• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Nov 14, 2006; 103(46): 17474–17479.
Published online Nov 6, 2006. doi:  10.1073/pnas.0605197103
PMCID: PMC1859953
Microbiology

An escort mechanism for cycling of export chaperones during flagellum assembly

Abstract

Assembly of the bacterial flagellar filament requires a type III export pathway for ordered delivery of structural subunits from the cytosol to the cell surface. This is facilitated by transient interaction with chaperones that protect subunits and pilot them to dock at the membrane export ATPase complex. We reveal that the essential export protein FliJ has a novel chaperone escort function in the pathway, specifically recruiting unladen chaperones for the minor filament-class subunits of the filament cap and hook-filament junction substructures. FliJ did not recognize unchaperoned subunits or chaperone-subunit complexes, and it associated with the membrane ATPase complex, suggesting a function postdocking. Empty chaperones that were recruited by FliJ in vitro were efficiently captured from FliJ-chaperone complexes by cognate subunits. FliJ and subunit bound to the same region on the target chaperone, but the cognate subunit had a ≈700-fold greater affinity for chaperone than did FliJ. The data show that FliJ recruits chaperones and transfers them to subunits, and indicate that this is driven by competition for a common binding site. This escort mechanism provides a means by which free export chaperones can be cycled after subunit release, establishing a new facet of the secretion process. As FliJ does not escort the chaperone for the major filament subunit, cycling may offer a mechanism for export selectivity and thus promote assembly of the junction and cap substructures required for initiation of flagellin polymerization.

Keywords: protein secretion, secretion pilots, type III export

Bacterial motility is commonly conferred by cell surface flagella. The long flagellar filament acts as a rotating helical propeller, and it is anchored to the basal body in the cell envelope via a flexible hook (1). The filament structure comprises four types of filament-class subunits. The major filament substructure comprises about 20,000 flagellin (FliC) subunits, which are polymerized under the distal filament cap, a FliD pentamer that is displaced farther from the cell as the filament grows. The filament is adapted to the flexible hook by a preformed hook-filament junction made up of 11 subunits each of FlgK and FlgL. The filament structure is assembled in strict sequence; the cap and junction substructures must be assembled before flagellin is accepted by the nascent structure.

The major subunit FliC and the minor subunits FliD, FlgK, and FlgL are delivered from the cytosol to the base of the nascent flagellum by a type III export pathway in which they are bound by the subunit-specific export chaperones FliS (for FliC), FliT (for FliD), and FlgN (for FlgK and FlgL). These export chaperones effect transition to the membrane by preventing premature polymerization of subunits, acting as “bodyguards” for the C-terminal amphipathic oligomerization domain (24) and by piloting the subunits to the export apparatus (5). The chaperoned subunits dock at the membrane-associated export ATPase FliI (5, 6), a hexameric ring with a central pore proposed to align with the central channel of the nascent flagellum (79). Transient intermediate complexes underlying the early stages of the export pathway have been identified, but there are few data about the series of events postdocking. In vitro studies of a related type III pathway have shown that ATP hydrolysis by the export ATPase facilitates chaperone release (10). However, nothing is known about the fate of chaperones once released from the filament-class subunits, and there is no evidence for subunit selectivity by the export apparatus despite the stringent delivery sequence and striking subunit stoichiometry in the completed flagellum.

To study further the sequence of events underlying the export pathway we have addressed the function of FliJ, which is essential for export of structural subunits (11). This unveiled an unpredicted and novel activity indicating cycling of export chaperones.

Results

FliJ Is Not an Export Chaperone for Hook- or Filament-Class Structural Subunits.

FliJ resembles subunit chaperones, e.g., in size, and has been suggested to act as a “general” cytosolic chaperone (12). We sought to isolate the predicted transient complexes of FliJ and subunits by performing affinity chromatography of E. coli extracts containing overexpressed untagged hook-and-filament-class subunits, using (His6)-tagged FliJ. This FliJ variant is active as it complements a fliJ null mutant. None of the flagellar hook-class subunits FlgD, FlgE, and FliK or filament-class subunits FlgM, FlgK, FlgL, FliD, and FliC was bound by (His6)FliJ, assayed following Ni2+-affinity copurification (Fig. 1Upper). As has been reported by other laboratories (13, 14), (His6)FliJ did bind to FliH (Fig. 1 Upper, extreme right), the FliI ATPase regulator. This indicated that FliJ is not a general subunit chaperone.

