Effects of salts and ionophores on proline transport in a moderately halophilic halotolerant bacterium

The effect of salt on proline uptake in a moderately halophilic halotolerant bacterium was studied. Cells were grown either on low salt or high salt media. A correlation was found between the salt concentrations in the growth media and the optimal concentration for uptake. The uptake rate was stimulated 2-3-fold by NaCl, as compared to KCl. The K_m, V and activation energies values for proline uptake, as well as the external pH effect, were similar in low-salt-grown cells and high-salt-grown cells. This suggests that the halotolerance of the transport system is not due to alterations of the system during growth at various conditions, but rather to its intrinsic ability to function under extreme environmental conditions. The uptake was inhibited by cyanide and carbonyl cyanide m-chlorophenylhydrazone, but not by arsenate, indicating that the electrochemical proton gradient (Δμ_H⁺), generated by respiration, is the main driving force for proline transport. In low-salt-grown cells, at pH 6.0, partial inhibition was exerted by nigericin or valinomycin, whereas at pH 8.0 the uptake was inhibited by valinomycin only. Similar, although less pronounced effects were found in high-salt-grown cells. The data suggest that at pH 6.0 proline transport is driven by Δμ_H⁺ (composed of electrical potential (Δψ) and pH gradient), whereas at pH 8.0 Δψ is the main driving force. Procedures of pretreatment with EDTA were developed to enable the penetration of the ionophores into the cells.