| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Universität Würzburg, Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
Correspondence
Christian Andersen
andersen{at}biozentrum.uni-wuerzburg.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Delepelaire, 2004). Channel-tunnels are pore-forming proteins located in the outer membrane, which interact with diverse inner-membrane complexes to form distinct export machinery (Andersen et al., 2002a; Koronakis et al., 2004). The type I secretion system is a channel-tunnel-dependent export system for proteins, such as toxins, S-layer proteins and diverse extracellular-acting enzymes. The paradigm for this export apparatus is the haemolysin secretion system of Escherichia coli (Wandersman & Delepelaire, 1990). In addition to protein secretion, channel-tunnels are also involved in export of noxious compounds, such as antibiotics, dyes, detergents, bile salts and heavy metals (Poole, 2005; Borges-Walmsley et al., 2003). These channel-tunnel-dependent efflux pumps play a major role in establishing the resistance of most Gram-negative bacteria to these compounds, and are becoming an increasing problem for the clinical use of antibiotics.
Currently, the structures of three channel-tunnels are known (Koronakis et al., 2000; Akama et al., 2004; Federici et al., 2005). The trimeric assembly forms a 140 Å (14 nm) long, cannon-shaped structure. It is anchored in the outer membrane by a 
-barrel domain and protrudes into the periplasm via a 100 Å (10 nm) long, 
-helical tunnel domain, which assembles with inner-membrane complexes to form a continuous export pathway across the cell envelope. In contrast to the wide-open, extracellular entrance, the periplasmic entrance is almost closed, which explains the very low single-channel conductance of the best-characterized representative TolC of E. coli (Andersen et al., 2002a). For TolC, the role of individual residues in electrophysiological characteristics and opening of the periplasmic entrance has been determined (Andersen et al., 2002b, c). It can be shown that an iris-like outwards movement of the inward-folded tunnel helices is necessary to open the periplasmic entrance, allowing export of substrates (Eswaran et al., 2003; Andersen et al., 2002b).
In Haemophilus influenzae, a human pathogen responsible for significant morbidity and mortality in young children (Turk, 1984; Funkhouser et al., 1991), a multidrug efflux pump has been identified, whose components are homologues of the E. coli AcrAB/TolC efflux pump (Sanchez et al., 1997; Trepod & Mott, 2004). The AcrB-homologous HI0895 is a transporter of the resistance nodulation cell division family, which forms a complex with the AcrA-homologous adaptor or membrane fusion protein HI0894 (Sanchez et al., 1997). This inner-membrane complex interacts with the TolC-homologous, outer-membrane channel-tunnel HI1462 to assemble a functional multidrug efflux pump (Trepod & Mott, 2004). Disruption of any of the coding genes causes hypersusceptibility to antibiotic agents (Dean et al., 2005; Sanchez et al., 1997; Trepod & Mott, 2004). It can also be shown that a loss of pump repression by mutation or disruption of the AcrR repressor gene is the origin of increased resistance of several clinical H. influenzae isolates, illustrating the importance of this efflux pump (Dean et al., 2005).
In this study, we have isolated the TolC-homologous HI1462 and characterized the biophysical properties of this pore-forming protein in lipid bilayer experiments. We present a computer model of the HI1462 channel-tunnel which explains its biophysical characteristics. Furthermore, we tested the ability of HI1462 to substitute the E. coli TolC in the AcrAB/TolC efflux pump and the HlyBD/TolC type I secretion system.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the same way, the pAra21 vector was made by substituting the IPTG-inducible T7 promoter region of pET-21a (Novagen) with araBADC. Then, the HI1462 gene, conjoined with the N-terminal His-tag, was cloned from pAraJS.2HI1462 into pAra21 using the primers 5'-GAATTACATATGCGGGCGAAAC-3' and 5'-GATTAATCCAATTTGATGAATTCAAATAATATCCCGCCATTG-3' (with restriction sites for NdeI and EcoRI, respectively). The resulting vector contained the HI1462 gene with an N-terminal His10-tag and a C-terminal His6-tag under the control of an arabinose-inducible promoter.