Fig. 1.
Recognition of flagellar subunits and chaperones by FliJ. Affinity chromatography of (Upper) cell lysates of E. coli C41 expressing (His6) FliJ (20 kDa) incubated with lysates of the same strain expressing one of the hook-class subunits FlgD (24 kDa), ...

FliJ Binds Chaperones for the “Minor” Subunits of the Filament Cap and Hook Junction.

Binding of FliJ to other flagellar components was screened by similar, glutathione Sepharose affinity copurification. This revealed that GST-FliJ recognizes the minor subunit-specific export chaperones FlgN and FliT (Fig. 1 Lower). In contrast, there was no recognition of a functional FliS chaperone [i.e., that bound its cognate subunit FliC subunit (2)], even when greater amounts of protein were tested or when His-tagged FliJ was used as bait in cell lysates, as in Fig. 1 Upper.

The FliJ-chaperone complexes were assembled in vitro and analyzed by gel-filtration chromatography. The three chaperones and FliJ each migrated alone as single species (Fig. 2AiAiii), the behavior of the 20-kDa (His6)FliJ protein being consistent with the aberrant migration of the putatively elongated monomer previously established by multiangle light scattering and analytical ultracentrifugation (13). Following incubation with FliJ, the FlgN chaperone shifted in its elution from 30 kDa to ≈42 kDa (Fig. 2Ai); similarly, FliT shifted from ≈25 kDa to ≈60 kDa (Fig. 2Aii). By contrast FliS did not shift, remaining at ≈16 kDa (Fig. 2Aiii). The results are consistent with assembly of FlgN-FliJ and FliT-FliJ complexes but not of FliS-FliJ, and this was confirmed by immunoblotting of the fractions (not shown). Although the FliJ-FlgN elution would be compatible with a 1:1 stoichiometry, this method is not appropriate for accurate stoichiometry assessment, especially as FliJ migrates aberrantly. Incubation of FliT with FlgN and FliJ together gave a single major elution peak at ≈62 kDa (Fig. 2Aiv), which immunoblotting showed contains FliJ and both chaperones (Fig. 2Aiv Lower). The location of the peak is not compatible with a mixture of binary complexes, indicating that a ternary complex can be formed.

Fig. 2.
Assembly of FliJ-chaperone complexes. (A) Gel-filtration chromatography after in vitro incubation of purified FliJ with FlgN (i), FliT (ii), FliS (iii), and both FlgN and FliT (iv) chaperones. Proteins were monitored spectrometerically at A280. Molecular-mass ...

Identification of the ternary FliJ-FlgN-FliT complex suggested that the chaperones can occupy the 147 residue FliJ simultaneously, presumably at distinct sites. To assess this, (His10)FliJ variants containing scanning 10-residue deletions (13) were tested for binding to each of the FlgN and FliT chaperones in the Ni2+-affinity copurification assay. These assays revealed (Fig. 2B) that binding of FlgN, but not FliT, was abolished by FliJ deletions between residues 21 and 50, within the predicted N-terminal coiled-coil (13). Conversely, binding of FliT, but not FlgN, was disrupted by deletions in the central region between residues 61 and 100. FliJ variants containing any “nonbinding” deletion did not complement a fliJ null mutant (13).

The results show that FlgN and FliT bind discrete nonoverlapping sites on FliJ and that these binding events are essential for export. This supports the size exclusion data, indicating how a ternary complex could be formed in the cell. Such simultaneous recruitment of both chaperones for all three minor subunits in a ternary complex may be significant in FliJ function. Our confirmation that FliJ interacts with FliH (Fig. 1) (13, 14) indicates that FliJ may function at the membrane ATPase complex. FliJ might recognize transient chaperone-subunit complexes en route to FliI docking, during docking, or even after docking but before subunit release. Therefore, we examined FliJ recognition of subunit-laden chaperones and the location of FliJ.

FliJ Recognizes Only Free Chaperones and Is Associated with the Membrane.

