Chemotactic signaling via carbohydrate phosphotransferase systems in Escherichia coli

Abstract

Chemotaxis allows bacteria to follow gradients of nutrients, environmental stimuli, and signaling molecules, optimizing bacterial growth and survival. Escherichia coli has long served as a model of bacterial chemotaxis, and the signal processing by the core of its chemotaxis pathway is well understood. However, most of the research so far has focused on one branch of chemotactic signaling, in which ligands bind to periplasmic sensory domains of transmembrane chemoreceptors and induce a conformational change that is transduced across the membrane to regulate activity of the receptor-associated kinase CheA. Here we quantitatively characterize another, receptor-independent branch of chemotactic signaling that is linked to the sugar uptake through a large family of phosphotransferase systems (PTSs). Using in vivo characterization of intracellular signaling and protein interactions, we demonstrate that signals from cytoplasmic PTS components are transmitted directly to the sensory complexes formed by chemoreceptors, CheA and an adapter protein CheW. We further conclude that despite different modes of sensing, the PTS- and receptor-mediated signals have similar regulatory effects on the conformation of the sensory complexes. As a consequence, both types of signals become integrated and undergo common downstream processing including methylation-dependent adaptation. We propose that such mode of signaling is essential for efficient chemotaxis to PTS substrates and may be common to most bacteria.

Fig. 1.

Interactions between the PTS and the chemotaxis pathway. (A) Overview of the PTS and the chemotaxis pathway (see text for details). Red arrows indicate interactions between components of both systems detected by an in vivo FRET screen (Fig. S1A). (B) Examples of a positive (Upper) and a negative (Lower) FRET measurement for CFP-CheA/EI-YFP pair in the wild-type and chemotaxis-negative (∆flhC) background, respectively. Red bar indicates a period of acceptor (YFP) photobleaching. AU, arbitrary units of fluorescence. Measurements for other positive FRET pairs and values of apparent FRET efficiency are shown in Fig. S2.

Fig. 2.

Characterization of the PTS-mediated response. Intracellular response of the chemotaxis pathway was measured using a CheY/CheZ FRET reporter of kinase activity in ∆(cheY cheZ) background [wild type (WT) for FRET] or in ∆trg ∆(cheY cheZ) background (∆trg). See Fig. S1 B and C for details of the assay. (A) Dose–response measurements. Buffer-adapted cells were stimulated by addition and subsequent removal of indicated concentration steps of glucose (Glc) or galactose (Gal). Kinase activity was plotted relative to the steady-state activity in the buffer. Zero activity was determined by a saturating stimulation with 100 μM α-methyl-DL-aspartate (MeAsp). Data were fitted using a Hill equation. Error bars here and throughout indicate SEs. (B) Dynamic range measurements. Cells were stimulated by stepwise addition of increasing amounts of attractant, allowing full adaptation before each subsequent stimulation. The response for each step was normalized to the response of buffer-adapted cells to 100 μM MeAsp. (C) Measurements of response sensitivity. Dose–response curves were measured as in A but for cells preadapted to an ambient concentration of 500 nM glucose or galactose, as indicated, and fitted using the Hill equation. Ligand concentration was normalized to the ambient concentration. (D) Response sensitivity at the peak of the dynamic range (S^R_P) for different attractants. S^R_P for the PTS-mediated response to glucose was calculated as the initial slope of the dependence in C. The values for ribose (Rib) and other attractants at the peaks of their respective dynamic range were measured previously and normalized to the fraction of ligand-specific receptors in the total receptor pool (19). (E) Effect of adaptation to glucose on the response to other ligands in ∆trg cells. The response was followed as a change in the ratio of YFP to CFP fluorescence due to FRET, with a higher ratio corresponding to higher FRET signal and therefore higher pathway activity. Addition and removal of mannitol (Mtl) and other attractants are indicated by down and up arrows, respectively. Gray bar indicates presence of glucose in the background, over which other stimuli were added. Gradual drift of the YFP/CFP ratio base line arises from a relatively faster loss of the CFP fluorescence over the time course of the measurement.

Fig. 3.

Involvement of chemoreceptor methylation in adaptation to PTS stimuli. (A) Dependence of adaptation time on the response amplitude for subsaturating stimulation with MeAsp and galactose in WT or glucose in ∆trg cells. Adaptation time was defined as the time required to regain 50% of the initial loss in FRET signal upon stimulation. Response amplitude was normalized to the maximal response elicited by 100 µM MeAsp. Inset shows an adaptation time course for MeAsp and glucose stimuli of similar strength, with down and up arrows indicating addition and removal of attractants, respectively. (B) PTS- and receptor-mediated responses in receptorless CheR⁺ CheB⁺ cells expressing Tar (Left) or Tsr (Right) from plasmids (Table S1). Note that incubation time with MeAsp or serine (Ser) was not long enough to allow full adaptation to saturating stimuli used here as a reference. (C) Response to glucose and MeAsp in receptorless ∆(cheR cheB) cells expressing single-modified Tar^QEEE. (D) Activity dependence of the PTS-mediated response. Receptorless ∆(cheR cheB) cells expressing half-modified Tar^QEQE were stimulated with glucose in the buffer or in presence of indicated concentrations of MeAsp (Fig. S5A). Response of cells expressing Tar^QEEE as in C is also shown. (E) Change of receptor methylation pattern in response to PTS- or receptor-mediated stimuli. Cells (∆trg ∆tsr) were stimulated by indicated amounts of MeAsp or glucose for 2 min and the distribution of Tar methylation levels was measured using immunoblotting (Fig. S6A). Intensity profiles reflect receptor mobility on the SDS/PAGE gel, with higher receptor mobility corresponding to higher methylation. For better comparison, each profile curve was normalized to the integral intensity of all bands within the respective lane after subtraction of the background.

Fig. 4.

CheB phosphorylation and adaptation to PTS stimuli. Comparison of FRET after addition of glucose in a Δtrg ΔcheB strain expressing wild-type CheB (A) or CheB_C, a constitutively active form of CheB lacking the regulatory domain (B), from plasmids (Table S1). The higher level of FRET in the glucose-adapted cells most likely results from CheA-independent phosphorylation of CheY (Fig. 3D and Fig. S4B).