The mutation of the arginine residue 396 to alanine was performed using the QuikChange Site-Directed Mutagenesis kit (Stratagene) on pAra21HI1462biHis with the mutation primers 5'-GCGTATCCGAATTGGCCGAATGGTTAGTTGC-3' and 5'-GCAACTAACCATTCGGCCAATTCGGATACGC-3', following the standard protocol.
tolC was cloned into pBAD/myc-His C using E. coli chromosomal DNA as template and the oligonucleotides 5'-CCACAAGGAATGCTCATGAAGAAATTGC-3' and 5'-GTCGTCATCAGTTACGGAATTCGTTATGACCG-3' as primers (with restriction sites for PagI and EcoRI, respectively). All constructs were confirmed by DNA sequencing (Seqlab).
Bacterial strains and growth conditions.
The E. coli strains and plasmids used in this study are listed in Table 1. The tolC knockout in AG100 and DC14 was performed according to the method of Datsenko & Wanner (2000). A TolC-KO_up primer (5'-CGCGCTAAATACTGCTTCACCACAAGGAATGCAAATGAAGAAGTGTAGGCTGGAGCTGCTTC-3'), a TolC-KO_down primer (5'-CCGTTACTGGTGTAGTGCGTGCGGATGTTTGCTGAACGACTGCATATGAATATCCTCCTTA-3') (underlined region binds to pKD3) and pKD3 as template were used for PCR to produce a tolC knockout fragment. The loss of tolC was verified by colony PCR using primers that bind up- and downstream of tolC. The resulting strains were denoted AG100TC and DC14TC, respectively.
|
Haemolysis was tested on blood-agar plates consisting of 25 ml LB agar supplemented with 0.75 ml defibrinated horse blood, spotted with 0.2 µl cell suspension (1x109 cells ml–1) and incubated for 18 h at 37 °C. MIC tests were performed in 96-well plates by serial dilution of the tested antimicrobial agent in LB medium with 2x104 cells per well as the inoculum. The plates were incubated overnight at 37 °C and analysed photometrically after 24 h with an ELISA reader (Molecular Devices) at 600 nm.
Protein expression.
E. coli BL21(DE3)Omp8 pAra21HI1462biHis was routinely grown at 37 °C with shaking (150 r.p.m.) in LB medium with ampicillin. Expression of HI1462 was induced by adding 0.02 % (w/v) arabinose at OD650 0.5–0.6. The culture was grown at 30 °C to prevent formation of inclusion bodies. After 3 h, cells were harvested by centrifugation (5000 g for 15 min) and washed twice in 10 mM Tris/HCl (pH 8.0). Cells were passed three times through a French pressure cell at 900 p.s.i. (6210 kPa). Unbroken cells were removed by centrifugation at 5000 g for 15 min. The cell envelope was obtained by centrifugation of the supernatant at 170 000 g for 60 min. The pellet was successively resuspended in 2 ml 1 % (v/v) Triton (twice), 1 % (v/v) lauryldimethylamine oxide (LDAO) and 2 % (v/v) LDAO followed by centrifugation at 170 000 g for 60 min, respectively. The 2 % (v/v) LDAO supernatant contained HI1462. For further purification, the supernatant was separated by FPLC using a MonoQ column (Pharmacia) and 0.5 % (v/v) LDAO 10 mM Tris (pH 8.0) as buffer. For elution of bound proteins, the KCl concentration in the buffer was raised continuously from 0 to 1 M. HI1462 eluted at 200 mM KCl. Finally, HI1462-containing fractions were applied to His-Select Ni-nitrilotriacetic acid (NTA) spin columns (Sigma).
SDS-PAGE and Western blotting.
SDS-PAGE was performed according to the Laemmli gel system (Laemmli, 1970). The gels were stained with Coomassie brilliant blue or with silver stain (Blum et al., 1987). Western blots were processed with the Bio-Rad Tank-Blot system with Protran nitrocellulose transfer membranes (Schleicher & Schuell BioScience) by applying a constant current of 350 mA for 30 min. The ECL Western Blotting Detection kit (Amersham Pharmacia Biotech) was used to detect binding of the Anti-His antibody according to the manufacturer's instructions.
