Demethylation of bacterial chemoreceptors is inhibited by attractant stimuli in the complete absence of the regulatory domain of the demethylating enzyme

The CheB methylesterase catalyzes the demethylation of membrane receptors during chemotaxis in Salmonella typhimurium. The kinetic properties of the full length product of the cheB gene are compared to those of the isolated C-terminal catalytic domain. The fragment has at least a 15-fold higher specific activity than the intact protein. In intact cells receptor demethylation is inhibited by attractants such as L-aspartate. We show here that both forms of the enzyme are similarly inhibited in vitro. Thus, the C-terminal catalytic domain of the CheB protein is sufficient for this aspect of esterase regulation. Inhibition by attractants appears to be caused by changes in receptor conformation rather than by changes in the activity of the demethylating enzyme.     

Transmembrane signaling in bacterial chemoreceptors

Bacterial chemoreceptors mediate chemotaxis by recognizing specific chemicals and regulating a noncovalently associated histidine kinase. Ligand binding to the external domain of the membrane-spanning receptor generates a transmembrane signal that modulates kinase activity inside the cell. This transmembrane signaling is being investigated by novel strategies, which have revealed a remarkably subtle conformational signal carried by a signaling helix that spans the entire length of the >350-A-long receptor. Multiple, independent lines of evidence indicate that, in the periplasmic and transmembrane domains, conformational signaling is a piston-type sliding of the signaling helix towards the cytoplasm.     

Physical responses of bacterial chemoreceptors

Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple "two-state" model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.     

Adaptational assistance in clusters of bacterial chemoreceptors

Sensory adaptation of low-abundance chemoreceptors in Escherichia coli requires assistance from high-abundance receptors, because only high-abundance receptors carry the carboxyl-terminal pentapeptide sequence NWETF that enhances adaptational covalent modification. Using membrane vesicles containing both high-abundance receptor Tar and low-abundance receptor Trg, we observed effective assistance in vitro for all three adaptational modifications: methylation, demethylation and deamidation. These results demonstrated that adaptational assistance involves not only the previously documented assistance for methylation but also assistance for the two CheB-catalysed reactions. We determined rates of assisted methylation and demethylation at many ratios of assisting to assisted receptor. Analysis by a model of assistance indicated one Tar dimer could assist seven Trg dimers in methylation or five in demethylation, defining assistance neighbourhoods. These neighbourhoods were larger than a trimer of homodimers, required only receptors and were minimally affected by formation of signalling complexes. Time courses of assisted Trg methylation in membranes with low amounts of Tar showed that assisting receptors did not diffuse beyond initial neighbourhoods for at least two hours. Taken together, these observations indicate that chemoreceptors can form stable neighbourhoods larger than trimers in the absence of other chemotaxis proteins. Such interactions are likely to occur in natural receptor clusters in vivo.     

Oxygen as attractant and repellent in bacterial chemotaxis.

Studies of bacterial chemotaxis to oxygen (aerotaxis) over a broad range of oxygen concentrations showed that at high concentrations, oxygen was a repellent of Salmonella typhimurium, Escherichia coli, and some bacilli, whereas it is known that at lower concentrations (less than or equal to 0.25 mM dissolved oxygen), oxygen is an attractant. In a temporal assay of aerotaxis, S. typhimurium in medium equilibrated with air (0.25 mM dissolved oxygen) and then exposed to pure oxygen (1.2 mM) tumbled continuously for approximately 20 s. The oxygen concentration that elicited a half-maximal negative (repellent) response was 1.0 mM for both S. typhimurium and E. coli. The receptor for the negative chemoresponse to high concentrations of oxygen is apparently different from the receptor for the positive chemoresponse to low concentrations of oxygen, since the oxygen concentration that elicits a half-maximal positive (attractant) response in S. typhimurium and E. coli is reported to be 0.7 microM. Adaptation to high concentrations of oxygen, like adaptation to low concentrations of oxygen, was independent of methylation of a transducer protein. Only the response to low oxygen concentrations, however, was altered by interaction with the amidated Tsr transducer in cheB mutants.     

The physical and functional thermal sensitivity of bacterial chemoreceptors

The bacterium Escherichia coli exhibits chemotactic behavior at temperatures ranging from approximately 20 °C to at least 42 °C. This behavior is controlled by clusters of transmembrane chemoreceptors made from trimers of dimers that are linked together by cross-binding to cytoplasmic components. By detecting fluorescence energy transfer between various components of this system, we studied the underlying molecular behavior of these receptors in vivo and throughout their operating temperature range. We reveal a sharp modulation in the conformation of unclustered and clustered receptor trimers and, consequently, in kinase activity output. These modulations occurred at a characteristic temperature that depended on clustering and were lower for receptors at lower adaptational states. However, in the presence of dynamic adaptation, the response of kinase activity to a stimulus was sustained up to 45 °C, but sensitivity notably decreased. Thus, this molecular system exhibits a clear thermal sensitivity that emerges at the level of receptor trimers, but both receptor clustering and adaptation support the overall robust operation of the system at elevated temperatures.     

