Bacteria surface charge in “layers”: revealed by wash buffers of different ionic strength

Bacteria surface charge derives its meaning from the cell’s environment; thus, there is no one specific surface charge. Determining the surface charge of bacteria in its native environment requires measuring the proxy variable, zeta potential, using cells obtained from field studies. However, lack of adequate cell mass and concerns over measurement of a mixed species consortia rather than a specific species meant that bacteria surface charge measurement require biomass obtained from laboratory shake flask pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed on the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass require repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes a systematic study on how wash buffers of different composition and salinity affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5a (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, proxy measurement of wash buffers’ efficacies showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlates with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl and phosphate buffer saline) seems to be the minimum below which there appeared to be little removal of nonspecifically adsorbed biomolecules (deionized water wash as control). On the other hand, high ionic strength of 0.6M (0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for the removal of ions intrinsic to the cell envelope; thus, indicating significant cell surface damage. Collectively, results obtained provided important pointers for wash buffer choice concerning preservation of cell envelope integrity. Finally, is there a true cell surface charge? Yes, but how to define it? How many “layers” of adsorbed biomolecules? Philosophically, cells in culture broth are coated with layers of metabolites, proteins and ions; hence, desire to reveal the true surface charge is essentially a decoating process, where wash buffers of increasing ionic strength could remove each layer through charge screening. Where is the endpoint? This research suggests that ionic strength of wash buffers chosen should be similar to that of the environment the research is seeking to address. Imagine a single bacterium suspended in LB medium, what is its relevant surface charge? It is the one where the loosely associated ions and metabolites can be removed, similar to the constant adsorption and desorption processes that the cell experiences in its growth environment. Thus, deionized water wash provides a good estimate of the bacteria surface charge as grown in specific medium.