Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan1
Author for correspondence: Ikuro Kawagishi. Tel: +81 52 789 2993. Fax: +81 52 789 3001. e-mail: i45406a{at}nucc.cc.nagoya-u.ac.jp
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ABSTRACT |
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Keywords: adaptation, chemotaxis, histidine kinase, methylation, revertant
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INTRODUCTION |
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Adaptation through covalent modification of receptors is essential for bacterial chemotaxis (for reviews, see Manson, 1992 ; Parkinson, 1993
; Blair, 1995
; Falke et al., 1997
; Stock & Surrette, 1996
). Escherichia coli and Salmonella enterica serovar Typhimurium have several chemoreceptors that are reversibly methylated upon adaptation. These chemoreceptors are homodimeric proteins (Milligan & Koshland, 1988
), each subunit of which consists of two membrane-spanning regions, a periplasmic ligand-binding domain and a cytoplasmic signalling and adaptation domain. In the adaptation process, four to five glutamate residues (present in the motif Glu-Glu-X-X-Ala-Ser/Thr, with the underlined Glu residue being the site of methylation) in two different helices in the cytoplasmic domain are methylated or demethylated. These reactions are catalysed by the methyltransferase CheR and the methylesterase CheB, respectively. CheR catalyses transfer of a methyl group from S-adenosylmethionine to the side-chain carboxyl of glutamate residues (Springer & Koshland, 1977
).
The receptor forms a ternary complex with a cytoplasmic histidine kinase (CheA) and an adaptor protein (CheW) (Gegner et al., 1992 ; Schuster et al., 1993
; Liu et al., 1997
). The unliganded (or repellent-bound) receptor stimulates the activity of CheA, which autophosphorylates itself and then transfers the phosphate group to the response regulators CheY and CheB (Hess et al., 1988
; Stock et al., 1988
; Wylie et al., 1988
). Phospho-CheY binds to FliM, which is a component of the flagellar motor, and induces the clockwise rotation of the motor, which causes tumbling (Barak & Eisenbach, 1992
; Welch et al., 1993
). Dephosphorylation of phospho-CheY is facilitated by CheZ. Phospho-CheB catalyses demethylation of the receptor (Anand et al., 1998
; Lupas & Stock, 1989
). When attractant binds to a chemoreceptor, it inhibits the activity of CheA (Borkovich et al., 1989
). Consequently, the concentration of phospho-CheY decreases and the flagella rotate counterclockwise, which results in smooth swimming (Barak & Eisenbach, 1992
; Welch et al., 1993
). The inhibition of the CheA kinase also decreases the concentration of phospho-CheB, the active form of the methylesterase, which reduces demethylation activity and hence increases methylation of the receptors. Methylation of a chemoreceptor stimulates CheA to counteract the inhibitory effect of the attractant (Borkovich et al., 1989
).
The structure of the cytoplasmic domain of Salmonella Tar and its relation to kinase regulation were thoroughly studied by cysteine and disulfide scanning (Bass & Falke, 1998 , 1999
; Bulter & Falke, 1998
; Danielson et al., 1997
). Recently, the three-dimensional structure of a cytoplasmic fragment of E. coli Tsr that can activate the CheA kinase was determined (Kim et al., 1999
). The cytoplasmic fragment (residues 286526, corresponding to residues 284528 of Tar) has a highly
-helical structure: each subunit consists of a coiled coil of two long, antiparallel helices (MH1 and MH2) connected by a U-turn, and the two subunits of a dimer form a quasi-four-helix bundle. Methylation is predicted to alter the interaction between these cytoplasmic helices to switch signalling states.
A single cell contains thousands of chemoreceptors, but only hundreds of CheR molecules (Simms et al., 1987 ). How can the receptors be effectively methylated? The C-terminal pentapeptide sequence of chemoreceptors is well conserved among the high-abundance receptors (Tsr, Tar and Tcp), with the consensus being Asn-Trp-Glu-Thr(Ser)-Phe. This motif is not conserved in the low-abundance receptors (Trg and Tap). CheR binds to this pentapeptide, which is distant from the methylation sites (Wu et al., 1996
; Djorjevic & Stock, 1998
). The low-abundance receptors have weaker abilities to be methylated and to allow adaptation when they exist in a cell as sole chemoreceptors (Springer et al., 1977
; Hazelbauer & Engström, 1980
; Yamamoto et al., 1990
; Feng et al., 1997
; Weerasuriya et al., 1998
). However, when high-abundance and low-abundance receptors are co-expressed in a cell, the methylation level of the latter is enhanced (Kondoh et al., 1979
; Yamamoto et al., 1990
). Furthermore, the addition of the NWETF sequence to Trg enhances the methylation and adaptation abilities of that receptor (Feng et al., 1999
). Finally, Barnakov et al. (1999
, 2001
) recently showed that CheB also binds to the NWETF motif. Taken together, the pentapeptide sequence seems to play a crucial role in receptor methylation and adaptation.
