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J. Biol. Chem., Vol. 280, Issue 2, 1448-1456, January 14, 2005
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From the
Department of Agricultural Chemistry, Kinki University, Nakamachi 3327-204, Nara 631-8505, ¶Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Laboratory of Molecular Microbiology, School of Agriculture, ||Nagoya University, Chikusa-ku, Nagoya 464-8601, and **Division of Molecular Biology, Nippon Institute for Biological Science, Ome, Tokyo 198-0024, Japan
Received for publication, September 2, 2004 , and in revised form, November 1, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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On the basis of Escherichia coli genome sequence, a total of 30 HK, each containing the conserved self-phosphorylation domain, and a total of 32 RR, each containing the conserved receiver domain, have been predicted (3). After detailed analysis of the genome sequence, two additional RR candidates, YgeK and YhjB, have been identified, both containing the conserved helix-turn-helix motif of the RR family.2 Recently, Oshima et al. (4) performed a microarray analysis for a total of 30 TCS mutants of E. coli and speculated that, at least for certain combinations, TCSs functionally interact each other to expand the signal transduction network so as to allow some genes to respond to various signals in the environment (4). To examine the specificity of HK-RR interaction in a more direct way, we have purified as many HK and RR proteins as possible, and we tested the self-phosphorylation of HK and the trans-phosphorylation of RR by phosphorylated HK in all possible combinations.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Protein Expression—To achieve high level expression of the target proteins, each plasmid was transformed into three different competent E. coli cells, two IPTG-inducible strains, BL21(DE3) and JM109(DE3), and one salt-inducible strain BL21(SI) and selected for one strain showing the maximum induction level among the three test strains. For large scale protein production, each transformant was grown in 200 ml of LB broth and at 0.8–0.9 of A600; IPTG was added to BL21(DE3) and JM109(DE3) cultures at the final concentration of 1 mM, or NaCl was added to BL21(SI) culture at the final concentration of 0.3 M. After 3 h, cells were harvested by centrifugation, washed with lysis buffer (50 mM Tris-HCl, pH 8.0, 4 °C, and 100 mM NaCl), and then stored at -80 °C until use.
Protein Purification—Frozen cells were suspended in 3 ml of lysis buffer containing 100 mM phenylmethylsulfonyl fluoride. After addition of 80 µl of lysozyme (10 mg/ml), the cell suspension was stored on ice for 30 min and then lysed by sonication. After centrifugation at 15,000 rpm for 20 min at 4 °C, the supernatant was mixed with 2 ml of 50% nickel-nitrilotriacetic acid-agarose suspension (Qiagen) and loaded onto a column. After washing with 10 ml of lysis buffer, the column was washed with 20 ml of lysis buffer containing 0.5% Triton X. The His-tagged HK or RR protein was then eluted with 2 ml each of lysis buffer containing 0.1, 0.2, or 0.5 M imidazole. The recovery and purity of HK or RR protein in each eluate were checked by SDS-PAGE. The purified HK or RR protein fractions were pooled and dialyzed against storage buffer (50 mM Tris-HCl, pH 7.6, 4 °C, 200 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, and 50% glycerol). The protein concentration was determined by the protein assay kit (Bio-Rad), and the purity was checked by SDS-PAGE.
Self-phosphorylation of HK—The purified HK was diluted to 1 µM with kinase buffer (50 mM Tris-HCl, pH 8.0, 37 °C, 50 mM KCl, and 10 mM MgCl2), and the phosphorylation reaction was initiated by adding 1 µCi of [
-32P]ATP at a final concentration of 2.5 µM. The reaction was carried out at 37 °C for various times and terminated by adding an equal volume of 2x sample buffer (120 mM Tris-HCl, pH 6.8, 4 °C, 20% glycerol, 4% SDS, 10% 
-mercaptoethanol, and 0.1% bromphenol blue). After SDS-PAGE, the gel was washed with 45% methanol, 10% acetic acid, dried, and exposed onto an image plate. The intensity of each band on the image plate was measured with BAS1000 (Fuji Film Co., Japan).
