Bacterial f lagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the f lagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.Many species of bacteria are propelled by flagella, each consisting of a thin helical propeller attached, via a flexible coupling, to a rotary motor in the cell membrane (for recent reviews, see refs. 1 and 2). The energy for rotation of the flagella comes from the transmembrane gradient of protons (3-5) or, in certain marine or alkaliphilic species, sodium ions (6). The molecular mechanism of torque generation is not understood. The proteins most closely involved are MotA, MotB, and FliG. MotA and MotB are integral membrane proteins (7-11) that function in proton conduction (12)(13)(14) and that are believed to form the stator, or nonrotating part, of the motor (9,(15)(16)(17). FliG is located on the cytoplasmic side of the membrane, and is thought to be a component of the rotor (18)(19)(20)(21)(22).In hypotheses for the mechanism of the flagellar motor, charged residues have often been suggested to play key roles, serving, for example, to regulate movements of the rotor or to control proton flow (5,(23)(24)(25). We recently identified charged residues essential for motor function in both the rotor protein FliG and the stator protein MotA in mutational analyses of these proteins (26,27). In FliG, the residues Arg-281, Asp-288, and Asp-289 are of primary importance for function, whereas residues Lys-264 and Arg-297 might have secondary roles [FliG residue numbers here are higher by two than those given in the previous study, owing to recent corrections to the Escherichia coli fliG sequence (28)]. In MotA, the charged residues most important for function are Arg-90 and Glu-98, whereas Glu-150 may have a secondary role. Mutant phenotypes suggest that charge is the most important feature of these residues and that the charged residues in each protein function redundantly. Single charge-neutralizing replacements had only mild effects on function, whereas double rep...

Pathogenic Yersinia species inject virulence proteins, known as Yops, into the cytosol of eukaryotic cells. The injection of Yops is mediated via a type III secretion system. Previous studies have suggested that YopE is targeted for secretion by two signals. One is mediated by its cognate chaperone YerA, whereas the other consists of either the 5′ end of yopE mRNA or the N‐terminus of YopE. In order to characterize the YopE N‐terminal/5′ mRNA secretion signal, the first 11 codons of yopE were systematically mutagenized. Frameshift mutations, which completely alter the amino acid sequence of residues 2–11 but leave the mRNA sequence essentially intact, drastically reduce the secretion of YopE in a yerA mutant. In contrast, a mutation that alters the yopE mRNA sequence, while leaving the amino acid sequence of YopE unchanged, does not impair the secretion of YopE. Therefore, the N‐terminus of YopE, and not the 5′ end of yopE mRNA, serves as a targeting signal for type III secretion. In addition, the chaperone YerA can target YopE for type III secretion in the absence of a functional N‐terminal signal. Mutational analysis of the YopE N‐terminus revealed that a synthetic amphipathic sequence of eight residues is sufficient to serve as a targeting signal. YopE is also secreted rapidly upon a shift to secretion‐permissive conditions. This ‘rapid secretion’ of YopE does not require de novo protein synthesis and is dependent upon YerA. Furthermore, this burst of YopE secretion can induce a cytotoxic response in infected HeLa cells.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot ؊ (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot ؊ defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.Many motile bacteria are propelled by thin helical filaments that are each driven at the base by a rotary motor embedded in the cell membrane (reviewed in references 1, 20, 25). The filament/motor organelle is called a flagellum. In contrast to eukaryotic molecular motors, which use nucleoside triphosphates as an energy source, bacterial rotary motors are powered by the transmembrane gradient of protons (19,21) or, in some species, sodium ions (14). The mechanism by which bacterial flagellar motors convert the chemical energy of the ion gradient into the mechanical energy of rotation is not yet understood.Approximately 50 genes are needed for the assembly and operation of the flagella of Escherichia coli or Salmonella typhimurium (20). Among these, only a few encode products thought to be directly involved in the process of torque generation. Genes whose products might participate in torque generation have been identified in extensive mutant screens as those giving the Mot Ϫ phenotype, in which flagella are assembled but do not rotate. The presumption has been that mot mutations affect torque-generating activities specifically, while preserving the features of a protein needed for flagellar assembly. By using this criterion, five proteins that could have direct roles in torque generati...

No abstract

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37°C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface.

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram‐negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersiniaouter protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non‐secreted TTSS chaperone LcrH (low‐calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild‐type levels of YopD. Furthermore, the YopD–LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho‐GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two‐hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone–substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co‐ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.