Phototactic and Chemotactic Signal Transduction by Transmembrane Receptors and Transducers in Microorganisms

Microorganisms show attractant and repellent responses to survive in the various environments in which they live. Those phototaxic (to light) and chemotaxic (to chemicals) responses are regulated by membrane-embedded receptors and transducers. This article reviews the following: (1) the signal relay mechanisms by two photoreceptors, Sensory Rhodopsin I (SRI) and Sensory Rhodopsin II (SRII) and their transducers (HtrI and HtrII) responsible for phototaxis in microorganisms; and (2) the signal relay mechanism of a chemoreceptor/transducer protein, Tar, responsible for chemotaxis in E. coli. Based on results mainly obtained by our group together with other findings, the possible molecular mechanisms for phototaxis and chemotaxis are discussed

High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor.

The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P

Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells

Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells. Cultured rat glomerular mesangial and epithelial cells and bovine glomerular endothelial cells were exposed to various concentrations of hydrogen peroxide (H2O2). Mesangial cells treated with 10 to 100 µM H2O2 for 24 hours showed a two- to ninefold increase in Mn-SOD mRNA expression associated with significantly (P < 0.005) increased Mn-SOD activity (22.2 ± 1.2 and 12.2 ± 0.7 µ/mg protein for H2O2 100 µM treated and untreated cells, respectively). In contrast, expression of Cu-Zn SOD and β-actin mRNA was not affected by H2O2. Induction of Mn-SOD mRNA by H2O2 was inhibited by actinomycin-D (4 µM) treatment. Glomerular endothelial cells also showed an increase in Mn-SOD mRNA expression following 100 µM H2O2 treatment, as did glomerular epithelial cells following treatment with 500 and 1000 µM H2O2 but not with 100 µM. Transcriptional activity of the Mn-SOD gene was assessed with a fusion reporter gene consisting of a luciferase gene (pGL2P) and a 1.2 kb fragment from the rat Mn-SOD genomic DNA (-806 to +408 bp of the transcription initiation site, -806:+408). The construct was transfected into rat glomerular mesangial and epithelial cells. Mesangial and epithelial cells transfected with pGL2P (-806:+408) and treated with H2O2 (100 µM and 1 mM for mesangial and epithelial cells, respectively) demonstrated some threefold increase in luciferase activity, whereas cells transfected with pGL2P lacking the Mn-SOD fragment did not show changes in luciferase activity following H2O2 treatment. Other oxidants, namely α- and β-naphthoflavone (50 µM to mesangial cells) and puromycin aminonucleoside (25 to 50 µg/ml to epithelial cells), also induced transcriptional activation (2- to 5-fold increase) in these cells. Thus, Mn-SOD levels in glomerular cells are enhanced when they are exposed to oxidant stress, and this up-regulation involves transcriptional activation. Further, the Mn-SOD gene contains enhancer element(s) which respond to diverse oxidant stress. The inducibility by oxidants of local Mn-SOD demonstrates that glomerular SOD may play a decisive role in the pathogenesis of glomerular injuries in which the balance between oxidants and antioxidants is critical

Mutations in the stator protein PomA affect switching of rotational direction in bacterial flagellar motor

The flagellar motor rotates bi-directionally in counter-clockwise (CCW) and clockwise (CW) directions. The motor consists of a stator and a rotor. Recent structural studies have revealed that the stator is composed of a pentameric ring of A subunits and a dimer axis of B subunits. Highly conserved charged and neighboring residues of the A subunit interacts with the rotor, generating torque through a gear-like mechanism. The rotational direction is controlled by chemotaxis signaling transmitted to the rotor, with less evidence for the stator being involved. In this study, we report novel mutations that affect the switching of the rotational direction at the putative interaction site of the stator to generate rotational force. Our results highlight an aspect of flagellar motor function that appropriate switching of the interaction states between the stator and rotor is critical for controlling the rotational direction

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

Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motor

Takekawa Norihiro, Nishikino Tatsuro, Kishikawa Jun-ichi, et al. Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motor. mBio, e01261-24 (2024); https://doi.org/10.1128/mbio.01261-24.The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF–FliG fusion proteins and solved the near-atomic resolution structure of the S-ring—the upper part of the MS-ring—using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio, whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella, such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species

Fhl1 W122S causes loss of protein function and late-onset mild myopathy.

A member of the four-and-a-half-LIM (FHL) domain protein family, FHL1, is highly expressed in human adult skeletal and cardiac muscle. Mutations in FHL1 have been associated with diverse X-linked muscle diseases: scapuloperoneal (SP) myopathy, reducing body myopathy, X-linked myopathy with postural muscle atrophy, rigid spine syndrome (RSS) and Emery-Dreifuss muscular dystrophy. In 2008, we identified a missense mutation in the second LIM domain of FHL1 (c.365 G>C, p.W122S) in a family with SP myopathy. We generated a knock-in mouse model harboring the c.365 G>C Fhl1 mutation and investigated the effects of this mutation at three time points (3–5 months, 7–10 months and 18–20 months) in hemizygous male and heterozygous female mice. Survival was comparable in mutant and wild-type animals. We observed decreased forelimb strength and exercise capacity in adult hemizygous male mice starting from 7 to 10 months of age. Western blot analysis showed absence of Fhl1 in muscle at later stages. Thus, adult hemizygous male, but not heterozygous female, mice showed a slowly progressive phenotype similar to human patients with late-onset muscle weakness. In contrast to SP myopathy patients with the FHL1 W122S mutation, mutant mice did not manifest cytoplasmic inclusions (reducing bodies) in muscle. Because muscle weakness was evident prior to loss of Fhl1 protein and without reducing bodies, our findings indicate that loss of function is responsible for the myopathy in the Fhl1 W122S knock-in mice

Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery

In Escherichia coli, chemoreceptor clustering at a cell pole seems critical for signal amplification and adaptation. However, little is known about the mechanism of localization itself. Here we examined whether the aspartate chemoreceptor (Tar) is inserted directly into the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar–GFP, fluorescent spots first appeared in lateral membrane regions, and later cell poles became predominantly fluorescent. Unexpectedly, Tar–GFP showed a helical arrangement in lateral regions, which was more apparent when a Tar–GFP derivative with two cysteine residues in the periplasmic domain was cross-linked to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domain of the double cysteine Tar–GFP mutant was replaced by that of the kinase EnvZ, which does not localize to a pole. Observation of GFP–SecE and a translocation-defective MalE–GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested that the general protein translocation machinery (Sec) itself is arranged into a helical array, with which Tar is transiently associated. The Sec coil appeared distinct from the MreB coil, an actin-like cytoskeleton. These findings will shed new light on the mechanisms underlying spatial organization of membrane proteins in E. coli

Characterization of FliL Proteins in <i>Bradyrhizobium diazoefficiens</i> : Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum inductionInstituto de Biotecnologia y Biologia Molecula