Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005? 200556410781086Original ArticleChemoreceptor clustering and adaptational modificationS. R. Lybarger et al. Molecular Microbiology (2005) 56(4), 1078–1086 doi:10.1111/j.1365-2958.2005.04593.x Clustering requires modified methyl-accepting sites in low-abundance but not high-abundance chemoreceptors of Escherichia coli Suzanne R. Lybarger,1† Usha Nair,1 Angela A. Lilly,2 Introduction Gerald L. Hazelbauer2 and Janine R. Maddock1* Many bacterial proteins occupy specific sub-cellular 1Department of Molecular, Cellular and Developmental niches (Lybarger and Maddock, 2001; Shapiro et al., Biology, University of Michigan, 830 North University, 2002). One well-characterized example is the ternary sig- Ann Arbor, MI 48109, USA. nalling complex that mediates chemotaxis, which is found 2Department of Biochemistry, University of Missouri- in clusters localized in the polar membranes of Escheri- Columbia, 117 Schweitzer Hall, Columbia, MO 65211, chia coli (Maddock and Shapiro, 1993; Sourjik and Berg, USA. 2000; Shiomi et al., 2002; Cantwell et al., 2003; Banno et al., 2004) and all other species which have been inves- Summary tigated (Alley et al., 1992; Maddock and Shapiro, 1993; Harrison et al., 1999; Gestwicki et al., 2000; Kirby et al., Chemotaxis signalling complexes of Escherichia coli, 2000). Although the functional consequences of this polar composed of chemoreceptors, CheA and CheW, form clustering remain largely uncharacterized, clustering and clusters located predominately at cell poles. As the polarity may be inherent features of the signalling systems only kind of receptor in a cell, high-abundance recep- that mediate bacterial chemotaxis and may be involved in tors are polar and clustered whereas low-abundance signal amplification and sensory adaptation (Le Moual chemoreceptors are polar but largely unclustered. We et al., 1997; Li et al., 1997; Bray et al., 1998; Duke and found that clustering was a function of the cytoplas- Bray, 1999; Duke et al., 2001; Sourjik and Berg, 2004). mic, carboxyl-terminal domain and that effective clus- Chemotaxis signalling complexes consist of a core tering was conferred on low-abundance receptors by complex of transmembrane chemoreceptors, the histidine addition of the ~20-residue sequence from the car- kinase CheA, and the coupling protein CheW (Gegner boxyl terminus of either high-abundance receptor. et al., 1992; Schuster et al., 1993; Liu et al., 1997). This These sequences are different but share a carboxyl- complex interacts with response regulator CheY, phos- terminal pentapeptide that enhances adaptational phatase CheZ, methyltransferase CheR, and methyl- covalent modification and allows a physiological esterase/deamidase CheB, creating a supramolecular balance between modified and unmodified methyl- complex (see Bren and Eisenbach, 2000; Hazelbauer, accepting sites, implying that receptor modification 2004). Signalling from receptor to flagellar motor occurs might influence clustering. Thus we investigated by modulation of CheY phosphorylation that is controlled directly effects of modification state on chemorecep- by autophosphorylation of CheA which, in turn, is con- tor clustering. As the sole receptor type in a cell, low- trolled by chemoreceptor conformation. An activating con- abundance receptors were clustered only if modified, formation is favoured by covalent modification at specific but high-abundance receptors were clustered inde- methyl-accepting glutamates in the cytoplasmic domain, pendent of extent of modification. This difference whereas an inhibiting conformation is favoured by occu- could mean that the two receptor types are fundamen- pancy at attractant binding sites in the periplasmic tally different or that they are poised at different posi- domain. The balance between ligand occupancy and tions in the same conformational equilibrium. Notably, modification of methyl-accepting glutamates by the action no receptor perturbation we tested altered a predom- of CheR and CheB mediates sensory adaptation, and the inant location at cell poles, emphasizing a distinction time lag between the two provides a molecular memory. between determinants of clustering and polar Chemoreceptors are homodimers of extended helical localization. bundles that can be divided into a periplasmic ligand- binding domain, a transmembrane domain, and a cyto- Accepted 26 January, 2005. *For correspondence. E-mail plasmic signalling and adaptation domain (Falke and [email protected]; Tel. (+1) 734 936 8068; Fax (+1) 734 647 0884. †Present address: University of Maryland School of Medicine, Hazelbauer, 2001). Dimers associate at their cytoplasmic, 15601 Crabbs Branch Way, Rockville, MD 20855. membrane-distal tips to form