Chaperone-subunit complexes are transient in vivo (5), so FlgN-FlgK, FliT-FliD, and FliS-FliC complexes were preassembled in vitro following purification of proteins by affinity chromatography (3, 7). FliJ binding was assessed by GST affinity chromatography and analyzed by SDS/PAGE (Fig. 3A). None of the assembled chaperone-subunit complexes copurified with GST-FliJ.

Fig. 3.
Recognition of chaperone-subunit complexes by FliJ. (A) Affinity chromatography of (GST) FliJ (41 kDa) incubated with in vitro preformed chaperone-subunit complexes FlgN-FlgK (FlgNK), FliT-FliD (FliTD), and FliS-FliC (FliSC), assayed by SDS/PAGE and stained ...

We assessed whether chaperone-subunit complexes were recognized in vivo by FliJ, assaying FliJ pull-down of FlgN-FlgK from S. typhimurium ΔfliJΔflgM in which class III flagellar genes are expressed at all stages of flagellum assembly (15). Cells expressing (His10)FliJ were disrupted using a French pressure cell to release membrane-associated flagellar components to the soluble fraction (7). From this release fraction (His10), FliJ was affinity purified, and coeluted proteins were assayed by immunoblotting. The subunit FlgK was not detected (Fig. 3B), but FlgN chaperone was consistently copurified with FliJ. The data showed that FliJ is unable to bind subunits whether chaperoned or not; they appear to preclude FliJ from being a cytosolic “suprachaperone” or, indeed, acting to release chaperone-docked subunits at the export apparatus. FliJ has been surmised to be a cytosolic chaperone (12), but the data (Fig. 1) (13) indicate FliJ interacts with FliH, which is complexed with FliI at the inner membrane (7). Indeed, FliJ has also been reported to interact with two other membrane-associated flagellar proteins: the export protein FlhA (13) and the C-ring protein FliM (16). To establish the location of FliJ, fractionation was carried out on cell lysates of a Salmonella ΔfliJ mutant producing complementing levels of (His10)FliJ. Immunoblotting with His-tag antisera revealed FliJ was predominantly (≈70%) in the membrane/insoluble fraction, with the lower proportion in the cytosol (Fig. 4A). The same result was obtained with a Salmonella flhDC null mutant producing (His10)FliJ in the absence of all other flagellar components. This suggests that like the membrane-associated FliH and FliI (7), FliJ has intrinsic membrane affinity.

Fig. 4.
Membrane localization of FliJ. (A) Cytosolic (cyt) and membrane/insoluble (mem) fractions of S. typhimurium ΔfliJ and ΔflhDC mutants expressing (His10) FliJ at ΔfliJ-complementing levels. (B) Membrane fractions of S. typhimurium ...

Cell membrane fractions were separated by density gradient centrifugation (17), and immunoblotting showed that (His10)FliJ colocalized with inner membrane fractions containing NADH oxidase activity and the FliH component of the ATPase complex (Fig. 4B). FliJ membrane affinity was also confirmed by an in vitro liposome flotation assay (Fig. 4C), in which FliJ colocalized with E. coli phospholipids in a manner analogous to the inner-membrane-associated ATPase FliI and its regulator FliH, with which FliJ interacts (Fig. 1) (13, 14), and in contrast to the cytosolic chaperone FlgN (5). FliJ interaction with the ATPase complex is also consistent with a 2.4-fold increase observed in in vitro FliI ATPase activity in the presence of FliJ (2.3–5.5 μmol·min−1·mg−1 ± <3%), whereas ATPase activity was unaffected by the FliJ-FlgN complex.

The accruing data had excluded a number of possibilities and suggested that FliJ might recruit newly unloaded chaperones at the ATPase complex (i.e., after subunits had been released into the translocation phase). Such an activity could increase the local chaperone concentration at the export machinery, effecting a cycle to enhance chaperone binding of new subunits and, thus, docking. To assess whether FliJ could direct such a chaperone transfer and cycle, an in vitro chaperone capture assay was developed.

Subunit Capture of Cognate Chaperone from FliJ-Chaperone Complexes.

