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1 Graduate School of Life Sciences, Toyo University, Oura-gun, Gunma 374-0193, Japan
2 Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan
3 Department of Pharmacology, School of Medicine, University of California, Davis, CA 95616, USA
4 Howard Hughes Medical Institute, Department of Cardiovascular Research, Children's Hospital and Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
5 Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, NY 10029, USA
6 Department of Frontier Bioscience, Faculty of Engineering, Hosei University 3-7-2 Kajino-cho, Koganei, Tokyo 184-8584, Japan
Correspondence
Masahiro Ito
ito{at}itakura.toyo.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2005; Kung & Blount, 2004). Recently, roles have been demonstrated for the voltage-gated sodium channel NaVBP from alkaliphilic Bacillus pseudofirmus OF4 in pH homeostasis, motility and chemotaxis at alkaline pH (Ito et al., 2004b). The current study probes cell biological properties of NaVBP that could underpin these physiological roles.
NaVBP is a member of the bacterial voltage-gated sodium channel superfamily NaVBac, whose founding member is the NaChBac channel from alkaliphilic Bacillus halodurans C-125 (Koishi et al., 2004; Ren et al., 2001). The strong resemblance of NaVBac channels to one of the six-transmembrane segment repeats of biomedically important eukaryotic voltage-gated channels has led to intense investigative interest in these bacterial channels (Ren et al., 2001). Electrophysiological and mutational analyses have focused largely on NaChBac, either in lipid bilayers or expressed in eukaryotic cells (Blanchet et al., 2007; Chahine et al., 2004; Kuzmenkin et al., 2004; Pavlov et al., 2005; Richardson et al., 2006; Zhao et al., 2004). Since B. halodurans C-125, the native host of NaChBac, is not genetically accessible, it has not been possible to evaluate the match between properties determined in non-native settings and conditions under which NaChBac can be shown to function physiologically. In our initial studies, we therefore turned to B. pseudofirmus OF4, in which the ncbA gene that encodes the NaChBac homologue NaVBP could be deleted in order to probe the physiological roles of the channel (Ito et al., 2004b).
B. pseudofirmus OF4 grows on non-fermentative carbon sources in a pH range from 7.5 to >11. At pH values above 7.5, robust Na+/H+ antiporter (exchanger) activity, together with the activity of the proton-coupled ATP synthase, support net uptake of protons in respiring cells, thus achieving a cytoplasmic pH that is lower than the external pH. Antiport action reduces the proton motive force and establishes an inwardly directed sodium motive force. Na+-coupled solute uptake and flagellar rotation take advantage of this sodium motive force to support solute uptake and motility (Fujinami et al., 2007; Ito et al., 2004a; Krulwich et al., 2007; Padan et al., 2005). The Na+ that enters the cytoplasm during this Na+-coupled bioenergetic work completes the Na+ cycle and plays an important role in supporting the ongoing Na+/H+ antiport activity for pH homeostasis (Fig. 1).
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, b). The ncbA mutant exhibits motility only after prolonged growth in liquid culture or on soft agar plates. The third, striking phenotype observed at high pH in both poorly motile and highly motile variant ncbA mutants is a high frequency of tumbling (Ito et al., 2004b). The tumbly ncbA mutants exhibit an ‘inverse chemotaxis’, i.e. the mutants move toward repellents and away from attractants.
