GTP-Dependent FlhF Homodimer Supports Secretion of a Hemolysin in Bacillus cereus

Abstract

The multidomain (B-NG) protein FlhF, a flagellar biogenesis regulator in several bacteria, is the third paralog of the signal recognition particle (SRP)-GTPases Ffh and FtsY, which are known to drive protein-delivery to the plasma membrane. Previously, we showed that FlhF is required for <i>Bacillus cereus</i> pathogenicity in an insect model of infection, being essential for physiological peritrichous flagellation, for motility, and for the secretion of virulence proteins. Among these proteins, we found that the L₂ component of hemolysin BL, one of the most powerful toxins <i>B. cereus</i> produces, was drastically reduced by the FlhF depletion. Herein, we demonstrate that <i>B. cereus</i> FlhF forms GTP-dependent homodimers <i>in vivo</i> since the replacement of residues critical for their GTP-dependent homodimerization alters this ability. The protein directly or indirectly controls flagellation by affecting flagellin-gene transcription and its overproduction leads to a hyperflagellated phenotype. On the other hand, FlhF does not affect the expression of the L₂-encoding gene (<i>hblC</i>), but physically binds L₂ when in its homodimeric form, recruiting the protein to the plasma membrane for secretion. We additionally show that FlhF overproduction increases L₂ secretion and that the FlhF/L₂ interaction requires the NG domain of FlhF. Our findings demonstrate the peculiar behavior of <i>B. cereus</i> FlhF, which is required for the correct flagellar pattern and acts as SRP-GTPase in the secretion of a bacterial toxin subunit.