FIGURE 4.

Arginine inhibits acidification of the B. burgdorferi cytoplasm and prevents induction of RpoS-regulated proteins independent of the histidine kinase Hk2. (A) Fluorescence microscopy or (B) quantitative microplate assay of spirochetes grown in media alone (untreated), or in the presence of 20 mM benzoate, benzoate and arginine, or benzoate and alanine and stained with the membrane-permeable pH-sensitive dye pHrodo Green. Increased fluorescence indicates a decrease in pH and the mean value from three biological replicates is shown with the standard deviation. Asterisks denote a statistically significant difference of the mean determined using a one-way ANOVA where p < 0.05. (C) Immunoblot of OspC and BBA66 in B. burgdorferi cells treated with benzoate, benzoate and arginine, or benzoate and alanine. (D) Immunoblot of cell lysates from B. burgdorferi B31-A3 (WT) and B. burgdorferi B3-A3Δhk2 (Δhk2) for RpoS-dependent virulence factors OspC, BBA66 and the BosR-regulated factor NapA/BB690 in response to growth in BSK-II or BSK-II supplemented with benzoate, or benzoate and arginine. Flagellin levels were used as a loading control and the position of molecular weight standards is indicated on the right in kDa. With the exception of the microplate assay which was performed in biological duplicate and technical triplicate, all experiments were performed with three biological replicates and representative immunoblots are presented in (C,D). Images for (A) were selected at random from images found in Supplementary Figure S2.