Noncanonical Sensing Mechanisms for Bacillus subtilis Chemoreceptors

ABSTRACT

Bodhankar et al. reported a noncanonical sensing mechanism that involves signal interaction with the McpA chemoreceptor signaling domain resulting in a chemorepellence response of Bacillus subtilis. The identified repellent binding site is analogous to that for attractant binding in McpB, another B. subtilis chemoreceptor.

INTRODUCTION

Bacteria have evolved a very large number of ligand binding domains (LBDs) that form part of different types of transmembrane signal transduction receptors (1). Typically, the interaction of a signal molecule with the extracytoplasmic LBD of a transmembrane receptor creates a molecular stimulus that is transduced across the membrane to modulate the activity of the cytosolic domain, triggering the signaling output. Chemoreceptor-based signaling circuits are found in about half of the sequenced bacteria (2) and mediate chemotaxis, control alternative cellular functions like the levels of second messengers, or be involved in type IV pili based motility and mechanosensing (3, 4). A canonical chemoreceptor contains an extracytosolic LBD and a cytosolic signaling domain (Fig. 1). Although there is evidence for the existence of alternative mechanisms for chemoreceptor stimulation that do not involve a signal-LBD interaction, our knowledge of the corresponding molecular details is very limited. In this issue, Bodhankar et al. (5) reported a noncanonical mechanism for sensing phenol and related aromatic compounds by Bacillus subtilis. The authors found that this bacterium was chemotactically attracted to low, micromolar concentrations of phenol, whereas this compound was a chemorepellent at higher, millimolar concentrations. Whereas chemoreceptors HemAT and McpC mediated chemoattraction to phenol, McpA was responsible for repellence from this compound (Fig. 1).

FIG 1.

The diversity of repellent and attractant sensing mechanisms in B. subtilis and Escherichia coli chemoreceptors. The figure is based on data reported in (5, 6, 13, 14, 20–24). The histidine kinases, adenylyl cyclases, methyl-accepting binding proteins and phosphatases (HAMP) and signaling domains are shown as green and light gray cylinders, respectively. LBD, ligand-binding domain; DHMA, 3,4-Dihydroxymandelic acid; AI-2, autoinducer-2. ArtP, MetQ, YckB, MBP, and LsrB are solute binding proteins that are present in the extracytosolic space. The question mark highlights sensing mechanisms that have not been verified by direct binding studies.

McpA is a canonical chemoreceptor comprising a dCache type LBD that was flanked by two transmembrane regions (Fig. 1). By combining microbiological experimentation with protein studies, the authors showed that chemoreceptor McpA employed a nonconventional sensing mechanism that did not involve the LBD. Instead, signal binding occurred at the cytosolic signaling domain. The corresponding binding pocket was identified and found to be in an analogous position as that for the recognition of the chemoattractants ethanol and benzene by McpB, another B. subtilis chemoreceptor with a dCache type LBD (Fig. 1) (5, 6). Of note, the ligand affinities for the McpA and McpB signaling domains were well below those typically observed for signal-LBD interactions, which are frequently in the range of 1 to 50 μM (1). Because the signaling domain sequence is well conserved among species, the demonstration of repellent and attractant binding to analogous sites at the signaling domains of McpA (5) and McpB (6), respectively, raises the question of whether chemoreceptors in other species are activated by a similar mechanism.

MULTIPLE ATTRACTANT AND REPELLENT SENSING MECHANISMS

Access to nutrients is generally considered the major driving force for the evolution of chemoattraction, whereas chemorepellence permits escape from niches with harmful conditions, such as the presence of toxic compounds (7). However, efflux pumps appear to be the primary mechanism to cope with toxic chemicals, which may explain why chemorepellence is less frequently observed than chemoattraction. Although the molecular mechanisms of chemorepellence are less studied than those of chemoattraction, it has been reported that some chemorepellents bind to the chemoreceptor LBDs (8–10), suggesting that repellent responses can be induced by mechanisms that resemble the canonical mechanism for chemoattraction. Importantly, the tactic responses to phenol have also been studied in Escherichia coli, showing certain parallelism with the data obtained in B. subtilis. All four E. coli transmembrane chemoreceptors comprising a periplasmic LBD were found to respond to phenol. Whereas Tar senses phenol as an attractant, Tsr, Tap, and Trg mediate repellent responses (11, 12). Tar and Tsr are important model receptors and their chemoattraction responses to aspartate and serine, respectively, are triggered by signal interaction with the LBD (13). However, Pham and Parkinson (14) have demonstrated that the Tsr- and Tar-mediated phenol responses do not require the LBD or the signaling domain. Based on multiple pieces of evidence, the authors suggested that phenol responses are caused by ligand diffusion into the cytoplasmic membrane where it perturbs the structural stability or position of the transmembrane helices, leading to receptor stimulation (14). Next to these two repellent sensing mechanisms that involve ligand interaction with the LBD and the transmembrane regions, the study of Bodhankar et al. (5) expanded the mechanistic diversity of repellent sensing by demonstrating that ligand interaction with the chemoreceptor signaling domain can induce repellence (5) (Fig. 1). The demonstration that the analogous binding sites in the signaling domain of McpA and McpB bind a chemorepellent (phenol) and chemoattractants (ethanol and benzene) (5, 6), respectively, suggests a universal nature of this mechanism. Taken together, these findings indicate that multiple mechanisms induce chemorepellence. Several chemoattractant sensing mechanisms have been reported (15), but data indicate that there is also a significant diversity in the repellent sensing mechanisms.

