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

Bacterial surface charge derives its meaning from the cell’s environment such as the solution in contact with the cell. 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 bacterial surface charge measurement require biomass obtained from pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed onto the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass incorporated repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes the results of a systematic study on how wash buffers of different composition and ionic strength affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5α (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium with 2 g/L glucose and a formulated medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, the results showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlated with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl) seemed to be the minimum below which there was incomplete removal of nonspecifically adsorbed biomolecules. On the other hand, high ionic strength of 0.6M (e.g., 0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for removal of ions intrinsic to the cell envelope. Collectively, results obtained inform wash buffer choice with regards to preserving cell envelope integrity, and avoidance of adsorption of buffer ions such as citrate. But, is there a true cell surface charge? Yes, but how do we define it in number of “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 remove each layer via charge screening. However, where is the endpoint? This research offers a different perspective and answer. Imagine a single bacterium suspended in LB medium, where there is constant adsorption and desorption of biomolecules as the cell grows: what is its relevant surface charge? It is the one where the loosely associated ions and metabolites are removed while retaining the nonspecifically adsorbed ions and biomolecules. Thus, deionized water wash provides a good estimate of the bacterial surface charge as grown in specific medium.