Lipid bilayer experiments.
The methods used for the lipid bilayer experiments have been described in detail by Benz et al. (1978). Black lipid bilayer membranes were obtained from 1 % (w/v) diphytanoyl phosphatidylcholine (Avanti Polar Lipids) in n-decane. The temperature was maintained at 20 °C during all experiments. Zero-current membrane potentials were measured by establishing a salt gradient across membranes containing 100–200 channels, as described by Benz et al. (1985).
Protein modelling.
The HI1462 structure was modelled using the Homology module of the InsightII software package (Accelrys) based on the structure of the closest homologous channel-tunnel, OprM of Pseudomonas aeruginosa (Akama et al., 2004). A refinement of the structure was performed using the CharmM module of the InsightII software package.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). When incubated at 100 °C prior to SDS-PAGE, the protein ran at 48 kDa, concordantly with the calculated value for the HI1462 monomer. Without a denaturing step, the band shifted to 
110 kDa, corresponding to HI1462 trimer. By Western blot analysis, we could clearly confirm that both bands represented HI1462.
|
). We assumed that the double His-tag on both ends of the protein had no influence on the single-channel conductance. It is very unlikely that the N-terminal His-tag, which was located at the 37-residue-long tail outside the conduit structure, had any influence on the channel-forming characteristics. The double-tagged version led to the same conductance steps as the single-tagged version, therefore, we concluded that the C-terminal His-tag, which was closer to the tunnel structure but still not part of it, also had no influence. The single-channel conductance in 1 M KCl was 43 pS. Measured in potassium acetate and LiCl, the single-channel conductance was 21 and 39 pS, respectively. This means that exchange of the cation had only a minor effect, while exchange of the anion led to a major decrease. This is the first hint that HI1462 is an anion-specific channel. Ion-specificity was quantified by zero-current potential measurements in the presence of salt gradients. A fivefold KCl gradient across a membrane with reconstituted HI1462 led to a membrane potential of 11.6 mV, negative on the diluted side. Analysis using the Goldman–Hodgkin–Katz equation resulted in a permeability ratio (PK/PCl) of 0.5. Thus, the HI1462 channel is anion-selective, as suggested by the single-channel recordings.
|
81 pS. Further biophysical characterization of HI1462 channels revealed that the pores were not voltage-dependent in a range between +100 and –100 mV, meaning that the conductance remained unchanged, and no opening or closing was observed. Unlike TolC of E. coli HI1462, channels could not be blocked by the divalent cation Zn2+.
Model of the HI1462 structure explains its biophysical characteristics
The biophysical analysis revealed that HI1462 is anion-specific. This is a major difference to TolC of E. coli, which is highly selective for cations (Andersen et al., 2002a). To obtain a structural insight at the molecular level, we modelled the structure of HI1462. At present, the structures of three channel-tunnels are known: TolC of E. coli, OprM of P. aeruginosa and VceC of Vibrio cholerae (Koronakis et al., 2000; Akama et al., 2004; Federici et al., 2005). A sequence alignment of the HI1462 sequence showed the best homology with the sequences of OprM (data not shown). Therefore, we used OprM as a template to model the HI1462 structure (Fig. 3a). Of particular interest are residues at the periplasmic end of the tunnel domain. It is known from E. coli TolC that residues lining the periplasmic entrance have a major effect on the electrophysiological behaviour of the channel-tunnel. Looking at the HI1462 model, there are valine residues (Val400) located on the second helical turn of the most inwardly directed helix, facing the lumen of the periplasmic entrance (Fig. 3b). These hydrophobic residues might be responsible for the low single-channel conductance observed for HI1462. Below this hydrophobic region, there are three charged residues per monomer, Glu394, Arg396 and Glu397, whereas the arginine residues are nearest to the lumen and are responsible for the electropositive lining of the periplasmic entrance. We suppose that these arginine residues are the origin of the anion-specificity of HI1462.