Flux detectors versus concentration detectors: two types of chemoreceptors

Dose-response curves relating the external stimulus concentration to receptor occupancy differ in two types of chemoreceptor organs. In 'concentration detectors' the receptor molecules at the receptor cell membrane are directly exposed to the external stimulus concentration; these organs exhibit the well-known hyperbolic dose-response relationship reflecting the association-dissociation of stimulus and receptor molecules. In contrast, 'flux detectors' accumulate the stimulus molecules in a perireceptor compartment. In flux detectors, deactivation of stimulus molecules may be in balance with arrival, as a prerequisite for producing a constant effective stimulus concentration at constant adsorptive flux of stimulus molecules. In a simple model of a flux detector in which receptor molecules themselves catalyze the deactivation, the dose-response relationship is linear. It reflects the rate of stimulus deactivation. If the deactivation is catalyzed by a separate enzyme, the dose-response relationship can be close to hyperbolic, or linear. In all cases, the receptor molecules are maximally occupied if the adsorptive flux equals or exceeds the maximum rate of stimulus deactivation. The time course of the receptor potential recorded from moths' pheromone receptors depends on the odor compound, which suggests that a peripheral process, possibly the stimulus deactivation, is the slowest, rate-limiting process of the transduction cascade. Further evidence comes from experiments with stimuli oversaturating the mechanism responsible for the decline of the receptor potential.      

Cell cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division

The master CtrA response regulator functions in Caulobacter to repress replication initiation in different phases of the cell cycle. Here, we identify an essential histidine kinase, CckA, that is responsible for CtrA activation by phosphorylation. Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. Removal of the membrane-spanning region of CckA results in loss of polar localization and cell death. We propose that polar CckA functions to activate CtrA just after the initiation of DNA replication, thereby preventing premature reinitiations of chromosome replication. Thus, dynamic changes in cellular location of critical signal proteins provide a novel mechanism for the control of the prokaryote cell cycle.     

Mutations that disrupt the morphogenesis and localization of a sperm-specific organelle in Caenorhabditis elegans

Nematode sperm contain unusual organelles, membranous organelles, which undergo dramatic morphological changes during spermatogenesis. Early in spermatogenesis, the membranous organelle functions to transport sperm specific components to the spermatids; later, during the formation of the crawling spermatozoa, it adds new components to the cell surface as it fuses with the plasma membrane. Genetic analysis of spermatogenesis in the nematode Caenorhabditis elegans has revealed mutations that specifically disrupt the proper cellular localization and morphogenesis of this organelle. In animals homozygous for the either the known deficiency hcDf1 or the probable deficiency h12, the membranes of the membranous organelles are aberrantly covered with ribosomes. A mutation in the spermatogenesis-defective spe-10 gene causes severe defects in the morphogenesis of a fibrous body-membranous organelle complex. In both cases, these mutations also disrupt the proper localization of both nuclei and membranous organelles in haploid spermatids and spermatozoa.     

Intracellular localization of the active process in polar transport of auxin

Summary The cytoplasm of maize coleoptile cells was displaced to either the apical or basal ends of the cells by centrifuging (1750xg for 10 min) segments in which protoplasmic streaming had been stopped by pretreatment with cytochalasin B. Centrifugation toward the base of the segment promotes the subsequent basipetal transport of indole-3-acetic acid, whereas apical centrifugation dramatically inhibits this transport. Apical centrifugation neither promotes acropetal transport nor reverses the polarity of auxin transport. Experiments in which the amyloplasts were separated from the bulk of the cytoplasm indicate that the basipetal transport is independent of both the position and pressure exerted by the amyloplasts but is strongly dependent on the amount of cytoplasm at the basal end of the cells. These effects of centrifugation on auxin transport lead to the conclusion that the metabolic component of the transport is a polar secretion of auxin localized in the basal plasma membrane of each cell.     

A mechanism for polar protein localization in bacteria

We investigate a mechanism for the polar localization of proteins in bacteria. We focus on the MinCD/DivIVA system regulating division site placement in the rod-shaped bacterium Bacillus subtilis. Our model relies on a combination of geometric effects and reaction-diffusion dynamics to direct proteins to both cell poles, where division is then blocked. We discuss similarities and differences with related division models in Escherichia coli and also develop extensions of the model to asymmetric polar protein localization. We propose that our mechanism for polar localization may be employed more widely in bacteria, especially in outgrowing spores, which do not possess any pre-existing polar division apparatus from prior division events.     

Identification of a chemoreceptor zinc-binding domain common to cytoplasmic bacterial chemoreceptors

We report the identification and characterization of a previously unidentified protein domain found in bacterial chemoreceptors and other bacterial signal transduction proteins. This domain contains a motif of three noncontiguous histidines and one cysteine, arranged as Hxx[WFYL]x(21-28)Cx[LFMVI]Gx[WFLVI]x(18-27)HxxxH(boldface type indicates residues that are nearly 100% conserved). This domain was first identified in the soluble Helicobacter pylori chemoreceptor TlpD. Using inductively coupled plasma mass spectrometry on heterologously and natively expressed TlpD, we determined that this domain binds zinc with a subfemtomolar dissociation constant. We thus named the domain CZB, for chemoreceptor zinc binding. Further analysis showed that many bacterial signaling proteins contain the CZB domain, most commonly proteins that participate in chemotaxis but also those that participate in c-di-GMP signaling and nitrate/nitrite sensing, among others. Proteins bearing the CZB domain are found in several bacterial phyla. The variety of signaling proteins using the CZB domain suggests that it plays a critical role in several signal transduction pathways.     

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure–activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.     

Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors.     

Ultrasound increases the rate of bacterial cell growth

Ultrasound was employed to increase the growth rate of bacterial cells attached to surfaces. Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli cells adhered to and grew on a polyethylene surface in the presence of ultrasound. It was found that low-frequency ultrasound (70 kHz) of low acoustic intensity (<2 W/cm(2)) increased the growth rate of the cells compared to growth without ultrasound. However, at high intensity levels, cells were partially removed from the surface. Ultrasound also enhanced planktonic growth of S. epidermidis and other planktonic bacteria. It is hypothesized that ultrasound increases the rate of transport of oxygen and nutrients to the cells and increases the rate of transport of waste products away from the cells, thus enhancing their growth.