Peter M. Wolanin

Influence of topology on bacterial social interaction

The environmental topology of complex structures is used by Escherichia coli to create traveling waves of high cell density, a prelude to quorum sensing. When cells are grown to a moderate density within a confining microenvironment, these traveling waves of cell density allow the cells to find and collapse into confining topologies, which are unstable to population fluctuations above a critical threshold. This was first observed in mazes designed to mimic complex environments, then more clearly in a simpler geometry consisting of a large open area surrounding a square (250 × 250 μm) with a narrow opening of 10–30 μm. Our results thus show that under nutrient-deprived conditions bacteria search out each other in a collective manner and that the bacteria can dynamically confine themselves to highly enclosed spaces.

Signal transduction: Receptor clusters as information processing arrays

The organization of transmembrane receptors into higher-order arrays occurs in cells as different as bacteria, lymphocytes and neurons. What are the implications of receptor clustering for short-term and long-term signaling processes that occur in response to ligand binding?

Bacterial Chemosensing: Cooperative Molecular Logic

Bacterial chemotaxis is mediated by transmembrane receptors that bind attractant and repellent chemicals and control an intracellular protein kinase. Each cell contains thousands of receptor subunits that form a tightly packed array at one pole. Recent studies of bacterial behavior have begun to reveal the molecular logic of this sensory architecture.

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Escherichia coli chemotaxis is mediated by membrane receptor/histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, lzTarC, that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from lzTarC. Thus, the organization of these receptor/signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo.

Transmembrane Signaling and the Regulation of Histidine Kinase Activity

Publisher Summary Transmembrane signal transduction plays a central role in biology. All cells transport information from surface receptors into the cytoplasm where it is processed and used to regulate virtually every aspect of biological activity. This is analogous to the uptake of nutrient molecules from the environment. In the case of membrane transporters and channels, the molecular basis for their function has become clear through high-resolution structural studies. This chapter focuses on functions of histidine protein kinase (HPK) and HPK-linked receptors in well-characterized prokaryotic systems that regulate gene expression and motility. In prokaryotes, histidine protein kinases (HPKs) transduce sensory inputs into protein phosphorylation outputs. Although all HPKs share homologous kinase catalytic domains, their activities are generally regulated by external stimuli via a wide variety of sensory input domains. Like tyrosine protein kinases, dimerization is important for HPK function. A possible insight into the mechanism of signaling by HPKs comes from observations of tight clustering by the chemotaxis receptors in Escherichia coli and other bacteria. The examination of the E. coli chemotaxis system reveals that changes in lateral interaction among hundreds or even thousands of receptors in large clusters play a key role in chemotaxis signal transduction.

Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism

Response regulator proteins of two‐component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY∼P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by α4, β5, α5, and α1. Our findings argue against the two‐state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.

Intramolecular rearrangements as a consequence of the dephosphorylation of phosphoaspartate residues in proteins

Aspartate phosphorylation induces changes in protein conformation that are used to regulate processes ranging from gene expression and cell differentiation to cell motility and the generation of electrochemical gradients across membranes. We show here that dephosphorylation of the phosphoaspartate in the chemotaxis response regulator CheY can result in the loss of a water molecule that may be due to formation of a succinimide intermediate.

Topologically Induced Bacterial Aggregation in A Microfabricated Random Maze

We have used techniques of soft lithography to fabricate complex but well controlled topological microenvironments in order to study the collective behavior of Escherichia coli. We have found that starting from a homogeneously distributed suspension of bacteria, they self-aggregate into confined geometries. This was first observed in PDMS mazes designed to mimic complex environments, then more clearly in a simple geometry consisting of a large open area surrounding a 250×250 micron square with a narrow 40 micron channel. Our results show that bacteria chemotactically communicate amongst themselves and search out collectively highly confined enyironments when they are under stressed conditions caused by local bacteria densities.