Recently, we systematically mutagenized this motif of Tar and showed that W550 and F553 (especially W550) play critical roles in the interaction with CheR, probably through hydrophobic interactions (Shiomi et al., 2000 ). Because CheR binding to Tar is almost completely eliminated by the W550A substitution, the methylation level of Tar-W550A is lower than that of wild-type Tar both in the absence and the presence of aspartate (Shiomi et al., 2000
). However, methylation of Tar-W550A is still slightly enhanced by aspartate (Shiomi et al., 2000
). The methylation level of Trg, which does not have a CheR-binding motif, is also regulated to some extent by its specific attractant ribose or galactose (Feng et al., 1999
). To study the physiological role of the NWETF motif further, we isolated intragenic suppressors of the W550A mutation. Six suppressors were isolated and were divided into two classes: five class I suppressors, which generated a compensatory tumbling bias (CheA kinase-activating) and one class II suppressor that restored the affinity of Tar for CheR.
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METHODS |
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Plasmids used in this work are listed in Table 1. The pBR322-based plasmid pNI101 (Nishiyama et al., 1997
) carries the wild-type tar gene. Plasmid pOKU121 (Shiomi et al., 2000
) derives from pNI101 and encodes Tar-W550A. Plasmid pNT201 (Borkovich et al., 1989
) carries the wild-type tar gene under the control of the tac promoter and was used to overexpress the Tar protein. Plasmid pKB23 (Borkovich et al., 1992
) is based on pACYC184 and carries the wild-type cheR gene placed downstream of the tac promoter.
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The 0·5 kb EcoRVEcoT22I fragments containing the W192R, A198E, V201E, V202L, H267D or T303I mutations were cloned into the corresponding region of pNI101 or pOKU121 to yield pDS390W, pDS390A, pDS391W, pDS391A, pDS392W, pDS392A, pDS393W, pDS393A, pDS394W, pDS394A, pDS395W or pDS395A, respectively.
Plasmid pNI101 encoding wild-type Tar has two NdeI sites: one is in the tar gene and the other is in the sequence derived from pBR322. The latter site was eliminated by blunt-ending and ligation to yield pNI101N. Site-directed mutagenesis of residue G278 was carried out by a two-step PCR method (Landt et al., 1990 ). The resulting 0·3 kb NdeIEcoT22I fragments containing the G278I, G278R, G278S or G278T substitution were cloned between NdeI and EcoT22I sites of pNI101N to yield pDS396W, pDS397W, pDS398W or pDS399W, respectively. The 0·5 kb EcoRVEcoT22I fragments from pDS396W, pDS397W, pDS398W or pDS399W were cloned into the corresponding region of pOKU121 to yield pDS396A, pDS397A, pDS398A or pDS399A, respectively.
Plasmids overproducing Tar-W550A or W550A-Op554W were constructed as follows. The parental plasmid pNT201, which has two AvaI sites, was partially digested with AvaI, blunt-ended and ligated to yield pDS101. The 1·4 kb XbaIAvaI fragments from pOKU121 (W550A) or pDS304 (W550A-Op554W) were cloned into the corresponding region of pDS101 to yield pDS103 (W550A) and pDS374 (W550A-Op554W), respectively.
Swarm assay for chemotaxis.
Swarm assays were performed with tryptone semisolid agar (1% tryptone, 0·5% NaCl, 0·3% agar) or minimal semisolid agar [10 mM potassium phosphate buffer (pH 7·0), 1 mM (NH4)2SO4, 1 mM MgSO4, 1 mM glycerol, 1 µg thiamine ml-1, 0·1 mM threonine, 0·1 mM leucine, 0·1 mM histidine, 0·1 mM methionine, 0·3% agar] supplemented with various concentrations of aspartate. When necessary, 50 µg ampicillin ml-1 and/or 25 µg chloramphenicol ml-1 was added. Cell suspensions (2 µl each, about 4x106 cells) were spotted onto swarm plates, which were then incubated at 30 °C for 1020 h.
