The Che system: a specialised two-component system for taxis

The two-component system mediating tactic responses is commonly referred to as the Che system. The Che system in Archaea is basically similar to the corresponding bacterial system, and the proteins involved are homologs of the respective bacterial proteins. However, there are minor and major variations in this system, both between different bacterial clades and between Bacteria and Archaea (for review seeSzurmant and Ordal, 2004). A universal scheme of the Che system is shown in Figure 6.1.

The overall workflow of taxis signalling can be divided into four steps – signal reception and transduction, excitation, adaptation, and signal termination. These steps are described in the next sections. An additional section is about fumarate, which is also involved in flagellar motor switching.

6.1.1.1 Signal reception and transduction

Signals are recognised and transduced by a certain class of proteins called halobacte-rial transducer proteins (Htrs) in H. salinarum or methyl-accepting chemotaxis pro-teins (MCPs) in other archaea and bacteria. The transducers control the activity of the histidine kinase CheA and thereby the level of CheY-P, the output molecule of the Che system. MCPs/transducers usually contain a cytoplasmic signalling re-gion where the histidine kinase CheA and the coupling protein CheW bind, a methy-lation/demethylation region that is critical for adaptation, and one or two HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phos-phatase) domains. The HAMP domains convey the signal of ligand binding from the sensor domain or protein to the output module. The way the signal is transduced

across the membrane is still not completely understood - most experimental data sup-port the sliding-piston model, in which ligand binding induces a piston-like sliding of the signalling helix toward the cytoplasm (Falke and Hazelbauer, 2001).

Figure 6.1:General model of prokary-otic chemotaxis systems. Biochemi-cal processes in the two-component chemo-taxis pathway are shown. Hexagons repre-sent response regulator domains. Compo-nents found throughout all species are in red, components found in almost all species are in orange; components which are only present in certain species are in yellow.

Figure and caption slightly modified from Szurmant and Ordal(2004).

In contrast to many eukaryotic receptors that dimerise upon ligand binding, prokaryotic transducers form stable dimers even in the ab-sence of ligand (Milligan and Koshland, 1988).

The receptors in prokaryotes are not distributed evenly around the cell body but form large clusters where thousands of sensory complexes are thought to come together (Maddock and Shapiro, 1993; Sourjik and Berg, 2000; for re-view seeKentner and Sourjik,2006;Hazelbauer et al., 2008). Whereas the clustering of recep-tors as essential feature of prokaryotic signal processing is widely accepted, the arrangement of receptors in the clusters is still under discus-sion. The crystal structure of the cytoplasmic fragment of theE. coli serine receptorTsr shows trimers of receptor dimers (Kim et al., 1999), which can be the basic building blocks of recep-tor arrays. Hexagonal cluster structures can be formed by connecting the trimers of dimers by CheA dimers (Shimizu et al., 2000). However, newer findings suggest that CheA and CheW

are not required for cluster formation (Kentner and Sourjik, 2006), and the crystal structure of the cytoplasmic part of a Thermotoga maritima receptor (Park et al., 2006) revealedhedgerows of dimers instead of trimers of dimers. The different models of receptor cluster formation are reviewed in Kentner and Sourjik(2006).

6.1.1.2 Excitation

The input, usually sensed by the receptors, influences the autophosphorylation activ-ity of the histidine kinase CheA. After autophosphorylation of a particular histidine

residue, the phosphoryl group is immediately transferred from CheA to the response regulator CheY. Phosphorylated CheY (CheY-P) is the output signal for the flagel-lar motor. Hence CheA integrates the different stimuli to generate an unambiguous output to the flagellar motor. In E. coli (Borkovich et al., 1989),S. meliloti (Schmitt, 2002), R. spheroides (Shah et al., 2000), and H. salinarum (Rudolph and Oesterhelt, 1996) attractants decrease and repellents increase CheA activity, in B. subtilis CheA regulation is reversed (Garrity and Ordal, 1997).