Trans-phosphorylation of RR by HK—The phosphorylated form of HK (1 µM) prepared as above was mixed on ice with a mixture of RR (1 µM) and excess cold ATP (0.5 mM) and then incubated at 37 °C for various times. The reaction was terminated by adding an equal volume of 2x sample buffer. The samples were analyzed by SDS-PAGE. The gel was washed with 45% methanol, 10% acetate, dried, and exposed onto an image plate. The intensity of each gel band was measured with BAS1000 (Fuji Film Co.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To achieve the maximum level of protein induction, we first checked the expression level by using three different competent strains, IPTG-inducible BL21(DE3) and JM109(DE3) and salt-inducible BL21(SI), and under various induction conditions with respect to the inducer concentration and the induction time. The host strain and the best conditions for maximum expression differed from protein to protein (data not shown). Three HK proteins, RcsC, YpdA (B2380), and QseC, were not induced in all the hosts used and under all the conditions tested. Except for these three HK proteins, 61 other proteins (27 HKs and 34 RRs) were subjected to large scale expression and purification. Most of the proteins were recovered in soluble fractions and purified to apparent homogeneity by a single step of affinity chromatography. Two HK proteins (AtoS and YehU) and one RR protein (FimZ) were recovered in pellet fractions after centrifugation and thus solubilized in lysis buffer containing 7 M urea. For these proteins, urea was removed after protein purification by dialyzing against storage buffer without urea. As a result, a total of 61 proteins, 27 truncated form HKs and 34 full-length RR, were purified, all of which apparently showed a single band on SDS-PAGE (data not shown).
Self-phosphorylation of HKs—A total of 27 purified HK was subjected to self-phosphorylation in the presence of radioactive ATP. As shown in Fig. 1, 25 species of the purified HK showed the self-phosphorylation activity, but a detectable level of phosphorylation was not observed for two HKs, CitA and YojN. The YojN protein is not an orthodox-type sensor because it lacks the conserved catalytic domain of His kinase, whereas the lack of self-phosphorylation activity for CitA is not yet clear. For all 25 catalytically active HKs, the self-phosphorylation took place rapidly, and the maximum level of phosphorylation was observed within 30 min. The phosphorylation level did not increase after prolonged incubation for up to 2 h (data not shown). The level of phosphorylation, however, decreased after addition of unlabeled ATP (see below), indicating that the self-phosphorylation of HK is a reversible reaction. The dissociation of radioactive phosphorus was also observed even in the absence of ATP. These observations suggest that the HKs used in this study were de-phosphorylated during purification and after prolonged storage in the absence of ATP.
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5% (the amount of phosphorylated form in the total amount of HK added), assuming that each HK carries a single site of phosphorylation (Fig. 2). The self-phosphorylation level was the highest (4%) for CheA, herein classified as group L1 (level 1). Group L2 with the high activity (higher than 0.03%) includes 14 members (YfhK, TorS, BaeS, RstB, PhoR, BasS, YedV, BarA, CreC, PhoQ, UhpB, CusS, KdpD, and DcuS in decreasing order) (Fig. 2). Six L3 group members (NtrB, HydH, CpxA, ArcB, NarX, and EnvZ in decreasing order) exhibited the phosphorylation level of lower than 0.02% and higher than 0.005%. The phosphorylation level was lower than 0.005% for L4 group HK members (NarQ, AtoS, EvgS, and YehU).
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Among the 25 HK species analyzed, a total of 17 members showed good correlation between the rate (R) and the level (L) of self-phosphorylation (shaded area in Fig. 2). The activity difference in this group might reflect a difference in the population of active HK molecules in the purified HK preparations. One of the novel findings in this study was the detection of dephosphorylation activity for HK. Even in the presence of [32P]ATP, for instance, the level of phosphorylated NtrB decreased after saturation (see Fig. 1). Five HK members, ArcB (spot A), NtrB (spot Q), HydH (spot M), NarQ (spot O), and EvgS (spot L), showed high rates of phosphorylation but low saturation levels (R > L, above the shaded area in Fig. 2). As in the case of NtrB, the HKs of this group may have high activities of dephosphorylation under the reaction conditions employed. On the other hand, three HK members, CheA (spot F), BaeS (spot C), and CreC (spot H) (R < L, below the shaded area in Fig. 2), showed high levels of self-phosphorylation even though the rates of phosphorylation were low.