Temporal assay of chemotactic response.
Temporal stimulation assays for chemotaxis were carried out as described previously (Okumura et al., 1998 ). Cells were grown at 30 °C in TG medium [1% tryptone, 0·5% NaCl, 0·5% (w/v) glycerol] supplemented, when necessary, with 50 µg ampicillin ml-1 and/or 25 µg chloramphenicol ml-1. Cells were harvested in late-exponential phase, washed with MLM medium [10 mM potassium phosphate buffer (pH 7·0), 0·1 mM EDTA, 10 mM DL-lactate, 0·1 mM methionine], resuspended in MLM medium and incubated at room temperature for 30 min. For doseresponse assays, the swimming patterns of the cells were observed with a dark-field microscope immediately after the addition of chemoeffector. For adaptation assays, a chemoeffector was added to the cell suspension, and small aliquots were taken at intervals for microscopic observation. The smooth-swimming fraction of the cells was measured using an Argus-10 image processor (Hamamatsu Photonics).
Analysis of receptor methylation by immunoblotting.
Taking advantage of the fact that methylation of a receptor increases its mobility in SDS-PAGE (Boyd & Simon, 1980 ; Chelsky & Dahlquist, 1980
; DeFranco & Koshland, 1980
; Engström and Hazelbauer, 1980
), receptor methylation was examined with immunoblotting. HCB339 (
MCPs) cells expressing the revertant Tar proteins were grown at 30 °C for 12 h in TG medium [1% tryptone, 0·5% NaCl, 0·5% (w/v) glycerol] supplemented with ampicillin. The culture was diluted 1:40 into fresh TG medium supplemented with ampicillin. Cells were grown with vigorous shaking, harvested in the late-exponential phase and resuspended in 100 µl distilled water. The samples were analysed by SDS-PAGE followed by immunoblotting with anti-Tsr (Iwama et al., 1997
), which cross-reacts with Tar.
Analysis of receptor methylation by radiolabelling.
Methylation of Tar was assayed by radiolabelling, as described previously (Nishiyama et al., 1997 ). Cells in medium consisting of 10 mM potassium phosphate buffer (pH 7·0), 0·1 mM EDTA, 10 mM DL-lactate, 3 µM methionine and 0·2 mg ml-1 chloramphenicol were incubated at 30 °C with vigorous shaking. After 30 min, 3·3 µM [methyl-3H] methionine [8·5 Ci mmol-1 (314·5 GBq mmol-1); Amersham] was added to the cells and this suspension was shaken for another 30 min. The cell suspension was divided into two aliquots, which were shaken for 15 min in the presence and absence of 10 mM aspartate, respectively. The reaction was terminated by adding trichloroacetic acid, and methyl-labelled proteins were analysed by SDS-PAGE followed by fluorography.
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RESULTS |
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Effects of kinase-activating mutations on the W550A receptor
The results described above raised the possibility that other tumbling-bias (kinase-activating) mutations may also serve as class I suppressors of the W550A substitution. We therefore introduced several known tumbling-bias mutations into the W550A receptor. First, we focused on residue G278 in the first methylation helix because this residue is a critical site of kinase regulation (Trammell & Falke, 1999 ). The residue was replaced by Ser, Thr, Arg or Ile. Cells expressing Tar-G278T-W550A or Tar-G278R-W550A formed a similar swarm to cells expressing the class I revertant Tar receptors, whereas cells expressing Tar-G278S-W550A formed a smaller swarm (Fig. 5B
). Cells expressing Tar-G278I-W550A failed to swarm (Fig. 5B
), which is consistent with the fact that the Tar-G278I mutant protein cannot regulate kinase activity in response to aspartate (i.e. it constitutively activates CheA kinase) (Trammell & Falke, 1999
).