Preformed (His6)FliJ-FlgN complex was bound to nickel beads and challenged with cognate (FlgK) subunit. Immunoblotting of FlgN that was released into the eluate or retained in the FliJ-FlgN complex showed that FlgK displaced FlgN from FliJ in a concentration-dependent manner (Fig. 5A), with ≈0.05 μM FlgK sufficient to reduce by 50% the amount of FlgN retained by FliJ and 10 μM FlgK dislodging >90% of the chaperone. In parallel assays the (His6)FliJ-FlgN complex was challenged by the same concentrations of the noncognate subunit FliC. By contrast, FliC was unable to displace the FlgN chaperone from FliJ (Fig. 5A).

Fig. 5.
Capture of FliJ-bound chaperone by cognate subunit. (A) FliJ-bound and subunit-eluted FlgN was assayed by immunoblotting following incubation of Ni2+ agarose-bound in vitro preformed (His6) FliJ-FlgN chaperone complex with subunit, either cognate FlgK ...

To show that the putative FlgK subunit-FlgN chaperone complexes were formed after chaperone capture from FliJ, gel-filtration chromatography was performed on elution fractions from an assay in which 10 μM FlgK was applied. The results (Fig. 5B) show that in the elution peak the FlgN chaperone and FlgK subunit coeluted with an apparent molecular mass of 70 kDa, with residual FlgK monomer (49 kDa) and FlgN dimer (≈25 kDa) also observed in the elution profile. The data show that FliJ-bound chaperones are transferred to their cognate filament subunits without requiring other proteins. As FliJ binds only empty chaperones and not subunits, this suggests that its displacement involves either transient binding of chaperone by both FliJ and subunit at different chaperone sites, effecting an allosteric change, or competition for the same binding site on the chaperone, with the subunit having greater affinity. We examined these possibilities by establishing the sites and strengths of the two chaperone interactions.

Chaperone Transfer Is Affected by Subunit Displacement of FliJ from a Common Binding Site.

To identify the region(s) of FlgN required for binding of FliJ and the two subunits FlgK and FlgL, variants of FlgN containing 10-residue scanning deletions in the C-terminal subunit-binding region were constructed and tested in affinity copurification assays (Fig. 6A). All the variants were expressed at levels comparable to the full length and were identically soluble (input, Fig. 6A). In contrast to the full-length FlgN and most deleted variants, which bound all three proteins, FlgN variants lacking residues 81–90 and 91–100 abolished binding of both subunits and of FliJ. This is consistent with both proteins binding to a common site on the chaperone. We sought to establish the relative affinity of the competing FliJ and FlgK proteins for the chaperone FlgN using isothermal titration calorimetry (ITC). The heat output measured over time and the derived integrated measure of heat transfer with respect to concentration depicted in both cases an exothermic interaction that allowed derivation of affinity values using a single-binding-site model. Stepwise titration of FlgN into FlgK (Fig. 6B) revealed that chaperone bound to subunit with a KD of 0.03 μM. Similar titration of FlgN into FliJ showed binding to be more than 700-fold weaker, with a KD of 22 μM. This indicates that chaperone transfer to subunit is achieved by direct competition and displacement at a common binding site.

Fig. 6.
Competitive binding of a common site on the chaperone by cognate subunit and FliJ. (A) Binding of FliJ and cognate subunits to the chaperone FlgN. Ni2+ affinity chromatography (as in Fig. 1) and Coomassie blue staining indicate recognition of internally ...

Discussion

Our screen for FliJ intermediate export complexes revealed that FliJ recruits unladen FlgN and FliT, which chaperone the minor filament-class subunits FlgK, FlgL, and FliD, but not with FliS, which chaperones the major component FliC (flagellin). FliJ formed binary and ternary complexes with the chaperones via distinct, binding sites that do not overlap the region determining interaction with FliH, the FliI ATPase regulator. Deletions within either chaperone binding site disabled flagellar subunit export (13). FliJ did not bind chaperone-subunit complexes, consistent with chaperoned subunit docking occurring in the absence of FliJ (5). This indicates that FliJ is not involved in transition of chaperoned subunits from the cytosol to the membrane. Like the ATPase complex components FliI and FliH (7), FliJ localizes predominantly to the inner membrane and has intrinsic affinity for phospholipids; it interacts with FliH (13, 14) and also the membrane export component FlhA (13) and C-ring protein FliM (16). FliJ increased FliI ATPase activity in vitro, but it does not copurify with FliI in affinity chromatography (not shown), suggesting that there might be a FliJ-FliI interaction that is transient and/or stabilized by other flagellar components. These data indicated that FliJ had a novel activity, binding empty chaperones at the export ATPase complex. By establishing an in vitro capture assay we were able to show that cognate (but not noncognate) subunits can sequester chaperones from FliJ complexes in a concentration-dependent manner. FliJ was unable to sequester chaperones from cognate subunit. FliJ and the subunits FlgK and FlgL bind the same 20-residue region of the FlgN chaperone, pointing to direct competition at a common binding site. This was confirmed by ITC, which revealed that FlgN chaperone has a ≈700-fold higher affinity for its cognate subunit FlgK than for FliJ. These data appear to explain how empty chaperones are recruited and held by FliJ before transfer to their cognate subunits.