Trans-phosphorylation of RRs by HKs—From both the previously characterized data of E. coli TCS systems and the paired location of HK and RR genes on the E. coli genome, a total of 26 TCS pairs including both CheA-CheB and CheA-CheY have been predicted (3). The genes for five HKs (arcB, barA, narQ, rcsC, and yojN) and six RRS (arcA, fimZ, narP, rcsB, rssB, and uvrY) are not linked to the genes for respective RR partners on the genome. Based on genetic and/or biochemical data, these orphan HKs and RRs have been considered to form the following cognate HK-RR pairs: ArcB-ArcA, BarA-UvrY, and NarQ-NarP (6, 17, 18). The RcsC-YojN and YojN-RcsB systems form a sequential pathway, RcsC-YojN-RcsB, of the His-Asp phospho-relay (19). Up to now, however, no HK partners have been identified for two RRs, FimZ and RssB.
By using a total of 25 functional HKs with self-phosphorylation activity (see Figs. 1 and 2), we performed the trans-phosphorylation assay for all possible combinations. A fixed amount of each HK was first incubated with [-32P]ATP for self-phosphorylation until saturation (see Fig. 1), and then mixed with an equal molar amount of the respective cognate RR and an excess amount of unlabeled ATP (100-fold molar excess over the radiolabeled ATP). First we examined the HK-RR cognate pairs. Among a total of 26 cognate HK-RR pairs, including both CheA-CheB and CheA-CheY pairs, a significant level of transphosphorylation was observed at least for 24 pairs (Fig. 3). EvgS failed to phosphorylate EvgA under the reaction conditions employed, whereas trans-phosphorylation level for AtoS-AtoC was hard to detect because these two components could not be separated onto SDS-PAGE.
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Cross-talks in Trans-phosphorylation between Non-cognate HK-RR Pairs—Phosphorylation in vivo of RR by non-cognate HK was suggested by genetic and microarray analysis of some HK mutants (4, 17, 20). Here we carried out more systematic and direct search for the cross-talk of trans-phosphorylation of RRs by non-cognate HKs. By using 21 species of the functional HK except for TorS and YehU and 34 species of RR, we examined possible cross-talks in trans-phosphorylation for 692 combinations (21 x 34 - 22 (cognate pairs)). HKs were first self-phosphorylated by incubation with [-32P]ATP for 30 min and then mixed with equimolar amounts of each non-cognate RR and excess unlabeled ATP. After 30 s of incubation, the mixtures were subjected to SDS-PAGE analysis.
Among a total of 692 non-cognate HK-RR pairs, trans-phosphorylation between non-cognate pairs was identified for a total of 22 combinations, as indicated by the red bars in Fig. 5. Seven species of HK (pBarA, pBaeS, pDcuS, pEnvZ, pRstB, pUhpB, and pYedV) phosphorylated non-cognate RR(s). Among these HKs, pUhpB phosphorylated 9 non-cognate RRs and 1 orphan RR (RssB), pBarA phosphorylated 4 non-cognate RRs, and pBaeS phosphorylated 2 non-cognate RRs, whereas pDcuS, pEnvZ, pRstB, and pYedV phosphorylated each non-cognate RR. The external signals sensed by these HKs must regulate, under certain conditions, the genes, which are under the control of another HK-RR system.
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Of the two orphan RRs (FimZ and RssB), of which the pairing HK partners have not been identified, RssB was found to be phosphorylated by three HKs, pArcB, pCheA, and pUhpB. RssB binds to the stationary phase-specific RNA polymerase 
S (RpoS) and transforms it susceptible to degradation by ClpXP proteases. The results described herein suggest that one of the three HKs is the cognate HK of RssB (phosphorylation by other two HKs must be due to cross-talk in trans-phosphorylation). All these HKs are, however, known to form TCS with the respective cognate RR partner (ArcA for pArcB; CheB and CheY for pCheA; and UhpA for pUhpB). Phosphorylation of FimZ was, however, not detected with use of any HKs used in this study.
Interference of TCS Signal Transduction by Non-cognate RR—For some specific combinations, the presence of non-cognate RR enhanced dephosphorylation of HK. The enhancement of HK dephosphorylation by non-cognate RR suggests the interference of one TCS signal transduction by another pathway. The combinations of interference are summarized in Fig. 5 (shown by blue lines). Among the RRs tested, five species of RR (CitB, CpxR, PhoB, RssB, and YhjB) enhanced dephosphorylation of more than two species of HK, and four species (CusR, HydG, PhoP, and YfhA) enhanced dephosphorylation of one non-cognate HK. In addition, YhjB, a NarL family orphan RR, stimulated dephosphorylation of 2 HKs, EnvZ and NtrB, even though HK for YhjB phosphorylation was not identified. It is worthwhile to note that RssB, the stability regulator of S (RpoS), interacts with and induces dephosphorylation of 6 HKs, including pBaeS, pCreC, pDcuS, pHydH, pNarQ, pNtrB, and pRstB.
Dephosphorylation of some HKs was enhanced by multiple species of non-cognate RR. For instance, dephosphorylation of pDcuS was enhanced by four non-cognated RRs (CitB, CpxR, PhoB, and RssB) and dephosphorylation of pEnvZ, pNtrB, and pRstB was stimulated each by three non-cognate RRs (Fig. 5). Enhancement of HK dephosphorylation by non-cognate RRs may indicate the interference of signal transduction between different TCSs, leading to expand the cross-talk within the signal transduction network.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In most cases, the HK with a high rate of self-phosphorylation showed a high level of phosphorylation (see Fig. 2). The activity difference might be related, to a certain extent, to the level of functional protein molecules in HK preparations. However, the amount of phosphorylated form in each HK preparation used might be low, if any, because the HK dephosphorylation takes place during purification and storage. Some HKs such as ArcB, NtrB, HydH, and NarQ showed high rates of phosphorylation but low levels of phosphorylation, presumably because these HKs have the high rate of dephosphorylation (Fig. 6) as demonstrated for NtrB (see Fig. 1). In contrast, another group including CheA, BaeS, and CreC showed high levels of self-phosphorylation even though the phosphorylation rate was low (see Fig. 6). The HKs of this group may be able to maintain the phosphorylated state (or the response memory) for long periods. The quick response for the HKs of this group, however, must be archived by another step such as the signal sensing of HK, conformational change of membrane-bound HK, and DNA-binding affinity of RR. The difference in kinetic parameters among the test HKs suggests that the in vitro assay of HK self-phosphorylation reflects, to certain extent, the in vivo situations.
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Both the rate and level of trans-phosphorylation differed between HK-RR pairs. Based on the rate of trans-phosphorylation, HK-RR pairs could be classified into two groups (see Fig. 4). Group A pairs showed high rates of trans-phosphorylation, but the rate was lower for group B pairs. As in the case of HK self-phosphorylation, the rate and the extent of trans-phosphorylation, herein observed, may reflect the nature of signal transduction of each TCS. The group A TCSs are able to respond quickly to changes in the environment (see Fig. 6). It is noteworthy that the three HKs (ArcB, NarQ, and NtrB) with a high rate but a low level of self-phosphorylation are all included in this group.
The group A pairs can be further classified into two subgroups with respect to the stability of phosphorylated RR. The phosphorylated RR is stable for group A1 pairs, whereas group A2 pairs are unstable and rapidly dephosphorylated. The group A1 TCSs are capable of maintaining the response memory for long periods. On the other hand, quick dephosphorylation (or inactivation) of the group A2 RRs may lead to rapid loss of the memory, thereby returning to the steady-state of gene expression after transient activation or repression upon exposure to external stresses (see Fig. 6). The instability of phosphorylated RRs could be intrinsic properties but not due to contamination of nonspecific phosphatases, because the addition of HK or RR of group A2 pairs do not accelerate dephosphorylation of RRs from different TCSs (data not shown). The accurate measurement of the rate and the saturation level of trans-phosphorylation are therefore more difficult than those for selfphosphorylation, because HK carries the phosphatase activity of its own phosphate moiety (this work) and the cognate RR-associated phosphate (21). For instance, the level of pArcB is significantly higher than the input pArcA, suggesting that loss of radioactive phosphate from pArcA prior to its transfer to ArcB.
Our systematic search for trans-phosphorylation of RRs by non-cognate HKs under the same reaction conditions as used for trans-phosphorylation between the cognate pairs detected the cross-talk in phospho-relay for at least 22 combinations (see Fig. 5). In addition, two of the three combinations, which were newly identified for RssB phosphorylation, might be arisen from the cross-talk. Most interestingly, YgeK, a NarL family RR, is unique, because it lacks the receiver domain, which generally contains the conserved Asp residue that is phosphorylated by HK. Most surprisingly, two HKs, pBarA and pUhpB, phosphorylated YgeK (see Fig. 5), suggesting the presence of novel phospho-relay other than the typical His 
Asp phosphorelay. Overall, the total number of cross-talks in E. coli TCS may be 21 (3.0%) out of a total of 692 non-cognate pairs examined. In certain cases, one RR can be phosphorylated by multiple species of HK. Most interestingly CheY, YfhA, and CusR, which showed high levels of phosphorylation (see "Results"), were phosphorylated by two, four, and three HKs, respectively (see Fig. 5). The target Asp residues on this group of RRs may be accessible by any HKs. In these cases, we cannot exclude that the results of cross-talks observed in vitro are artifacts arising from partial denaturation of HKs and/or RRs. However, significant levels of the cross-talk in trans-phosphorylation were observed for a group of RRs (NarP, CpxR, NarL, and HydG), which showed the low level of trans-phosphorylation by the cognate HKs. In these cases, the observed cross-talk may be more specific. For trans-phosphorylation to take place, HKs must carry the conserved segments to function as anchors for their attachment to the conserved segments close to the target Asp residue within RRs (22). The specificity of each cognate HK-RR pair seems to be determined by the variable region(s) close to this HK-RR contact domain. The search for the recognition and interaction sequences for both cognate and noncognate pairs awaits further analyses.
When one RR is phosphorylated by multiple species of HK, the target genes under the control of this particular RR may respond to various stresses. Likewise, one HK may phosphorylate multiple RRs. In these cases, the intracellular content of HKs and/or RRs is a major factor affecting the selection of the most favorite RR for the trans-phosphorylation. An excess amount of the activated RR, however, might lead to the autorepression of its own synthesis. The auto-regulation is known to operate for some TCS genes (23–26). In any case, we will be able to focus on these 19 (or 22) combinations for confirmation of the in vivo cross-talks between the TCS systems.
The phosphorylated RssB is known to bind the stationary phase-specific RNA polymerase S (RpoS) and to control its degradation by ClpXP protease (27–29). Here we found that RssB, the orphan RR, could be phosphorylated by three HKs, pArcB, pCheA, and pUhpB (see Fig. 5). ArcB is involved in the adaptation to an anaerobic state by switching the expression of genes for the generation of metabolic energy in the absence of oxygen. Trans-phosphorylation of RssB by the pArcB must induce the degradation of S. In fact, Sugiura et al. (30) reported that in the arcB-null mutant, the level of RpoS increased even during exponential growth phase under aerobic conditions. RssB is also activated by pCheA, which enhances the flagella-dependent behavior for chemotaxis. The appearance of RpoS during the transition from exponential growth to stationary phase represses the formation of flagella because of the competition between RpoS (for stationary-phase gene transcription) and RpoF (for flagella gene transcription) (31). It is reasonable that environmental signals, which enhance flagella function, also induce the formation of flagella by selective utilization of RpoF after degradation of competitive RpoS by RssB. Likewise, in the presence of external glucose 6-phosphate, selective degradation of RpoS is effective for enhancement of RpoD-dependent transcription of the genes for utilization of glucose 6-phosphate (32). At present, it is hard to choose the cognate HK for RssB from these three HK candidates.
Some of the non-cognate RR was found to stimulate the dephosphorylation of HKs, which are organized in other TCSs (see Fig. 5). In the functional differentiation of E. coli RNA polymerase, seven species of the factor compete for binding to a fixed number of the RNA polymerase core enzyme molecules (33). The intracellular concentration of the sum of all seven factors is 2–3-fold higher than that of the core enzyme (34), leading to the competition (35). The intracellular concentrations of transcription factors vary depending on the culture conditions and/or cell growth phases.3 In addition, in the case of RRs, the level of functional forms is controlled by phosphorylation. Both the level and the activity controls of transcription factors lead to governing the global pattern of genome transcription. One attractive hypothesis is that the induced HK dephosphorylation by certain non-cognate RRs is an interference system against competing TCSs.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Tables I and II.
To whom correspondence should be addressed. Tel.: 81-742-43-7274; Fax: 81-742-43-1445; E-mail: kyamam{at}nara.kindai.ac.jp.
1 The abbreviations used are: TCS, two-component system; HK, histidine kinase; RR, response regulator; IPTG, isopropyl 1-thio--D-galactopyranoside.
2 N. Fujita, personal communication.
3 A. Ishihama, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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