|
). A quantitative determination of ion selectivity by zero-current measurements supported the assumption that exchange of Arg396 by alanine reversed ion selectivity. The permeability ratio (PK/PCl) was 8.6, showing that HI1462RA was cation-selective.
|
280 pS, we also observed a second type of pore for HI1462RA with a single-channel conductance between 1.9 and 2.1 nS in 1 M KCl. The pores were rarely observed and their lifetime was very short. They closed immediately and adopted a conformation with a single-channel conductance of 280 pS corresponding to the small pores (Fig. 4). This observation let us conclude that both pore types can be attributed to the HI1462RA protein adopting two different conformations, and not to another co-purified, pore-forming protein. In lower salt concentrations, the higher conductance state was not observed.
|
). Channel-tunnels TolC and HI1462 were expressed from an arabinose-inducible plasmid in the tolC-deficient strain AG100TC. An arabinose concentration of 0.1 % (w/v) was chosen for induction of channel-tunnels because it resulted in the same zone of clearance as AG100 cells expressing TolC from the chromosome. Cells lacking TolC or HI1462 had a small zone of clearance due to haemolysin release by cell lysis. Interestingly, cells expressing HI1462 wild-type, as well as the HI1462 mutant, were able to secrete haemolysin. However, the halo was much smaller compared to cells expressing TolC. When HI1462 or TolC were induced by higher arabinose concentration, the size of the halo increased, but cells expressing HI1462 always had a smaller zone of clearance.
|
). Therefore, we conclude that HI1462 is not able to form a functional, or at least an effective, efflux pump with the E. coli AcrAB complex.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Out of these, AcrAB/HI1462 has been characterized as the primary efflux pump, exporting a broad variety of toxic compounds (Sanchez et al., 1997; Trepod & Mott, 2004). A decreased susceptibility of clinical isolates due to overexpression of efflux pump genes has confirmed the role of this efflux pump as a major determinant for drug resistance of H. influenzae (Dean et al., 2005). Analysis of the chromosome has revealed that HI1462 is the only functional channel-tunnel in H. influenzae. A phylogenetic analysis separates the channel-tunnel family into three groups, corresponding to the export processes in which the proteins are involved: protein secretion, drug efflux and cation efflux (Andersen et al., 2000). HI1462-like OprM of P. aeruginosa belongs to the group of channel-tunnels involved in drug efflux, whereas TolC of E. coli is a representative of the channel-tunnels involved in protein secretion. HI1462 reconstituted in planar lipid membranes revealed a single-channel conductance of 43 pS in 1 M KCl, almost half that of TolC, but 20-fold smaller than that of OprM (Andersen et al., 2002a; Wong et al., 2001). The low single-channel conductance of HI1462 accords well with our modelled structure (Fig. 3
). The periplasmic entrance of HI1462 is lined by three valine residues (Val400) forming a hydrophobic constriction, which is most likely responsible for the lower single-channel conductance of HI1462 compared to that of TolC. In TolC, there is an aspartate at the corresponding position (Asp374). Together with a second aspartate (Asp371), which is located one helical turn closer to the periplasmic entrance, it is responsible for the cation selectivity of TolC (Andersen et al., 2002c). In HI1462, the second central position is also covered by a negatively charged residue (Glu397). However, our measurements revealed that HI1462 is the first known TolC homologue which is anion-selective. This also explains why divalent cations do not block the channel as observed for TolC (Andersen et al., 2002c). We showed that the anion selectivity originates from the Arg396, which also lines the tunnel entrance. The dominant role of Arg396 was investigated in the mutant HI1462RA. Substitution of arginine by alanine reversed the ion selectivity of the HI1462 channel, implying that the tunnel entrance became electronegative due to the glutamate residues at positions 397 and 394 (Fig. 3). Additionally, the HI1462RA mutant had an increased single-channel conductance. However, the almost sevenfold higher value (282 pS), compared to wild-type, cannot be explained solely by the absence of the bulky arginine residues. Compared with the single-channel conductance of TolC (80 pS), which has no arginine residue at the corresponding position, the HI1462RA entrance needs to be opened further. When looking at the HI1462 model, it becomes evident that Arg396 is involved in stabilization of the closed conformation of the HI1462 tunnel entrance. Arg396 is in close proximity to Glu397 of the adjacent monomer, allowing the formation of a circular network of salt bridges, which keeps the inward-folded helices in position. The absence of these connections in HI1462RA might allow the inner helices to move outwards, explaining the higher single-channel conductance. Interestingly, these were not the only conductance steps seen for HI1462RA. We also observed insertion events with a six- to sevenfold higher single-channel conductance, which could be undoubtedly assigned to the HI1462RA mutant protein. This high conductance level