Next, we examined the mutations that were initially isolated as suppressors of the A19K substitution that abolishes the signalling ability of Tar without impairing the aspartate-binding ability (Oosawa & Simon, 1986 ). All of them are single-residue substitutions in the second transmembrane helix (TM2; W192R, A198E, V201E and V202L) or in the first methylation helix (MH1; H267D and T303I) that cause a wide range of tumbling biases (Tatsuno et al., 1996
; Umemura et al., 1998
). In the absence of chemoeffector, cells expressing Tar-A198E-W550A or Tar-T303I-W550A tumbled incessantly, whereas cells expressing the other mutant receptors showed various degrees of signal bias (Fig. 5C
). In swarm assay, cells expressing Tar-A198E-W550A or Tar-T303I-W550A formed a similar swarm to cells expressing the class I revertant receptor, whereas cells expressing any of the other constructs failed to swarm (Fig. 5D
). Thus, the A198E (in TM2) and T303I (in MH1) substitutions, but not the other substitutions which caused weaker tumbling biases, can serve as suppressors of the W550A substitutions. Taken together, a strong (but not constitutive) tumbling bias seems to be required (and probably sufficient) for suppression of the W550A substitution.
Analysis of the class II suppressor
The results described above suggest that the class II suppressor restores the affinity of Tar-W550A for CheR. We tried to detect binding of CheR to inverted membrane vesicles containing Tar-W550A-Op554W. However, the level of binding was much lower than that to membranes containing wild-type Tar (data not shown). The class II revertant Tar receptor has 11 additional residues (WRVFTNAVIAA) due to the mutation of the termination codon (Op554W). This carboxy-terminal extension contains an xWxxF motif that is especially important for the interaction with CheR (Djordjevic & Stock, 1998 ; Shiomi et al., 2000
) and therefore might serve as a (weak) binding site for CheR. To examine this possibility, we first examined the effect of the Op554W mutation on the Tar receptor with the F553A mutation, which is less severe than W550A (Shiomi et al., 2000
), probably because the phenylalanine residue is less critically involved in the interaction with CheR than the tryptophan residue (Djordjevic & Stock, 1998
). Cells expressing Tar-F553A-Op554W swarmed as fast as cells expressing Tar-W550A-Op554W (Fig. 6
), indicating that the Op554W mutation can also suppress the defect caused by the F553A mutation. We next examined the effect of Op554A substitution (i.e. the loss of the xWxxF motif in the 11 additional sequences) on the W550A receptor. Cells expressing Tar-W550A-Op554A failed to swarm, suggesting that the tryptophan residue at position 554 is critical for suppression. These results are consistent with the interpretation that the class II suppressor partially restores binding of CheR to the receptor by creating an xWxxF motif in the carboxy-terminal additional sequence.
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DISCUSSION |
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Of the class I suppressors, G278A and G278C had already been characterized by Trammell & Falke (1999) : the receptors with these mutations superactivate CheA autophosphorylation in the absence of aspartate, but in the presence of aspartate the mutant receptors fully and partially inhibit the kinase, respectively. The receptors with the other class I suppressors (T334A and A398T) also exhibited tumbling-biased signalling and their methylation levels were lower than that of Tar-W550A, suggesting that these mutations also render the signal bias toward the activation of CheA. We also showed that some other tumbling-bias mutations, which allow regulation of the activity of CheA kinase in response to aspartate, are also able to suppress the W550A suppression regardless of the place of the second site mutation. However, among the tumbling-bias mutations tested, those which cause weaker tumbling biases or which cause constitutive activation of CheA failed to suppress the W550A mutation. Therefore, we conclude that the class I suppressors compensate for an excessively smooth-swimming (i.e. CheA-inactivating) bias caused by decreased methylation of Tar-W550A.
Wild-type Tar inhibits CheA activity in the presence of aspartate, but increased methylation of Tar during adaptation to aspartate restores the CheA- stimulating activity. Methylation of Tar-W550A can also be stimulated to a certain extent by aspartate, but this enhancement of methylation may be too slight to reactivate CheA in the presence of aspartate. The class I revertant Tar receptors can inhibit the CheA activity in response to aspartate, but they still can stimulate CheA when their methylation is enhanced by aspartate (i.e. when the cell adapts to aspartate) although their absolute levels of methylation are lower than that of wild-type Tar, presumably because of constitutive activation of CheA, and hence the methylesterase CheB. This difference in CheA activation between the methylated and unmethylated ligand-occupied revertant receptors may be enough to support chemotaxis to some extent.
