POSTERS - BLAST X - University of Utah

BLAST X Poster #24
ANALYSIS OF THE PEPTIDOGLYCAN-BINDING DOMAIN OF THE FLAGELLAR STATOR
PROTEIN MotB USING SYSTEMATIC MUTAGENESIS AND CHIMERIC PROTEIN IN
ESCHERICHIA COLI
Yohei Hizukuri 1 , John Frederick Morton 1 , Toshiharu Yakushi 1, 2 , Seiji Kojima 1 and Michio
Homma 1
1
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho,
Chikusa-Ku, Nagoya 464-8602, Japan
2
Present address: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi
University, Yamaguchi 753-8515, Japan
The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought
to be anchored to the peptidoglycan (PG) layer by the C-terminal conserved peptidoglycanbinding
(PGB) motif of MotB. To clarify the role of the C-terminal PGB region of MotB, we
performed systematic cysteine mutagenesis of this region in the Escherichia coli MotB.
Furthermore, we constructed three chimeric MotB proteins whose PGB regions were replaced
with corresponding regions of other PGB proteins, E. coli Pal (peptidoglycan-associated
lipoprotein), PomB (Vibrio MotB homolog) or MotY (Vibrio flagellar T-ring protein). Although
these chimeric proteins did not complement the motB mutant, we were able to isolate two
independent motile revertants from cells producing the MotB-Pal chimera. One is the F172V
mutation in the Pal region of the chimera, and this mutation did not affect the ability to bind to
PG when introduced into native Pal. The other is the P159L mutation in the MotB region, and
Pro159 mutation in native MotB has been reported to affect a spatial positioning of the
MotA/MotB stator complex relative to the motor switch complex. We speculated that the PGB
region of MotB-Pal chimera was properly aligned by the mutations and the stator and rotor could
interact properly. We tried to interpret phenotype of MotB Cys mutants by using the crystal
structure of the E. coli Pal, and found that MotB mutants that affected motility were nearly
overlapped with the predicted PG-binding residues of Pal. Our results indicate that the PGB
regions from functionally distinct proteins, MotB and Pal, works similarly to anchor the stator
complex.
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