was unstable and observed only rarely at high salt concentrations. We assume that the lack of the circular network allows the tunnel entrance to temporarily adopt a fully open state. At lower salt concentrations, the high conductance state was absent. This could be explained by additional ionic interactions which keep the tunnel entrance in a narrow conformation. At high ionic strength, these interactions are destabilized, allowing transition into the fully open state, whereas at lower salt concentrations, they are stable enough to keep the entrance in the narrow conformation. For TolC, it is known that the open state can be achieved by disturbance of a circular network of hydrogen bonds and salt bridges close to the periplasmic entrance (Andersen et al., 2002b). Sequence alignment shows that the aspartate (Asp153 in TolC) located at the outer coiled coil, which is central to the formation of the circular network, is not conserved in HI1462, which possesses a serine (Ser190) at the corresponding position. Therefore, it seems reasonable that there must be other connections, which keep the channel-tunnel in a closed conformation, to prevent unwanted influx or efflux through a wide-open channel-tunnel in the outer membrane. The electrophysiological characterization of the HI1462RA mutant shows that the salt bridge, which can be formed between Arg396 and Glu397 according to the modelled HI1462 structure, is most likely responsible for keeping the tunnel entrance in a tightly closed conformation.
It is surprising that OprM of P. aeruginosa, which shows a higher homology to HI1462 than TolC, has a single-channel conductance of 850 pS in 1 M KCl (Wong et al., 2001), which is almost 20-fold higher than that of HI1462. However, the high single-channel conductance seems to conflict with the solved crystal structure of OprM, which shows a nearly closed periplasmic entrance (Akama et al., 2004). It should be mentioned that the reconstituted channels are not stable and appear to form substates (Wong et al., 2001), a characteristic also observed for the open state of TolC (Andersen et al., 2002b). Additionally, the pore-forming activity of OprM in planar lipid bilayers is very low compared to other reconstituted pore-forming proteins (R. Benz, personal communication), which lets us assume that only a minority of the OprM molecules reconstitute as pores in a transient open state, whereas the majority of the molecules insert into the membrane as closed pores, which allows no passage of ions and cannot therefore be detected by electrophysiological measurements.
The genomic organization of the multidrug efflux pump in H. influenzae resembles that of E. coli to the extent that the gene encoding the channel-tunnel is not linked to those encoding the proteins of the inner-membrane complex. Among the six predicted multidrug efflux pumps in H. influenzae, there is another export mechanism coded by a gene for the membrane fusion protein EmrA (HI0898) and a transporter of the major facilitator superfamily EmrB (HI0897), which also needs a channel-tunnel protein to assemble a functional efflux pump according to the known homologues in E. coli (Lomovskaya & Lewis, 1992). Therefore, the sole channel-tunnel in H. influenzae HI1462 needs to be compatible with two different inner-membrane complexes. This is in accordance with TolC of E. coli, which is known to interact with at least eight different inner-membrane complexes (Andersen, 2003). Among these is the HlyBD complex, which with TolC forms a secretion apparatus for haemolysin (Wandersman & Delepelaire, 1990). Genes encoding a type I secretion system are absent in H. influenzae. Therefore, it is surprising that HI1462 can substitute for TolC in the E. coli protein secretion apparatus. The secretion was not as efficient as that of the wild-type system, which suggests either that the amount of HI1462 is lower compared to TolC, or that the two proteins HlyD and HI1462 are not fully compatible. By higher induction of HI1462, it was possible to increase the amount of secreted haemolysin. However, we never reached the maximal secretion levels observed with cells expressing TolC. Therefore, reduced haemolysin secretion is probably caused by compatibility problems between HlyD and HI1462 in the hybrid system. A slightly bigger halo, observed for the hybrid system including the HI1462RA mutant, might lead to the supposition that facilitated opening of the tunnel entrance by interaction with HlyD plays a role in enhanced secretion. Other hybrid, type I secretion systems have exchanged the outer-membrane component with highly homologous channel-tunnels, e.g. TolC of E. coli, HasF of Serratia marcescens, and PrtF of Erwinia chrysanthemii. They can be interchanged between the different secretion systems without loss of functional secretion (Binet & Wandersman, 1996; Letoffe et al., 1994; Akatsuka et al., 1997). Recently, it has been shown that RaxC, a channel-tunnel of the plant pathogen Xanthomonas oryzae pv. Oryzae, complemented an E. coli tolC mutant in a type I secretion system (da Silva et al., 2004). However, RaxC is also a channel-tunnel belonging to the protein secretion family. Thus, HI1462 is the first example of a channel-tunnel belonging to the drug efflux family which is able to act as an outer-membrane component for a type I secretion system.
