Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP.

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttle-mediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students' understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice.

Identification of an anchor residue for CheA-CheY interactions in the chemotaxis system of Escherichia coli.

Transfer of a phosphoryl group from autophosphorylated CheA (P-CheA) to CheY is an important step in the bacterial chemotaxis signal transduction pathway. This reaction involves CheY (i) binding to the P2 domain of P-CheA and then (ii) acquiring the phosphoryl group from the P1 domain. Crystal structures indicated numerous side chain interactions at the CheY-P2 binding interface.

Kinetics of ATP and TNP-ATP binding to the active site of CheA from Thermotoga maritima.

The mechanism of nucleotide binding to the active site of Thermotoga maritima CheA was investigated using stopped-flow fluorescence experiments that monitored binding of ATP and TNP-ATP to the catalytic domain (P4) of CheA that had been engineered to include a tryptophan residue as a fluorescent reporter group at the active site (P4(F487W)). Rapid decreases in protein intrinsic fluorescence and increases in TNP-ATP fluorescence were observed during binding reactions, and time courses were analyzed to define the kinetic mechanisms for ATP and TNP-ATP binding. This analysis indicated that binding of ATP(Mg(2+)) to P4(F487W) involves a single reversible step with a k(on) of 0.

Protein histidine kinases: assembly of active sites and their regulation in signaling pathways.

Protein histidine kinases (PHKs) function in Two Component Signaling pathways utilized extensively by bacteria and archaea. Many PHKs participate in three distinct, but interrelated signaling reactions: autophoshorylation, phosphotransfer (to a partner Response Regulator (RR) protein), and dephosphorylation of this RR. Detailed biochemical and structural characterization of several PHKs has revealed how the domains of these proteins can interact to assemble the three active sites that promote the necessary chemistry and how these domain interactions might be regulated in response to sensory input: the relative orientation of helices in the PHK dimerization domain can reorient, via cogwheeling (rotation) and kinking (bending), to effect changes in PHK activities that probably involve sequestration/release of the PHK catalytic domain by the dimerization domain.

The two active sites of Thermotoga maritima CheA dimers bind ATP with dramatically different affinities.

CheA is a central component of the chemotaxis signal transduction pathway that allows prokaryotic cells to control their movements in response to environmental cues. This dimeric protein histidine kinase autophosphorylates via an intersubunit phosphorylation reaction in which each protomer of the dimer binds ATP, at an active site located in its P4 domain and then catalyzes transfer of the gamma-phosphoryl group of ATP to the His(45) side chain within the P1 domain of the trans protomer. Here we utilize the fluorescent nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] to investigate the two ATP-binding sites of the Thermotoga maritima CheA dimer (TmCheA) and the single site of the isolated TmP4 domain (a monomer).

Analysis of ATP binding to CheA containing tryptophan substitutions near the active site.

Signal transduction in the chemotaxis system of Escherichia coli involves an autophosphorylating protein histidine kinase, CheA. At the active site of CheA, phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket, immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change ("lid closure") that has been characterized previously in X-ray crystal structures [Bilwes et al. (2001) Nat.

Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein.

In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA. Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)). Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest.

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA.

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA.

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.

CheA kinase and chemoreceptor interaction surfaces on CheW.

Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar.

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli.

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated.