1 School of Dentistry, University of California, Los Angeles, CA 90095-1668, USA
2 Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095-1668, USA
3 Molecular Biology Institute, University of California, Los Angeles, CA 90095-1668, USA
Correspondence
Renate Lux
lux{at}ucla.edu
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ABSTRACT |
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INTRODUCTION |
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The genome of T. denticola contains all the components necessary for flagellated motility and a complete chemotaxis signal transduction chain (Seshadri et al., 2004). Gene inactivation mutagenesis has confirmed the role of some of these proteins in T. denticola motility and chemotaxis (Kataoka et al., 1997
; Li et al., 1996
, 1999
; Limberger et al., 1999
; Lux et al., 2002
). In particular, homologues to methyl-accepting chemotaxis proteins (MCPs), DmcA and DmcB (Kataoka et al., 1997
; Li et al., 1999
), have been characterized. Recent whole-genome analysis has indicated the presence of an additional 18 putative MCP-encoding ORFs, as well as an operon containing cheR and cheB, which are required for adaptation (Seshadri et al., 2004
). Most interestingly, the operon encoding homologues to the key components of the chemotaxis signalling pathway, CheA, CheW and CheY, contains an additional gene, cheX, which is localized between cheW and cheY. Since cheX is part of the cheAWXY transcriptional unit, a potential role in chemotaxis has been assumed (Greene & Stamm, 1999
). Similar chemotaxis operons have been discovered in the genomes of Treponema pallidum and Borrelia burgdorferi (Fraser et al., 1997
, 1998
), the causative agents of syphilis and Lyme disease, respectively. Phenotypic analysis of Bor. burgdorferi and T. denticola mutant strains lacking cheX confirmed the involvement of this novel chemotaxis protein in the directed motility of spirochaetes, thus rendering CheX the most intriguing component of the spirochaete chemotaxis pathway (Charon & Goldstein, 2002
).
Homologues to CheX can be found in a number of organisms (Lux et al., 2000), but a direct connection to the chemotaxis pathway is only apparent in spirochaetes and Bacillus subtilis. CheC, the CheX homologue in B. subtilis, is involved in adaptation and possibly motor control. Interaction of CheC with its antagonist CheD and MCPs (Rosario & Ordal, 1996
), as well as CheA and the motor protein FliY, has been demonstrated (Kirby et al., 2001
). In addition, a role for CheC in dephosphorylation of CheY
P has been suggested (Szurmant et al., 2004
). During the review process for this manuscript, a study revealing the crystal structure of the CheC and CheX homologues in Thermotoga maritima demonstrated CheY phosphatase activity for both proteins (Park et al., 2004
), further substantiating their role in chemotactic signalling. In addition, this study by Park et al. confirmed that both proteins fold into similar structures, even though their primary sequences do not exhibit a striking homology to each other.
To further elucidate the role of the novel spirochaete chemotaxis protein CheX, the renowned yeast two-hybrid system (Fields & Song, 1989) was used to investigate potential interactions with known proteins in the chemotactic signal transduction pathway. In addition, established interactions between CheA, CheW and the MCPs (Borkovich et al., 1989
; Gegner et al., 1992
; Liu & Parkinson, 1989
; Schuster et al., 1993
), as well as interactions of CheY with CheA (Schuster et al., 1993
; Swanson et al., 1995
) or the motor switch proteins (McEvoy et al., 1999
; Welch et al., 1993
) were examined.
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METHODS |
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DNA manipulation and plasmid construction.
Bacterial genomic and plasmid DNA isolation, PCR amplification, cloning and transformation procedures were performed according to standard protocols (Sambrook et al., 2001). Primer pairs that were used to PCR-amplify full-length cheA, cheW, cheY, cheX, fliY, fliG and fliM genes of T. denticola, in addition to a dmcB fragment that encodes a truncated DmcB (DmcB) corresponding to a stable cytoplasmic fragment of the E. coli chemoreceptor Tsr (Ames & Parkinson, 1994
), are listed in Table 1
. The indicated external restriction sites were used to clone each of the amplified fragments into both pGAD and pGBD vectors (James et al., 1996
) to generate plasmids that encode fusion proteins of the individual chemotaxis components with the GAL4 transcription activation domain (GAL4-AD) and the GAL4 DNA-binding domain (GAL4-BD), respectively.
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-Galactosidase assay.
Yeast transformants carrying the plasmid combination chosen for more detailed analysis were grown overnight at 30 °C in liquid selective SD medium. The cultures were then transferred into fresh medium, grown to mid-exponential phase, pelleted and resuspended in 50 µl STES buffer [0·2 M Tris/HCl (pH 7·6), 0·5 M NaCl, 0·1 % (w/v) SDS, 0·01 M EDTA]. Glass beads (0·5 mm, Fisher Scientific) and 20 µl TE buffer (pH 7·6) were added to each tube. This mixture was vortexed vigorously (4 min) at room temperature in a Turbo Mixer (Scientific Industries) and centrifuged at maximum speed for 5 min. The supernatant was transferred into 1·1 ml Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7·0), supplemented with 50 mM -mercaptoethanol. One hundred microlitres of this suspension was used to determine the protein concentration of the sample according to Bradford (1976)
. The remaining sample was used to measure
-galactosidase activity (Miller, 1972
) with ONPG (4 mg ml1 in Z buffer) as a substrate. One unit (U) of enzyme activity corresponds to 1 nmol ONPG hydrolysed min1 (mg protein)1. The statistical relevance of the data obtained was determined using the t test.
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RESULTS AND DISCUSSION |
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The plasmids encoding protein fusions of CheA, CheW, CheX, CheY and DmcB with Gal4-AD or Gal4-BD were then transformed in all possible combinations into the yeast strain pJ69-4A to examine the respective one-on-one interactions. The resulting transformants were tested on plates containing different dropout mixes with increasing degrees of selective stringency allowing qualitative evaluation of the proteinprotein interactions tested (Table 2). In this initial screen the novel chemotaxis protein CheX exhibited a strong interaction with itself, as indicated by growth of the yeast strain expressing both the Gal4-AD and Gal4-BD CheX protein fusions on high-stringency dropout plates, suggesting homodimer or higher-order complex formation of the protein. This finding is in very good agreement with a recently published study that demonstrated dimerization of CheX from Th. maritima (Park et al., 2004
). The same study suggested that dimerization of CheX is required to form the active centre for CheY dephosphorylation. Since the yeast two-hybrid approach chosen for this study does not enable detection of the complex interactions between a protein dimer and a phosphorylated protein, this could be one of the reasons why an interaction between CheX and CheY was not found.
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Furthermore, most of the established interactions between CheA, CheW, MCPs and CheY could be confirmed for the T. denticola chemotaxis pathway (Table 3). In agreement with previous findings in other bacterial systems (Gegner & Dahlquist, 1991
; Gegner et al., 1992
; McNally & Matsumura, 1991
) demonstrating complex formation of CheA and CheW, both construct combinations yielded growth on high-stringency plates. Binding between CheA and CheY, which is known to be transient, resulted in growth on medium-stringency plates and formation of pinkish colonies under high-stringency conditions. For some of the constructs tested, however, only one combination indicated binding, but not the corresponding counterpart (CheW with DmcB), or the interaction was weaker than expected (CheA with itself) or completely absent (CheA with DmcB). Other research groups have reported similar results, to the effect that not all known interactions can always be detected (Marykwas et al., 1996
). Among other effects, steric hindrance (especially when larger proteins are involved) or insufficient domain folding of the fusion protein could be responsible for this phenomenon. Nevertheless, the yeast two-hybrid system remains a very well-established and powerful tool to detect novel proteinprotein interactions.
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Since the interactions identified on medium-stringency dropout plates resulted only in an approximately twofold increase above background levels of -galactosidase activity, a statistical analysis (t test) was performed to evaluate the significance of the apparent proteinprotein interactions. All interactions shown in Table 3
were considered significant as they displayed P values of <0·05. Interactions of CheX with Gal4-AD/BD fusion proteins of CheW or CheY were also tested using the quantitative
-galactosidase assay. Even though values above background level were obtained, statistical analysis revealed that they were not significant (data not shown). In conclusion, the interactions revealed in the initial qualitative screen using selective dropout medium plates with different stringencies were confirmed by the quantitative measurements.
CheX was found to bind CheA at a level comparable to the well-established CheA/CheY interactions (McNally & Matsumura, 1991; Schuster et al., 1993
), implying a more transient nature of CheA and CheX recognition. CheY that alternates between CheA and the flagellar motor switch changes its affinity for CheA upon phosphorylation of CheY, resulting in its release from CheA (Schuster et al., 1993
; Swanson et al., 1995
). In a similar manner to CheC, its counterpart present in B. subtilis, CheX, does not contain any apparent conserved phosphorylation or other modification sites, leaving the nature of CheX interaction with CheA to be elucidated. Since the novel chemotaxis protein CheX demonstrated a strong interaction with itself, dimerization or even the formation of higher-order complexes may be an important part of CheX function. The CheX dimer/oligomer could comprise the active state of the protein that is controlled by CheA or other proteins in the signal transduction chain. The recent study by Park et al. (2004)
demonstrated that CheX of Th. maritima dimerizes to form a putative active centre similar to the one found in a single molecule of the larger CheC protein.
Model for chemotactic signal transduction in T. denticola
The results presented here introduce a new player, CheX, in the chemotaxis signal transduction chain of the oral spirochaete T. denticola. A role for CheX in the directed movement of spirochaetes was already suggested by previous observations that inactivation of cheX resulted in an altered motility and chemotaxis phenotype in T. denticola as well as Bor. burgdorferi (Charon & Goldstein, 2002).
Most interestingly, CheC, the CheX homologue present in the complex B. subtilis chemotaxis system, was shown to employ similar interactions to those found in this study for CheX. In particular, CheC interacted with CheA, the central kinase in the chemotactic signal transduction pathway, but not CheY, strongly supporting the novel interactions described here for CheX. CheC was also suggested to interact with the putative motor switch protein of B. subtilis, FliY, a combination that could not be tested for CheX of T. denticola due to self-activation of the FliY constructs. Since previous studies grouped CheC and CheX together with the motor proteins FliY and FliM, to form the CYX protein family (Kirby et al., 2001; Szurmant et al., 2004
) and homologous domains are known to mediate proteinprotein interactions (Bilwes et al., 1999
; Mathews et al., 1998
), interaction of CheX with FliY remains a possibility. Furthermore, CheC of B. subtilis was recently found to enhance CheY dephosphorylation in a similar manner to FliY (Szurmant et al., 2004
), even though direct binding to CheY was not found. Since it is a member of the CYX family, which includes CheC and FliY, the authors suggested a putative similar function for CheX. This assumed ability of CheX to dephosphorylate CheY has recently been confirmed for CheX of Th. maritima.
Since T. denticola does not contain a homologue to CheD, the antagonist of CheC function in receptor modulation and adaptation (Rosario & Ordal, 1996; Rosario et al., 1995
), an involvement of CheX in similar functions appears unlikely. In addition, no interaction between CheX and a cytoplasmic fragment of the chemoreceptor DmcB was observed in this study.
T. denticola appears to follows the well-characterized basic general pathway present in most motile bacteria. In addition to the established chemotaxis proteins MCPs, CheA, CheW and CheY, whose interactions have been studied extensively on a molecular basis, the chemotaxis system of spirochaetes contains an extra player, CheX. Here, based on the interactions revealed in this study, we propose a model for the chemotaxis signal transduction pathway of T. denticola (Fig. 1) integrating CheX. These results are supported by similar findings for the CheX homologue CheC, a component of the far more complex chemotaxis system of B. subtilis, in addition to those of a novel study revealing structural and biochemical features of CheX and CheC of Th. maritima.
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Furthermore, in analogy to the observations reported for B. subtilis CheC, CheX could receive a signal from CheA and potentially transmit this signal to the motor switch protein FliY, which is encoded by the genomes of most spirochaetes that have been sequenced so far. The possibility that CheX acts as an additional messenger between the signalling complex and the flagella motor is especially intriguing, since spirochaetes require a mechanism allowing coordination of flagellar rotation at both cell poles in response to chemotactic stimuli. This coordination problem is distinctive for spirochaetes. Hence a unique chemotaxis protein such as CheX could play a role in this process, as suggested earlier by Charon & Goldstein (2002).
We are currently constructing a comprehensive yeast two-hybrid library of T. denticola to identify other potentially CheX interacting proteins. This approach, in addition to a detailed phenotypic analysis of mutant strains lacking CheX and/or CheY, will provide novel insights into the function of CheX in T. denticola chemotaxis and motility.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 September 2004;
revised 2 February 2005;
accepted 18 February 2005.
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