Substitution of TolC by HI1462 as an outer-membrane component of the E. coli AcrAB efflux pump did not restore resistance. Similar results have been observed when trying to complement TolC by OprM of P. aeruginosa (Tikhonova et al., 2002). It should be mentioned that MIC values are generally much lower for H. influenzae than for E. coli (Trepod & Mott, 2004). If HI1462 is responsible for the less efficient efflux pump of H. influenzae, the possibility cannot be excluded that the hybrid efflux pump AcrAB/HI1462 in E. coli is functional but does not give rise to the same level of resistance as the wild-type efflux pump. However, there are no reports in the literature which point towards the channel-tunnel as a rate-limiting factor for the efflux. Therefore, we conclude that HI1462 is not compatible with the inner-membrane complex of E. coli. This means that interaction between the inner-membrane complexes of drug efflux pumps and the corresponding channel-tunnels is more specific than the interaction of the inner and outer components of type I secretion systems. Further research is needed to understand these interactions at the molecular level. This is essential for developing drugs that inhibit these export mechanisms and disarm harmful bacteria.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akatsuka, H., Binet, R., Kawai, E., Wandersman, C. & Omori, K. (1997). Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters. J Bacteriol 179, 4754–4760.
Andersen, C. (2003). Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria. Rev Physiol Biochem Pharmacol 147, 122–165.[Medline]
Andersen, C., Hughes, C. & Koronakis, V. (2000). Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep 1, 313–318.[CrossRef][Medline]
Andersen, C., Hughes, C. & Koronakis, V. (2002a). Electrophysiological behavior of the TolC channel-tunnel in planar lipid bilayers. J Membr Biol 185, 83–92.[CrossRef][Medline]
Andersen, C., Koronakis, E., Bokma, E., Eswaran, J., Humphreys, D., Hughes, C. & Koronakis, V. (2002b). Transition to the open state of the TolC periplasmic tunnel entrance. Proc Natl Acad Sci U S A 99, 11103–11108.
Andersen, C., Koronakis, E., Hughes, C. & Koronakis, V. (2002c). An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations. Mol Microbiol 44, 1131–1139.[CrossRef][Medline]
Benz, R., Janko, K., Boos, W. & Lauger, P. (1978). Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli. Biochim Biophys Acta 511, 305–319.[Medline]
Benz, R., Schmid, A. & Hancock, R. E. (1985). Ion selectivity of gram-negative bacterial porins. J Bacteriol 162, 722–727.
Binet, R. & Wandersman, C. (1996). Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue. Mol Microbiol 22, 265–273.[CrossRef][Medline]
Blum, H., Beier, H. & Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99.[CrossRef]
Borges-Walmsley, M. I., McKeegan, K. S. & Walmsley, A. R. (2003). Structure and function of efflux pumps that confer resistance to drugs. Biochem J 376, 313–338.[CrossRef][Medline]
da Silva, F. G., Shen, Y., Dardick, C., Burdman, S., Yadav, R. C., de Leon, A. L. & Ronald, P. C. (2004). Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response. Mol Plant Microbe Interact 17, 593–601.[Medline]
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.