Our findings offer a new view of the sequence of events underlying export. We envisage that chaperone-piloted subunits dock transiently at the membrane ATPase complex, which catalyzes subunit unfolding and translocation, and effects chaperone release. FliJ then acts as a chaperone escort protein, clearing the ATPase of unloaded chaperones, which it transfers to new subunits. This view is compatible with our observed stimulation of FliI ATPase activity by free FliJ but not FliJ-chaperone complex. This escort activity of FliJ at the export ATPase complex would provide a chaperone sink to enhance export by allowing cycling of chaperones to increase the frequency of productive subunit-chaperone complex formation and docking. Our data reveal an entirely novel function for FliJ in the type III export of flagellar subunits: that of chaperone escort.

Whether such a chaperone escort mechanism occurs in the related type III systems that secrete virulence effectors is not obvious, but one can identify genes (e.g., Salmonella invI/spaM, Yersinia yscO, Shigella spa13, and E. coli orf15) immediately downstream of the export ATPase gene that encode proteins of similar size (18–20 kDa) and charge to FliJ.

The current view of the late stage of flagellar assembly does not encompass any notion of filament-class subunit export hierarchy. However, the flagellum stoichiometry suggests that minor components of the hook-filament junction (11 monomers of FlgK and FlgL per filament) and the filament cap (5 monomers of FliD) may have to compete for export with an excess of flagellin monomers. This view is strengthened by measurements that indicate comparable ratios of unincorporated subunits in the extra cellular medium (18) and cytosol (our unpublished data). This apparent problem would be compounded by the need for all three minor substructures to assemble before flagellin is incorporated (19), and suggests that there might be a mechanism to favor export of the minor subunits, especially if successive rounds of selection are required once each substructure begins to assemble. Selectivity could best act at individual nascent assemblies during or after docking. Our data could indicate a basis for such a selectivity process, in which FliJ recruits and escorts chaperones for the minor subunits as part of a cycle to promote the early formation of the adjacent hook-filament junction and cap structures.

Methods

Bacterial Strains and Plasmids.

Bacteria were cultured at 37°C to late exponential phase (A6001.0), unless stated, in Luria-Bertani (LB) broth containing, where appropriate, ampicillin, chloramphenicol, or kanamycin (each at 20 μg·ml−1). Wild-type S. typhimurium SJW1103 is motile (20), and derived mutants carry lesions in the cheW-flhD locus [SJW1368 (21)]. Mutants ΔfliJ::KmR, ΔflgM::KmR, and ΔfliJ::FRTflgM::KmR, in which genes were replaced by a kanamycin resistance cassette, were constructed using P22 transduction and/or the λ Red recombinase system (22). Recombinant proteins were expressed in E. coli C41 (23) from IPTG inducible plasmids pET15b (24), pACT7 (25), pGEX-4T-3 (26), or pTrc99-FF4 (27). To construct recombinant plasmids encoding, individual flagellar genes S. typhimurium flgD, flgE, flgK, flgL, flgM, flgN, fliC, fliD, fliH, fliJ, fliK, fliS, and fliT were each amplified from chromosomal DNA by PCR using Pfu turbo DNA polymerase. PCR products were inserted NdeI/BamHI into pACT7 or pET15b. For GST fusion constructs, genes were amplified by PCR, and products inserted BamHI/XhoI into pGEX-4T-3. FlgN internal 10-residue deletions were created by overlap-extension PCR and products inserted NdeI/BamHI into pACT7. Inserts were verified by DNA sequencing (Department of Genetics, University of Cambridge).

