Michael D. Manson

A sensitive, versatile microfluidic assay for bacterial chemotaxis

We have developed a microfluidic assay for bacterial chemotaxis in which a gradient of chemoeffectors is established inside a microchannel via diffusion between parallel streams of liquid in laminar flow. The random motility and chemotactic responses to l-aspartate, l-serine, l-leucine, and Ni2+ of WT and chemotactic-mutant strains of Escherichia coli were measured. Migration of the cells was quantified by counting the cells accumulating in each of 22 outlet ports. The sensitivity of the assay is attested to by the significant response of WT cells to 3.2 nM l-aspartate, a concentration three orders of magnitude lower than the detection limit in the standard capillary assay. The response to repellents was as robust and easily recorded as the attractant response. A surprising discovery was that l-leucine is sensed by Tar as an attractant at low concentrations and by Tsr as a repellent at higher concentrations. This assay offers superior performance and convenience relative to the existing assays to measure bacterial tactic responses, and it is flexible enough to be used in a wide range of different applications.

Holins kill without warning

Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis “clock,” in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.

Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium : characterization of the dipeptide permease (Dpp) and the dipeptide-binding protein

The dipeptide permease (Dpp) is one of three genetically distinct peptide‐transport systems in enteric bacteria. Dpp also plays a role in chemotaxis towards peptides. We have devised three selections for dpp mutations based on resistance to toxic peptides (bacilysin, valine‐containing peptides, and biala‐phos). All dpp mutations mapped to a single chromosomal locus between 77 and 78 min in Salmonella typhimurium and at 79.2 min in Escherichia coli. Expression of dpp was constitutive in both species but the absolute level of expression varied widely between strains. At least in part this difference in expression levels is determined by c/s‐acting sequences. The dpp locus of E. coli was cloned. The first gene in the operon, dppA, encodes a periplasmic dipeptide‐binding protein (DBP) required for dipeptide transport and chemotaxis. Downstream of dppA are other genes required for transport but not for chemotaxis. The dipeptide‐binding protein was found to share 26.5% sequence identity with the periplasmic oligopeptide‐binding protein OppA.

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells.

The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.

Energetics of flagellar rotation in bacteria

We have measured the rotation rates of tethered cells of Streptococcus strain V4051 as a function of dynamic load and protonmotive force. The cells do not spin in the absence of an exogenous energy source, but they start spinning 20 to 30 seconds after exposure to glucose. The angular velocity of metabolizing cells is inversely proportional to the viscosity of the medium. Starved cells spin within ten seconds after valinomycin is added to induce a potassium diffusion potential (cell interior negative) or after the medium is acidified to generate a transmembrane pH gradient (cell interior alkaline). In either case, the angular velocity of the cells is a linear function of the protonmotive force. These results imply that the passage of a fixed number of protons carries the flagellar motor through one revolution. The threshold protonmotive force for rotation, although not determined directly, appears to lie close to 0 mV. Starved cells also spin when protons move out of the cells in response to a diffusion potential or upon alkalinization of the medium. Thus, flagellar rotation can be driven by a protonmotive force of either sign.

CheZ Phosphatase Localizes to Chemoreceptor Patches via CheA-Short

We have investigated the conditions required for polar localization of the CheZ phosphatase by using a CheZ-green fluorescent protein fusion protein that, when expressed from a single gene in the chromosome, restored chemotaxis to a DeltacheZ strain. Localization was observed in wild-type, DeltacheZ, DeltacheYZ, and DeltacheRB cells but not in cells with cheA, cheW, or all chemoreceptor genes except aer deleted. Cells making only CheA-short (CheA(S)) or CheA lacking the P2 domain also retained normal localization, whereas cells producing only CheA-long or CheA missing the P1 and P2 domains did not. We conclude that CheZ localization requires the truncated C-terminal portion of the P1 domain present in CheA(S). Missense mutations targeting residues 83 through 120 of CheZ also abolished localization. Two of these mutations do not disrupt chemotaxis, indicating that they specifically prevent interaction with CheA(S) while leaving other activities of CheZ intact.

Bacterial motility and chemotaxis

Publisher Summary This chapter summarizes the basic features of bacterial motility and chemotaxis. Bacterial chemotaxis represents one of the simplest behaviors that can be studied. Its popularity as an experimental system stems from several sources. The genetic and biochemical tools have been developed to investigate the biology of enteric bacteria. The motility and chemotaxis can be virulence factors for intestinal and urogenital-tract pathogens. Flagella constitute one of the most effective bacterial antigens and the immune system directs its counterattacks against it. Chemotaxis also occurs in bacteria within the rhizosphere and in aquatic environments. Polar flagella arise at the morphologically definable ends of rod-shaped or curved bacterial cells. Bacteria can move in two or in three dimensions. Those that move on surfaces without flagella exhibit gliding motility, whereas those that rely on flagella to move on surfaces exhibit swarming motility. Any movement in three dimensions is called swimming. Bacteria have chemoreceptors, namely, proteins that bind to the effector ligands in a stereospecific way. The bacteria sense spatial gradients by means of a temporal mechanism. In addition to being attracted to or repelled by specific chemicals, bacteria exhibit phototaxis, magnetotaxis, osmotaxis, galvanotaxis, and thermotaxis. The chapter also describes the chemoreceptor structure and ligand binding and outlines the subsequent steps in signal transduction.

Attractant Signaling by an Aspartate Chemoreceptor Dimer with a Single Cytoplasmic Domain

Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood. The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA. The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro. Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate. Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients

ABSTRACT Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.

Chemotaxis to the Quorum-Sensing Signal AI-2 Requires the Tsr Chemoreceptor and the Periplasmic LsrB AI-2-Binding Protein

AI-2 is an autoinducer made by many bacteria. LsrB binds AI-2 in the periplasm, and Tsr is the l-serine chemoreceptor. We show that AI-2 strongly attracts Escherichia coli. Both LsrB and Tsr are necessary for sensing AI-2, but AI-2 uptake is not, suggesting that LsrB and Tsr interact directly in the periplasm.