Dean, C. R., Narayan, S., Daigle, D. M. & 8 other authors (2005). Role of the AcrAB-TolC efflux pump in determining susceptibility of Haemophilus influenzae to the novel peptide deformylase inhibitor LBM415. Antimicrob Agents Chemother 49, 3129–3135.
Delepelaire, P. (2004). Type I secretion in gram-negative bacteria. Biochim Biophys Acta 1694, 149–161.[Medline]
Eswaran, J., Hughes, C. & Koronakis, V. (2003). Locking TolC entrance helices to prevent protein translocation by the bacterial type I export apparatus. J Mol Biol 327, 309–315.[CrossRef][Medline]
Federici, L., Du, D., Walas, F., Matsumura, H., Fernandez-Recio, J., McKeegan, K. S., Borges-Walmsley, M. I., Luisi, B. F. & Walmsley, A. R. (2005). The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 Å resolution. J Biol Chem 280, 15307–15314.
Funkhouser, A., Steinhoff, M. C. & Ward, J. (1991). Haemophilus influenzae disease and immunization in developing countries. Rev Infect Dis 13, S542–S554.[Medline]
Jellen-Ritter, A. S. & Kern, W. V. (2001). Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone. Antimicrob Agents Chemother 45, 1467–1472.
Koronakis, V., Sharff, A., Koronakis, E., Luisi, B. & Hughes, C. (2000). Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 914–919.[CrossRef][Medline]
Koronakis, V., Eswaran, J. & Hughes, C. (2004). Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu Rev Biochem 73, 467–489.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Letoffe, S., Ghigo, J. M. & Wandersman, C. (1994). Secretion of the Serratia marcescens HasA protein by an ABC transporter. J Bacteriol 176, 5372–5377.
Lomovskaya, O. & Lewis, K. (1992). Emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci U S A 89, 8938–8942.
Nikaido, H. (1994). Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382–388.
Paulsen, I. T., Nguyen, L., Sliwinski, M. K., Rabus, R. & Saier, M. H., Jr (2000). Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes. J Mol Biol 301, 75–100.[CrossRef][Medline]
Poole, K. (2005). Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 56, 20–51.
Prilipov, A., Phale, P. S., Van Gelder, P., Rosenbusch, J. P. & Koebnik, R. (1998). Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol Lett 163, 65–72.[CrossRef][Medline]
Sanchez, L., Pan, W., Vinas, M. & Nikaido, H. (1997). The acrAB homolog of Haemophilus influenzae codes for a functional multidrug efflux pump. J Bacteriol 179, 6855–6857.
Schulein, R., Gentschev, I., Mollenkopf, H. J. & Goebel, W. (1992). A topological model for the haemolysin translocator protein HlyD. Mol Gen Genet 234, 155–163.[Medline]
Tikhonova, E. B., Wang, Q. & Zgurskaya, H. I. (2002). Chimeric analysis of the multicomponent multidrug efflux transporters from gram-negative bacteria. J Bacteriol 184, 6499–6507.
Trepod, C. M. & Mott, J. E. (2004). Identification of the Haemophilus influenzae tolC gene by susceptibility profiles of insertionally inactivated efflux pump mutants. Antimicrob Agents Chemother 48, 1416–1418.
Turk, D. C. (1984). The pathogenicity of Haemophilus influenzae. J Med Microbiol 18, 1–16.[Medline]
Wandersman, C. & Delepelaire, P. (1990). TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A 87, 4776–4780.
Wong, K. K., Brinkman, F. S., Benz, R. S. & Hancock, R. E. (2001). Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance. J Bacteriol 183, 367–374.
Received 23 December 2005;
revised 1 March 2006;
accepted 6 March 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
TolC). Channel-tunnels TolC, HI1462 or HI1462RA were expressed from an arabinose-inducible plasmid in AG100TC cells. Plates were inoculated with 2x105 cells and grown for 18 h at 37 °C. Bar, 1 cm.