Purification of Proteins.

(His6)-tagged proteins FlgN, FliT, FliS, and FliJ were purified as described (2, 3). Chaperone-subunit complexes were prepared from cells coexpressing (His6)-tagged chaperone and untagged subunit. Cells resuspended in buffer A (50 mM NaH2PO4, pH 7.4/150 mM NaCl/1 mM DTT/5 mM imidazole) were lysed by French pressure cell (Aminco). Membranes, unlysed cells, and insoluble proteins were removed by centrifugation (40,000 × g, 1 h), and the cleared cell lysate was passed over nickel nitrilotriacetic acid (Ni2+) agarose (Qiagen). After washing with 50 column volumes (buffer A with 30 mM imidazole), complexes were eluted (buffer A with 700 mM imidazole), dialyzed against buffer A, and further purified by Superdex 200 (GE Healthcare) gel filtration. FliI was purified as described (8).

Affinity Chromatography Copurification Assays.

Copurification (pull-down) of protein complexes was achieved with either Ni2+ agarose or glutathione Sepharose 4B (3, 5). In vitro mixed purified proteins or cleared cell lysates were incubated for 1 h with affinity resin. After extensive washing [buffer A (±10–60 mM imidazole)], proteins were eluted in buffer A containing either 700 mM imidazole or 20 mM glutathione (no detergent was used in these assays). For in vivo studies, soluble lysates of S. typhimurium strains expressing (His10)FliJ at a complementing level were prepared as above, incubated for 1 h with Ni2+, washed three times with buffer A (60 mM imidazole), and proteins eluted in SDS loading buffer. Using untagged FliJ was precluded by nonspecific binding to resin.

Analytical Gel-Filtration Chromatography.

Gel-filtration chromatography used Superdex 200 HR 10/30 (GE Healthcare). Protein samples (0.1–5 mg·ml−1, 1% bed volume) were resolved at a flow rate of 0.5 ml·min−1; 0.3 ml elution fractions were precipitated [10% (wt/vol) TCA] and resuspended in SDS loading buffer.

Salmonella Cell Fractionation and Membrane Separation.

Cultures were separated into membrane and cytosolic fractions by chemical lysis (5, 7). Membranes were analyzed on a 16-ml stepwise sucrose gradient (1–2 M) centrifuged for 16 h at 75,000 × g (17).

Liposome Flotation Assay.

Purified proteins (1–2 μg) were mixed with 40 μl of 10 mg·ml−1 E. coli total phospholipids (Avanti Polar Lipids) in TBS (20 mM Tris, pH 7.4/150 mM NaCl) (7) and incubated at room temperature for 15 min. Sucrose was added to 50% (wt/vol in 1 ml of TBS), and samples were overlaid with 3.5 ml of 40% sucrose (wt/vol in TBS) and 0.5 ml of TBS. After centrifugation at 75,000 × g for 16 h at 16°C, 10 fractions (0.5 ml) were collected and precipitated [10% (wt/vol) TCA]. Fractions 1–4, 5–7, and 8–10 were pooled to give top (T), middle (M), and bottom (B) fractions, respectively.

Steady-State ATPase Assay.

FliI ATP hydrolysis activity was measured at A340 by enzyme-coupled ATP/NADH oxidation (28) at 37°C in reaction buffer (50 mM triethanolamine, pH 8.0/10 mM magnesium acetate/1 mM DTT) in the presence of 1 mM phosphoenolpyruvate (PEP), 0.15 mM NADH, pyruvate kinase (5 units), lactate dehydrogenase enzymes (3.5 units) (rabbit muscle; Sigma), 0.1 mg/ml E. coli total phospholipids, ATP (3–0.01 mM), and FliJ (0.1–3 μM). Reactions were initiated with the addition of FliI (50 μg).

ITC.

ITC was performed at 25°C using a VP-ITC system and Origin software (Microcal Inc). Proteins were dialyzed into 50 mM NaH2PO4 (pH 7.4), 150 mM NaCl, and 1 mM DTT. The heat evolved following each injection was obtained from the integral of the calorimetric signal. The heat due to the binding reaction is the difference between the heat of reaction and the heat of dilution.