CheA consists of five domains (P1-P5) (Bilwes et al.,1999). The P1, or Hpt (histi-dine phosphotransfer), domain contains the histi(histi-dine residue that is phosphorylated.

P2 is the docking site for CheY and CheB, which receive the phosphoryl group from CheA-P. P3 is the dimerisation domain. Dimerisation of CheA is crucial since CheA autophosphorylates in trans. On P4 (HATPase_c: Histidine kinase-like ATPases) ATP binding and catalysis occurs, and P5 (also called CheW domain due to homol-ogy to this protein) is where CheA binds the receptors and the coupling protein CheW.

For activation of CheA at the receptors, the coupling protein CheW is required.

CheW proteins are found in all bacterial and archaeal species with a chemotaxis system (judged by the presence of CheA and receptors). H. salinarum contains two CheW paralogues. The exact role of these is not clear, but deletion of CheW1 and CheW2 results in different phenotypes (Aregger,2003).

As implied above, the output of the chemotaxis signalling system is CheY-P. Ele-vated CheY-P levels cause CCW flagellar rotation (smooth swimming) in B. subtilis and CW flagellar rotation (tumbling) in E. coli. Therefore the overall outcome is the same in both species – attractants cause smooth swimming, repellents cause tumbling and reorientation. In H. salinarum, CheY-P causes reversals and is required for CCW swimming as were shown by the constantly CW swimming phenotype of CheY and CheA deletion strains (Rudolph and Oesterhelt, 1996).

In flagellated bacteria, the target of CheY-P is the protein FliM (Welch et al., 1993), which builds together with FliN and FliG the flagellar motor switch complex.

The binding site of CheY-P is the highly conserved N-terminal peptide of FliM (Bren and Eisenbach, 1998). Archaea, although using CheY-P as switch factor in a similar fashion, do not possess FliM homologs. Also no equivalent to the CheY-P binding peptide has been identified. The site of interaction of CheY-P in Archaea is unknown (Nutschet al., 2003; Szurmant and Ordal, 2004; Ng et al., 2006).

6.1.1.3 Adaptation

Prokaryotic taxis requires a memory to decide whether during a move the conditions improved or worsened. That means that the actual stimulus strength is permanently compared to the stimulus strength as it was before (Koshland,1977). This is achieved by the adaptation system(s). The best understood adaptational mechanism is the methylation system of CheR and CheB, but other systems, e. g. involving CheC and CheD (Muff and Ordal,2007), exist.

The methyltransferase CheR transfers methyl groups from S-adenosylmethionine to certain glutamate residues in the methylation region of the receptors (Kehry and Dahlquist,1982;Nowlinet al.,1987). Methylation of receptors increases and demethy-lation decreases the activity of the signalling complex (Ninfa et al., 1991; Borkovich et al.,1992). CheB is a methylesterase that demethylates the same residues which are methylated by CheR. The methyl groups are released as methanol. Whereas CheR is constitutively active, the activity of CheB is regulated via phosphorylation of its response regulator domain by CheA: CheB-P is 100fold as active as unphosphorylated CheB (Lupas and Stock, 1989). That means that CheB forms a feedback loop be-tween CheA and the receptors. In H. salinarum and E. coli, but not in B. subtilis, CheB functions also as receptor glutamine deamidase. Glutamines have roughly the same effect on the signalling complex activity as methyl esterified glutamates (Rollins and Dahlquist, 1981;Kehry et al.,1983;Koch, 2005;Kochet al., 2008). In B. subtilis, this reaction is catalysed by CheD (Kristich and Ordal, 2002).

Several bacteria like B. subtilis and H. pylori contain CheV, a two-domain protein consisting of a N-terminal domain homologous to CheW and a C-terminal response regulator domain (Fredrick and Helmann, 1994). In CheV, phosphorylation of the response regulator domain seems to affect the conformation of the coupling domain, thereby decoupling CheA and the receptors. Thus the signalling complexes of ligand-bound receptors can reassume their prestimulus activity (Karatan et al., 2001). A gene coding for CheV was not yet found in any archaeal genome.

