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