Acknowledgments

We thank the Macnab laboratory (Yale University, New Haven, CT) for providing strains and plasmids and R. Hayward for critical reading of the manuscript. This work was supported by a Wellcome Trust Program grant (to C.H.) and a Commonwealth Scholarship Commission Studentship (to S.A.).

Abbreviation

ITC
isothermal titration calorimetry.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission. O.S. is a guest editor invited by the Editorial Board.

References

1. Macnab RM. Annu Rev Microbiol. 2003;57:77–100. [PubMed]
2. Auvray F, Thomas J, Fraser GM, Hughes C. J Mol Biol. 2001;308:221–229. [PMC free article] [PubMed]
3. Bennett JC, Thomas J, Fraser GM, Hughes C. Mol Microbiol. 2001;39:781–791. [PMC free article] [PubMed]
4. Fraser GM, Bennett JC, Hughes C. Mol Microbiol. 1999;32:569–580. [PubMed]
5. Thomas J, Stafford GP, Hughes C. Proc Natl Acad Sci USA. 2004;101:3945–3950. [PMC free article] [PubMed]
6. Gauthier A, Finlay BB. J Bacteriol. 2003;185:6747–6755. [PMC free article] [PubMed]
7. Auvray F, Ozin AJ, Claret L, Hughes C. J Mol Biol. 2002;318:941–950. [PMC free article] [PubMed]
8. Claret L, Calder SR, Higgins M, Hughes C. Mol Microbiol. 2003;48:1349–1355. [PMC free article] [PubMed]
9. Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K. Nature. 2001;410:331–337. [PubMed]
10. Akeda Y, Galan JE. Nature. 2005;437:911–915. [PubMed]
11. Minamino T, Macnab RM. J Bacteriol. 1999;181:1388–1394. [PMC free article] [PubMed]
12. Minamino T, Chu R, Yamaguchi S, Macnab RM. J Bacteriol. 2000;182:4207–4215. [PMC free article] [PubMed]
13. Fraser GM, Gonzalez-Pedrajo B, Tame JR, Macnab RM. J Bacteriol. 2003;185:5546–5554. [PMC free article] [PubMed]
14. Gonzalez-Pedrajo B, Fraser GM, Minamino T, Macnab RM. Mol Microbiol. 2002;45:967–982. [PubMed]
15. Gillen KL, Hughes KT. J Bacteriol. 1991;173:2301–2310. [PMC free article] [PubMed]
16. Gonzalez-Pedrajo B, Minamino T, Kihara M, Namba K. Mol Microbiol. 2006;60:984–998. [PubMed]
17. Osborn MJ, Munson R. Methods Enzymol. 1974;31:642–653. [PubMed]
18. Komoriya K, Shibano N, Higano T, Azuma N, Yamaguchi S, Aizawa SI. Mol Microbiol. 1999;34:767–779. [PubMed]
19. Homma M, Kutsukake K, Iino T, Yamaguchi S. J Bacteriol. 1984;157:100–108. [PMC free article] [PubMed]
20. Yamaguchi S, Fujita H, Taira T, Kutsukake K, Homma M, Iino T. J Gen Microbiol. 1984;130:3339–3342. [PubMed]
21. Ohnishi K, Ohto Y, Aizawa S, Macnab RM, Iino T. J Bacteriol. 1994;176:2272–2281. [PMC free article] [PubMed]
22. Datsenko KA, Wanner BL. Proc Natl Acad Sci USA. 2000;97:6640–6645. [PMC free article] [PubMed]
23. Miroux B, Walker JE. J Mol Biol. 1996;260:289–298. [PubMed]
24. Studier FW, Moffatt BA. J Mol Biol. 1986;189:113–130. [PubMed]
25. Thanabalu T, Koronakis E, Hughes C, Koronakis V. EMBO J. 1998;17:6487–6496. [PMC free article] [PubMed]
26. Kaelin WG, Jr, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, Chittenden T, Li Y, Farnham PJ, Blanar MA, et al. Cell. 1992;70:351–364. [PubMed]
27. Ohnishi K, Fan F, Schoenhals GJ, Kihara M, Macnab RM. J Bacteriol. 1997;179:6092–6099. [PMC free article] [PubMed]
28. Trentham DR, Bardsley RG, Eccleston JF, Weeds AG. Biochem J. 1972;126:635–644. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...