Yohei Hizukuri

The peptidoglycan-binding (PGB) Domain of the Escherichia coli Pal Protein can also Function as the PGB Domain in E. coli Flagellar Motor Protein MotB

The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan by the C-terminal conserved peptidoglycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal region, we performed systematic cysteine mutagenesis and constructed a chimeric MotB protein which was replaced with the peptidoglycan-associated lipoprotein Pal. Although this chimera could not restore motility to a motB strain, we were able to isolate two motile revertants. One was F172V in the Pal region and the other was P159L in the MotB region. Furthermore, we attempted to map the MotB Cys mutations in the crystal structure of Escherichia coli Pal. We found that the MotB mutations that affected motility nearly overlapped with the predicted PG-binding residues of Pal. Our results indicate that, although the functions of MotB and Pal are very different, the PGB region of Pal is interchangeable with the PGB region of MotB.

Role of the Intramolecular Disulfide Bond in FlgI, the Flagellar P-Ring Component of Escherichia coli

The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our DeltaflgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of DeltadsbB cells. Additionally, the low level of FlgI observed in the DeltadsbB strain increased in the presence of l-cystine, an oxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient self-assembly into the P ring.

Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor

The flagellar motor is composed of the stator and the rotor, and the interaction between the stator and the rotor at the cytoplasmic region is believed to produce mechanical force for the rotation of flagella. The periplasmic region of the stator has been proposed to play an important role in assembly around and incorporation into the motor. In this study, we provide evidence suggesting that the periplasmic region of the stator component MotB interacts with the P-ring component FlgI, which functions as a bearing for the rotor along with the L-ring protein FlgH, from a site-directed disulphide cross-linking approach. First, we prepared four FlgI and three MotB cysteine-substituted mutant proteins and co-expressed them in various combinations in Escherichia coli. We detected cross-linked combinations of FlgI G11C and MotB S248C when treated with the oxidant Cu-phenanthroline or bismaleimide cross-linkers. Furthermore, we performed Cys-scanning mutagenesis around these two residues and found additional combinations of cross-linked residues. Treatment with a protonophore CCCP significantly reduced the cross-linking efficiency between FlgI and MotB in flagellated cells, but not in non-flagellated cells. These results suggest a direct contact between MotB and FlgI upon assembly of the stator into a motor.

Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli

The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted Escherichia coli P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts of five FlgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The five residues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FlgI proteins, the highly conserved region in the N terminus, residues 1–120, of FlgI is speculated to play important roles in the stabilization of FlgI structure and the formation of the P ring by interacting with FlgI molecules and/or other flagellar components.