cells was -0.23 muC/cm(2) versus -20.0 muC/cm(2) based on the titration cur ve. The differences in surface charge estimates,between the EFM images and titration data are consistent not only with charge development throughout t he entire-volume of the bacterial cell wall (i.e., in association with func tional groups that are not directly exposed at the cell surface) but also w ith the presence of a thin structural layer of water containing charge-comp ensating counterions. In combination, the pK(a) spectra and EFM data demons trate that a particularly high degree of electrochemical heterogeneity exis ts within the cell wall and at the cell surface of S. putrefaciens.