A bifurcated pathway for arginine-induced morphogenesis.

A. Scheme of arginine catabolic pathway. B. Growth-based assays. Five microliters (5 μl) of serially diluted cells were spotted onto the surface of SXD (X = Am (Ammonium sulfate), Arg, Orn, Pro or Urea) and then grown for 48 h at 30°C. Strains used: wildtype (WT; PMRCA18), car1-/- (CFG077), dur1,2-/- (CFG091), put1-/- (A) (CFG122), put1-/- (B) (CFG155), and put1-/- dur1,2 -/- (CFG158). C. Arginine rapidly derepresses proline catabolic genes. PUT1, PUT2, PUT3 and DUR1,2 expression in wildtype (SC5314) cells 1 h at 37 °C after shifting from YPD (t = 0) to YNB+Arg (pH = 6.0). Gene expression was determined by qRT-PCR using the levels of RIP1 to normalize expression. D. Proline catabolic pathway is required for growth in arginine as a sole nitrogen and carbon source. Cells with the indicated genotypes were harvested from log phase YPD cultures and diluted in YNB+Arg+BCP medium (pH = 4.0) to OD600 ≈ 0.01, cultures were incubated for 16 h at 37 °C. Alkalization (shift to purple) correlates with growth. Strains used: WT (SC5314), dur1,2-/- (CFG246), put1-/- (CFG149), and put2-/- (CFG143). Identical results were obtained using PMRCA18-derived mutants. E. Rapid activation of proline catabolism in the presence of arginine, ornithine, and proline. Immunoblot analysis of Put2-HA in whole cell lysates prepared from CFG185 (PUT2/PUT2-HA) cells grown at 37 °C in the specified SXD media for the indicated times. Cells were pre-grown in YPD and inoculated at an OD600 of 0.5. Levels of α-tubulin were used as loading control. F. Pharmacological inhibition of proline dehydrogenase (Put1) reduced filamentous growth of C. albicans in the presence of arginine. Macrocolonies of wildtype (WT; PMRCA18) grown at 37 °C for 72 h on SRD medium supplemented with L-THFA as indicated. G. Filamentous growth of strains on SXD media (X = Arg, Urea, or Arg + Urea as sole nitrogen sources). Macrocolonies were grown at 37 °C and photographed at 24 and 48 h. Strains used: wildtype (WT; PMRCA18), car1-/- (CFG077), dur1,2-/- (CFG091), and put1-/- (CFG122).