Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor

Abstract

SummaryMotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFPFliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotA C), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotAC with FliG is required for the efficient assembly of the stators around the rotor.

Assembly and Activation of the MotA/B Proton Channel Complex of the Proton-Driven Flagellar Motor of Salmonella enterica

“…Since the stall torque is dependent on the number of functional stators in the motor (Fig. 4), this suggests that MotA occupies the stator-binding sites of the motor and reduces the number of functional stators (Morimoto et al, 2010b). In agreement with this, fluorescent spots of MotA-mCherry have been observed at the same position as those of GFP-FliG even in the absence of MotB (Fig.…”

“…In agreement with this, fluorescent spots of MotA-mCherry have been observed at the same position as those of GFP-FliG even in the absence of MotB (Fig. 6) (Morimoto et al, 2010b). Thus, MotA alone can be installed into a motor.…”

“…Two charged residues of MotA, Arg-90 and Glu-98, are involved in electrostatic interactions with charged residues of FliG for torque generation (Zhou & Blair, 1997;Zhou et al, 1998a). The motA(R90E) and motA(E98K) alleles are recessive and hence do not exert a negative dominance when their expression levels were the same as those of wild-type cells (Zhou & Blair, 1997;Morimoto et al, 2010b). Interestingly, however, an increase in the expression level of the MotA(R90E)/B complex by more than 10-fold allows 70% of the cells to swim in liquid media (Morimoto et al, 2010b).…”

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

“…After fusing the fluorescent proteins YPet, eGFP and Dendra2 directly to the N-terminus of MotB (FPs-MotB), as done in several studies with (e) 15,18,19,21,29,38 , we verified that the constructed strains were motile in soft agar at the population level (Fig. 1B).…”

Bioinformatic analysis of the flagellar system of the rickettsia-like alphaproteobacteria Candidatus Hepatobacter penaei

“…De igual forma, Arg90 y Glu98 son aminoácidos altamente conservados (Morimoto et al 2010) y ambos fueron detectadas en la proteína deducida MotA de Candidatus Hepatobacter penaei. La sustitución de estos aminoácidos por mutaciones puede tener un efecto negativo en el motor y con ello en la motilidad de la bacteria, debido a que tiene un efecto directo en la rotación del flagelo.…”