Figure 3

Schematic representation of Hklep and Rrlep proteins tested for phosphorylation assays and complementation of L. biflexa mutants. (A) The L. biflexa Hklep protein is composed of three domains. Hklep is putatively anchored into the inner membrane through two transmembrane segments (TM1-TM2) separated by a periplasmic loop (PL) that could correspond to the sensing domain. The residue histidine 98 is predicted to be the site of phosphorylation. The asterisk marks its mutation into an alanine residue in Hklep(H98A). In HklepΔ, the periplasmic loop is replaced by another sequence (see Materials and Methods). In Hklep (80–253), the putative sensing domain is deleted and the original promoter of hklep is replaced by a leptospiral promoter. (B) The L. biflexa Rrlep protein is composed of two domains. The residue aspartate 53 is predicted to be the site of phosphorylation. The asterisk marks its mutation into an alanine residue in Rrlep(D53A). Lbi and Lint refer to the L. biflexa and L. interrogans alleles, respectively. "-" indicates no in vitro phosphorylation or absence of complementation of the L. biflexa mutant strain. "+" indicates in vitro phosphorylation or complementation of the L. biflexa mutant strain. ND: not determined. The expression of HklepH98A, Hklep(80–253), Rrlep(D53A), or Rrlep-Lint (LB015) failed to complement their respective mutant and was characterized by an absence of growth (-) in EMJH medium like observed for hklep and rrlep mutants. The expression of HklepΔ or Hklep-Lint (LB014) complemented hklep mutant restoring a wild type growth (+) in EMJH medium.