InB. subtilis, a third way of adaptation is described involving the proteins CheC and CheD (Muff and Ordal,2007). CheC is a CheY-P phosphatase (Szurmantet al.,2003), CheD catalyses the deamidation of glutamines at the receptors (Kristich and Ordal, 2002). Both proteins were shown to form a heterodimer (Rosario and Ordal, 1996).

This interaction increases the CheC phosphatase activity (Szurmantet al.,2004) and

inhibits the deamidation activity of CheD (Chao et al.,2006). CheY-P was shown to stabilise the CheC:CheD interaction, and thus CheC and CheD form a third feedback loop to the receptors (Muff and Ordal, 2007). H. salinarum possesses both a CheD and CheC homologs. However, receptor deamidation activity in this organism has been demonstrated for CheB and not CheD (Koch, 2005;Koch et al., 2008), and the function of CheD is unclear. It remains to be elucidated if other adaptation systems than the CheR/CheB methylation system play a role in archaeal chemotaxis.

6.1.1.4 Signal termination

The chemotaxis system must be able to respond to changing stimuli within seconds to effectively direct the movement towards the best places. This is in part achieved by a short half-life of CheY-P, which is, depending on the species, in the range of a few seconds (Rudolph et al., 1995) to almost one minute (Hess et al., 1988; Stock et al., 1988). CheY was found to actively catalyse autodephosphorylation (Lukatet al.,1990;

Silversmith et al.,1997).

To further accelerate signal removal, several chemotactic organisms express CheY-P phosphatases. In the γ- andβ-proteobacteria, this is done by the protein CheZ (Hess et al., 1988; Zhao et al., 2002). In other chemotactic eubacteria and all chemotactic archaea, CheC in combination with CheD seems to be involved in CheY-P dephospho-rylation (Szurmantet al.,2004). Furthermore, inB. subtilis the flagellar motor switch protein FliY, a distinct homolog of CheC and the CheX protein (a CheY-P phos-phatase present for example in T. maritima), was found to have CheY-P phosphatase activity (Szurmantet al., 2004).

In α-proteobacteria like S. meliloti, a third mechanism of signal termination is present. A second CheY acts as phosphate sink and thereby possibly assists the phosphate removal from the “main” CheY (Sourjik and Schmitt, 1998).

6.1.1.5 Fumarate as switch factor

The first evidence for fumarate as switch factor was found in H. salinarum, where it restored wild-type behaviour in a straight-swimming mutant (Marwanet al.,1990). It was demonstrated that fumarate is released to the cytoplasm from membrane-bound pools after light stimulation (Marwan et al., 1991; Montrone et al., 1993). Almost to the same time, Barak and Eisenbach (1992b) observed that fumarate and CheY

are required for switching in cell envelopes of E. coli and S. typhimurium. Using an E. coli strain with increased cytoplasmic fumarate concentrations due to a deletion of succinate dehydrogenase (SDH), which acts on fumarate, a correlation between the cytoplasmic fumarate level and both the switching frequency and the fraction of cells rotating clockwise could be established (Montrone et al., 1996, 1998; Prasad et al., 1998). Prasad et al. (1998) also demonstrated that the target of fumarate is the switch and not CheY, and that it acts, at least in part, by lowering the free energy difference between the CW and the CCW state of the motor. In a recent work, fu-marate reductase (FRD) was identified as target of fufu-marate at the motor in E. coli (Cohen-Ben-Luluet al.,2008). This enzyme, otherwise functioning in anaerobic respi-ration, interacts with the flagellar motor switch protein FliG. However, H. salinarum does neither code for FRD (which is mainly found in obligate or facultative anaerobic bacteria) nor for FliG. So in this species fumarate must act by a different, till now unknown, mechanism. The excitation part of fumarate signalling, i. e. when and how it is released, has not yet been identified either.