The mechanism by which the class I mutations suppress the defect introduced by the W550A substitution demonstrates the robustness of the chemotaxis system (Barkai & Leibler, 1997 ; Alon et al., 1999
). Alon et al. (1999)
showed that chemotaxis is largely independent of the basal swimming bias and the adaptation time. Although the revertants failed to support swarming in minimal semisolid agar containing 10-5 M aspartate, the requirement for higher concentrations of aspartate is readily explained by the suppression mechanism described above. The tumbling-biased signals produced by the class I receptors may be too strong to be suppressed by 10-5 M aspartate.
Presumably, the class II revertant receptor partially restores binding of CheR to the receptor. When expressed as a sole chemoreceptor in a cell, this receptor supports chemotaxis and a wild-type signal bias, and it is methylated almost as effectively as wild-type Tar. We examined binding of CheR to inverted membrane vesicles containing the class II receptor, but the level of binding was much lower than to membranes containing wild-type Tar (data not shown), suggesting that the affinity of the revertant protein for CheR is weaker than that of wild-type Tar. This revertant Tar receptor has 11 foreign residues (WRVFTNAVIAA) due to the mutation of the termination codon (Op554W). The results presented here suggest that the class II suppressor partially restores binding of CheR to the receptor by creating an xWxxF motif in the carboxy-terminal additional sequence. This means that the CheR-binding sequence may not have to be located at the extreme carboxy terminus of the chemoreceptor.
The current understanding of the regulation of methylation of chemoreceptors is as follows. Binding of an attractant to a chemoreceptor leads to inhibition of CheA (Borkovich et al., 1989 ), and hence decreases phosphorylation of CheB (Hess et al., 1988
; Lupas & Stock, 1989
) to decrease the rate of demethylation. The attractant-bound receptor becomes a better substrate for CheR and a poorer substrate for CheB, resulting in a net increase in methylation of the liganded receptor (Springer et al., 1977
; Wang & Koshland, 1980
). Since the methylation levels of the class I revertant Tar proteins were lower than the methylation level of Tar-W550A, we conclude that the affinity of Tar for CheR was not restored by the class I suppressors. However, the methylation levels of these receptors were slightly but significantly enhanced by aspartate, suggesting that binding of CheR to the NWETF sequence is not strictly required for the regulation of the methylation level of the chemoreceptor.
What is the role of the pentapeptide sequence then? It is likely that the pentapeptide motif is required for maintaining an appropriate level of receptor methylation by increasing the local concentration of CheR around the receptor cluster in the cell. This interpretation is consistent with the following facts: (i) CheR is much less abundant than the receptor molecules (Simms et al., 1987 ); (ii) the overproduction of CheR suppresses the defect of the mutations in the pentapeptide sequence (Okumura et al., 1998
; Shiomi et al., 2000
); (iii) the receptor molecules form a cluster with CheW and CheA at a cell pole (Maddock & Shapiro, 1993
; Lybarger & Maddock, 2000
); (iv) a receptor dimer with the CheR-binding sequence can help methylation of a neighbouring receptor dimer without the CheR-binding sequence (Le Moual & Koshland, 1997
; Li et al., 1997
); (v) GFP-CheR localized to cell poles in the presence, but not in the absence, of a chemoreceptor with the NWETF sequence (Shiomi et al., 2002
).
Okumura et al. (1998) showed that cells expressing mutant Tcp (NWESLA) respond to, but do not adapt to the repellent glycerol. Barnakov et al. (1999
, 2001
) demonstrated that CheB also binds to the NWETF peptide. We also found that the W550A receptor serves as a poorer substrate of CheB (unpublished results). These findings suggest that the NWETF sequence serves also as a modulator of CheB activity. The intragenic suppressors of the W550A mutation isolated in this study are either CheA-activating mutations or a mutation that seemingly partially restores the affinity of Tar for CheR. Moreover, the same original mutation can be suppressed by the overproduction of CheR (Shiomi et al., 2000
). Therefore, at least under the conditions tested, binding of the receptor to CheR may be more important (or rate-limiting) to support chemotaxis than that to CheB. It is also possible that the W550A mutation has a more severe effect on binding of CheR than that of CheB. Further analyses will be required to address these issues.
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ACKNOWLEDGEMENTS |
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Received 28 February 2002;
revised 4 June 2002;
accepted 21 June 2002.
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