The parallel electrophysiological studies of the channel in eukaryotic cells have demonstrated that NaVBP is an Na+-specific voltage-gated channel whose activation is potentiated at high pH (Ito et al., 2004b). The electrophysiological characteristics indicate that in the natural alkaliphile host, the role of high external pH (and secondary elevation of cytoplasmic pH) in channel opening is supplemented by additional triggers (Ito et al., 2004b). Such an additional trigger could result from an interaction between the channel and either the chemotaxis signalling machinery or the flagellar motor (Fig. 1
). A particularly attractive hypothesis is that NaVBP interacts with transmembrane chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), accounting for both triggering and channel effects in chemotaxis. The membrane-associated MCPs have periplasmic ligand-binding domains that monitor attractants and repellents on the outside of the cell, and have signalling/adaptation domains in their cytoplasmic segments that communicate with the flagellar motor via a two-component signalling pathway (Szurmant & Ordal, 2004). Signalling is initiated by a signalling complex that is formed at conserved cytoplasmic domains of the MCPs (Wadhams & Armitage, 2004; Zhulin, 2001). In Escherichia coli and Salmonella, MCPs form a complex with the histidine kinase CheA, which mediates the signalling triggered at the MCP receptor, and with adaptor protein CheW; this complex localizes to the cell poles (Gegner et al., 1992; Liu et al., 1997; Maddock & Shapiro, 1993; Sourjik & Berg, 2000); weaker lateral clusters are also observed (Lybarger & Maddock, 1999; Skidmore et al., 2000). In Bacillus subtilis, the core elements that mediate chemotaxis resemble those of the E. coli–Salmonella systems (Lamanna et al., 2005; Rao et al., 2004; Szurmant & Ordal, 2004; Weis, 2006). As in the enteric bacteria, MCPs are localized to the polar end of other rod-shaped bacteria, including Bacillus species (Gestwicki et al., 2000; Kirby et al., 2000; Lamanna et al., 2005). Polar localization and/or clustering of the complexes has been proposed to play a critical role in signal amplification in E. coli (Bray et al., 1998; Duke & Bray, 1999; Irieda et al., 2006; Lybarger & Maddock, 2001; Shapiro et al., 2002). If NaVBP interacts with polar MCPs and/or the kinase/adaptor proteins that are complexed with them in B. pseudofirmus OF4, the channels should also localize to the cell poles. On the other hand, if NaVBP interacts with flagellar motors, a delocalized pattern would be expected, consistent with a peritrichous pattern of flagella in this alkaliphile (Fig. 1) (Fujinami et al., 2007; Sturr et al., 1994). We conducted an immunofluorescence microscopy (IFM) analysis of B. pseudofirmus OF4 cells to confirm the polar localization of MCPs and to compare it with the localization pattern of NaVBP. MCP localization was assessed using mAbs against NaVBP, and polyclonal antibodies that were raised against B. subtilis McpB, a chemoreceptor for Asn, Asp, Gln, Glu and His (Hanlon & Ordal, 1994). The alkaliphile gene whose product cross-reacts with this antibody has not yet been identified; we refer to this putative MCP as McpX. Channel localization was also probed using a plasmid expressing an NaVBP–cyan fluorescent protein (CFP) fusion protein in B. pseudofirmus OF4. We further examined whether disruption of the alkaliphile cheAW locus decreases localization of McpX, NaVBP or both.
The results indicate that NaVBP co-localizes at cell poles with an alkaliphile protein that is recognized by antibodies raised against B. subtilis McpB. They further show that mutational loss of the channel leads to McpX delocalization, while a cheAW-disrupted mutant leads to significant delocalization of both the channel and McpX.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli and B. subtilis strains were grown routinely in LB or LBK medium (Goldberg et al., 1987). Alkaliphilic B. pseudofirmus OF4 wild-type (strain 811M) (Clejan et al., 1989) and its derivative strains were grown aerobically in semi-defined malate–yeast extract (MYE) medium (pH 10) (Sturr et al., 1994) at 37 °C. pH 10 medium replaced the pH 10.5 medium used in most of our studies because the SC34 strain that lacked a functional NaVBP exhibited a significant deficit in mid-exponential growth at the higher pH value; earlier studies of SC34 growth had examined yield at stationary phase and a defect at high pH was not observed (Ito et al., 2004b). Nifedipine-treated wild-type (811M) cells were prepared by adding 50 µM nifedipine to mid-exponential phase cells (OD600 0.6). Growth was continued for 1 h, and the cells were then used for IFM. Antibiotics were added at 5 µg ml–1 for chloramphenicol and 0.3 µg ml–1 for erythromycin.
