George H. Wadhams

MAKING SENSE OF IT ALL: BACTERIAL CHEMOTAXIS

Bacteria must be able to respond to a changing environment, and one way to respond is to move. The transduction of sensory signals alters the concentration of small phosphorylated response regulators that bind to the rotary flagellar motor and cause switching. This simple pathway has provided a paradigm for sensory systems in general. However, the increasing number of sequenced bacterial genomes shows that although the central sensory mechanism seems to be common to all bacteria, there is added complexity in a wide range of species.

Stoichiometry and turnover in single, functioning membrane protein complexes

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP–MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains ∼22 copies of GFP–MotB, consistent with ∼11 stators each containing two MotB molecules. We also observed a membrane pool of ∼200 GFP–MotB molecules diffusing at ∼0.008 µm2 s-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP–MotB between the membrane pool and motor with a rate constant of the order of 0.04 s-1: the dwell time of a given stator in the motor is only ∼0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.

Signal processing in complex chemotaxis pathways

Bacteria use chemotaxis to migrate towards environments that are better for growth. Chemoreceptors detect changes in attractant levels and signal through two-component systems to control swimming direction. This basic pathway is conserved across all chemotactic bacteria and archaea; however, recent work combining systems biology and genome sequencing has started to elucidate the additional complexity of the process in many bacterial species. This article focuses on one of the best understood complex networks, which is found in Rhodobacter sphaeroides and integrates sensory data about the external environment and the metabolic state of the cell to produce a balanced response at the flagellar motor.

Signal-dependent turnover of the bacterial flagellar switch protein FliM

Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.

Targeting of two signal transduction pathways to different regions of the bacterial cell.

Components of bacterial chemosensory pathways which sense via transmembrane receptors have been shown to localize to the cell poles. Many species, however, have operons encoding multiple putative chemosensory pathways, some including putative cytoplasmic receptors. In‐genome fusions to single or multiple genes encoding components of two chemosensory pathways in Rhodobacter sphaeroides, cheOp2 and cheOp3, revealed that while sensory transducing proteins associated with transmembrane receptors and encoded on cheOp2 were targeted to the cell poles, the proteins associated with putative cytoplasmic receptors and encoded on cheOp3 were all targeted to a cytoplasmic cluster. No proteins were localized to both sites. These data show that bacteria target components of related pathways to different sites in the cell, presumably preventing direct cross‐talk between the different pathways, but allowing a balanced response between extracellular and cytoplasmic signals. It also indicates that there is intracellular organization in bacterial cells, with specific proteins targeted and localized to cytoplasmic regions.

Localization of MreB in Rhodobacter sphaeroides under conditions causing changes in cell shape and membrane structure

MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R. sphaeroides shape was therefore investigated. Attempts at deleting or insertionally inactivating mreB were unsuccessful under all growth conditions. Immunofluorescence microscopy showed MreB localized to mid-cell in elongating cells under both aerobic and photoheterotrophic conditions. Three-dimensional reconstruction showed that MreB formed a ring at mid-cell. MreB remained at mid-cell as septation began but localized to new sites in the daughter cells before the completion of septation. MreB localized to putative septation sites in cephalexin-treated filamentous cells. Genomic single-copy mreB was replaced with gfp-mreB, and green fluorescent protein (GFP)-MreB localized in the same pattern, as seen with immunofluorescence microscopy. Some of the cells expressing GFP-MreB were abnormal, principally displaying an increase in cell width, suggesting that the fusion was not fully functional in all cells. GFP-MreB localized to swellings at mid-cell in cells treated with the penicillin-binding protein 2 inhibitor amdinocillin. These data suggest that MreB is essential in R. sphaeroides, performing a role at mid-cell in elongating cells, and in early septation, putatively in the cytoplasmic control of the peptidoglycan synthetic complexes.

Rhodobacter sphaeroides: complexity in chemotactic signalling

Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins. The roles of these homologues have been extensively investigated using a combination of deletion, subcellular localization and phosphorylation assays. These studies have shown that the homologues have specific roles in the sensory pathway, and they differ in their cellular localization and interactions with other components of the pathway. The presence of multiple chemosensory pathways might enable bacteria to tune their tactic responses to different environmental conditions.

The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in‐frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non‐chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl‐accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.

TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm

TlpC is encoded in the second chemotaxis operon of Rhodobacter sphaeroides. This protein shows some homology to membrane‐spanning chemoreceptors of many bacterial species but, unlike these, is essential for R. sphaeroides chemotaxis to all compounds tested. Genomic replacement of tlpC with a C‐terminal gfp fusion demonstrated that TlpC localized to a discrete cluster within the cytoplasm. Immunogold electron microscopy also showed that TlpC localized to a cytoplasmic electron‐dense region. Correct TlpC–GFP localization depended on the downstream signalling proteins, CheW3, CheW4 and CheA2, and was tightly linked to cell division. Newly divided cells contained a single cluster but, as the cell cycle progressed, a second cluster appeared close to the initial cluster. As elongation continued, these clusters moved apart so that, on septation, each daughter cell contained a single TlpC cluster. The data presented suggest that TlpC is either a cytoplasmic chemoreceptor responding to or integrating global signals of metabolic state or a novel and essential component of the chemotaxis signalling pathway. These data also suggest that clustering is essential for signalling and that a mechanism may exist for targeting and localizing proteins within the bacterial cytoplasm.

Constitutive Dimerization of the G-Protein Coupled Receptor, Neurotensin Receptor 1, Reconstituted into Phospholipid Bilayers

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.