CONVENIENCE OF USING SATURATION-TRANSFER DIFFERENCE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (STD-NMR) TO CHARACTERIZE LOW-AFFINITY SIGNAL BINDING

In vitro protein-ligand binding studies were found to be a very convenient approach to detect the interaction of signals with receptors. Such experiments are necessary to determine whether a receptor is activated by direct ligand binding or by alternative modes such as the recognition of signal-loaded solute binding proteins. Isothermal titration calorimetry (ITC) is the gold-standard technique to monitor ligand interaction (16) and has been extensively used to study the interaction of signal molecules with individual LBDs or full-length chemoreceptors (17, 18). However, due to the limitation imposed by ligand dilution heats, ITC is well-suited to characterize high-affinity interaction (K_D in the nM and μM range) but fails frequently to detect lower affinity binding (K_D in the mM range). The recent studies conducted in the Rao laboratory (5, 6) have made use of STD-NMR to monitor low-affinity binding of signal molecules to purified chemoreceptor signaling domains that have permitted to estimate dissociation constants. In both studies (5, 6) the data derived from these ligand-protein interaction studies agree to a large degree with the microbiological studies. It, therefore, appears that STD-NMR is a complementary technique to ITC permitting not only the detection of low-affinity binding to the signaling domain but potentially also to the LBD.

A SIGNIFICANT NUMBER OF CHEMORECEPTORS RESPOND TO DIFFERENT TYPES OF SIGNAL MOLECULES EMPLOYING DIFFERENT MECHANISMS

The signals that stimulate many chemoreceptors in different species have been identified over the last decades (1), and it is, therefore, tempting to refer to these proteins as amino acid, organic acid, purine, and polyamine chemoreceptors. However, the more detailed studies of chemoreceptors in model species like B. subtilis and E. coli (Fig. 1) have shown that they frequently respond to very different signals employing frequently different sensing mechanisms. This notion was well illustrated by Bodhankar et al. (5). The authors showed that responses to aromatic compounds are mediated by four chemoreceptors, namely, HemAT, McpA, McpB, and McpC. However, these receptors were not unknown and had been previously studied. HemAT binds oxygen at a heme-containing LBD and mediates aerotaxis (19, 20). McpA was found to mediate taxis to glucose and α-methylglucosides by unknown mechanisms (21). McpB and McpC bind amino acids at their LBD causing chemoattraction (22, 23). In addition, McpC senses carbohydrates at its cytosolic fragment (24) (Fig. 1). Similar observations have been made for the E. coli Tar and Tsr receptors (Fig. 1). The study of Bodhankar et al. (5) thus reinforces the notion that chemoreceptors are frequently multifunctional mediating responses to multiple types of signals employing different mechanisms.

OUTLOOK

A significant number of studies have investigated the molecular mechanism of signal transduction in chemoreceptors, i.e., the nature of the molecular consequences induced by signal recognition at the LBD that cause ultimately an alteration of CheA activity and chemoattraction. Although this mechanism is still subject to debate, several studies show that ligand binding causes a piston shift movement in the second transmembrane region that is then relayed to the signaling domain (25). The demonstration that the binding of phenol to a site at the signaling domain of McpA causes chemorepellence permits studies to cast light into the repellence signal transduction mechanism (5). These data could then be compared to the changes induced by the binding of the chemoattractants benzene and ethanol to the analogous binding site at the signaling domain in McpB (6), which would provide information on the molecular basis resulting in either chemorepellence or attraction.

Apart from chemoreceptors, bacteria contain an array of other transmembrane signal transduction receptors that sense extracytosolic signals and generate different types of responses, such as, for example, altered gene expression or changes in second messenger levels (1). Major receptor families are sensor histidine kinases, diguanylate cyclases, and phosphodiesterases or different protein kinases (26). In analogy to chemoreceptors, the canonical model of receptor activation consists of signal interactions with the LBD. The existence of multiple noncanonical activation mechanisms for chemoreceptors hence raises the question as to whether similar mechanisms might be observed in other receptor families.

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