trimers of dimers (Kim et al., © 2005 Blackwell Publishing Ltd Chemoreceptor clustering and adaptational modification 1079 1999; Studdert and Parkinson, 2004) and trimers are examined localization of hybrids of the two receptor thought to form larger complexes. Such wider interactions classes to identify the location of determinants that medi- are suggested by the extent of cooperativity in signalling ate chemoreceptor clustering. The results of those studies (Li and Weis, 2000; Sourjik and Berg, 2004) and by the prompted us to investigate more closely the effect of size of neighbourhoods of adaptational assistance (M. Li chemoreceptor modification at methyl-accepting sites on and G.L. Hazelbauer, submitted). Interactions that tran- clustering and polar localization. scend a trimer imply that at least several trimers associate functionally and presumably structurally, and such con- tacts are likely to occur in receptor clusters (Maddock and Results Shapiro, 1993; Lybarger and Maddock, 2001). The carboxyl-terminal, cytoplasmic domain carries There are two classes of methyl-accepting chemore- determinants for efficient clustering ceptors in E. coli: the high-abundance receptors, Tsr and Tar, make up ~90% of the cellular complement of recep- To determine whether the difference in clustering of high- tors, whereas the low-abundance receptors, Trg and Tap, and low-abundance receptors could be correlated with a represent only a few per cent of cellular chemoreceptors particular chemoreceptor segment, we produced previ- (Li and Hazelbauer, 2004). Both high-abundance recep- ously constructed hybrids of a high- and a low-abundance tors mediate adaptation and efficient chemotaxis in the receptor (Fig. 1A) as the only receptor type in cells other- absence of other receptors (Springer et al., 1977). A low- wise wild-type for chemotaxis. The Tsr-Trg hybrids join the abundance receptor can signal in the absence of other amino-terminal domain of Trg to the carboxyl-terminal receptors (Hazelbauer and Engström, 1980), but requires (cytoplasmic) domain of Tsr (Trsr) or the amino-terminal the presence of a high-abundance receptor for effective domain of Tsr to the carboxyl-terminal domain of Trg adaptation and chemotaxis (Springer et al., 1977; (Tsrg) (Feng et al., 1997; Feng et al., 1999); likewise, the Hazelbauer and Engström, 1980; Feng et al., 1997; Tar-Tap hybrids are fusions between the amino-terminal Weerasuriya et al., 1998). High- and low-abundance domain of Tap and the carboxyl-terminal domain of Tar chemoreceptors are structurally distinct. Low-abundance (Tapr) or the amino-terminal domain of Tar and the car- receptors are almost 20 residues shorter at their carboxyl boxyl-terminal domain of Tap (Tarp) (Weerasuriya et al., ends than the high-abundance proteins and lack a pen- 1998) (Table 1). Immunogold microscopy revealed that all tapeptide sequence, NWETF, found at the extreme car- hybrid receptors were localized at cell poles. Hybrids with boxyl terminus of high-abundance receptors. The a cytoplasmic domain from a high-abundance receptor sequence serves as an interaction site for CheR and (Trsr and Tapr) were efficiently clustered; those with the CheB, substantially enhancing the efficiency of adapta- cytoplasmic domain of a low-abundance receptor (Tsrg tional modification (Wu et al., 1996; Djordjevic and Stock, and Tarp) were not (Fig. 1B, Table 2, Figs S1, S2 and 1998; Barnakov et al., 1999; Barnakov et al., 2001). The Table S1). Thus the carboxyl-terminal domains of high- pentapeptide sequence is necessary for efficient adapta- abundance receptors were sufficient to mediate cluster- tional modification of high-abundance receptors (Wu ing. Hybrids with amino-terminal halves from high- et al., 1996; Le Moual et al., 1997; Li et al., 1997; Barna- abundance receptors and carboxyl-terminal halves from kov et al., 1999) and for their ability to mediate effective low-abundance receptors (Tsrg and Tarp) were slightly chemotaxis (Le Moual et al., 1997; Li et al., 1997; Feng more clustered than the respective full length low- et al., 1999). Furthermore, addition of the final 19 residues abundance receptors, indicating that some feature in the of Tsr, which included the pentapeptide, to the carboxyl- amino-terminal half of a high-abundance receptor also terminal end of Trg created a low-abundance receptor that promoted clustering. was efficiently modified and mediated effective chemot- axis in the absence of other chemoreceptors (Feng et al., Efficient clustering is conferred by a short carboxyl- 1999), and addition of a comparable segment of Tar to a terminal sequence containing the pentapeptide that slightly truncated form of Tap produced intermediate activ- enhances adaptational modification ity (Weerasuriya et al., 1998).