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). Primers used are shown in Table 2. Two independent PCRs were performed on B. pseudofirmus OF4 wild-type chromosomal DNA template with the primer sets del-AW-F1-BH1 and del-AW-R1, and del-AW-F2 and del-AW-R2-ER1. Another PCR was performed on pC194 (Horinouchi & Weisblum, 1982) as DNA template with the primer set del-AW-F-CAT and del-AW-R-CAT to amplify the cat gene. The three purified PCR products were used as templates for a second PCR with primer sets del-AW-F1-BH1 and del-AW-R2-ER1. The purified product of this reaction was digested with BamHI and EcoRI and cloned into BamHI- and EcoRI-digested pG+host4, yielding pG4
cheAW : : CAT. The plasmid was transformed into B. pseudofirmus OF4-811M protoplasts. The protocol for isolation of single-crossover candidates has been described previously (Ito et al., 1997). From the two kinds of single-crossover candidates, analyses of the cheAW disruption mutant indicated that the recombination occurred in the region upstream of the cheA gene (region between del-AW-F1-BH1 and del-AW-R-CAT), and this was confirmed by PCR using primer sets OF4cheA24 and OF4cheA25, OF4cheA3 and OF4cheA25, and OF4cheA24 and OF4cheA15. The cheAW disruption mutant was designated strain 811M-cheAW.
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) was ligated with XbaI- and HindIII-digested pMW118, yielding pYM1. Plasmid pYM1 is a shuttle vector in E. coli and Bacillus species that is low copy in both hosts (about six copies per cell) (Ito et al., 2004a). The ncbA–ecfp fragment that enhanced the CFP gene (ecfp) was fused just before the stop codon of the ncbA gene using the gene SOEing method (Horton, 1997). A schematic diagram of the ncbA–ecfp fragment is shown in Fig. 2(a). The primers used for constructions are shown in Table 2. Two independent PCRs were performed on B. pseudofirmus OF4 wild-type chromosomal DNA template with the primer sets NC-F1-SC2 and NC-R1-1, and NC-F2-1 and NC-R2-BH1. Another PCR was performed on the pECFP plasmid DNA template with the primer set NC-F-CFP1 and NC-R-CFP1 to amplify the ecfp gene. The three purified PCR products were used as templates for a second PCR with the primer set NC-F1-SC2 and NC-R2-BH1. The purified product of this reaction was digested with EcoRI and cloned into EcoRI-digested pMW118, yielding pMWSC-CFP. pMWSC-CFP was digested with BamHI and HindIII, and ligated with the large fragment (5.0 kb) of BamHI- and HindIII-digested pYM1, yielding pSC-CFP. In this plasmid, NaVBP–CFP is expressed under the control of the native ncbA promoter.
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), pMWCFP and pCFP were constructed by using a parallel method. In pCFP, CFP is expressed under the control of the native ncbA promoter. The inserts of pSC-CFP and pCFP were mutation-free. The plasmids were transformed into B. pseudofirmus OF4-SC34 protoplasts (Ito et al., 1997).
Antibody preparation.
Rabbit polyclonal antibodies against the abundant McpB of B. subtilis (Hanlon & Ordal, 1994; Szurmant & Ordal, 2004) were obtained from G. W. Ordal (University of Illinois). An anti-NaVBP mouse mAb (L30/34; IgG2b isotype) was generated against a recombinant, bacterially expressed full-length NaVBP protein. Mice were immunized and hybridomas generated and screened by standard methodology (Bekele-Arcuri et al., 1996; Trimmer et al., 1985). L30/34 hybridomas were grown in roller bottles containing Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco). Supernatants were collected and mAbs purified with Protein G Agarose (Amersham Pharmacia) following the manufacturer's protocol.
Immunoblot analysis.
B. pseudofirmus OF4 wild-type and its derivative mutant strains were grown overnight on MYE medium as described above. Overnight culture (1 ml) was inoculated into 100 ml fresh MYE medium, pH 10, and grown to OD600 0.6. Cells were harvested and washed in TSE buffer (50 mM Tris/HCl, pH 8.0, 10 % sucrose, 1 mM EDTA). Cells were suspended in the same buffer and a protease inhibitor cocktail (Sigma-Aldrich) and DNase (50 µg ml–1) were added. Cells were disrupted by sonication and unbroken cells were removed by centrifugation at 9100 g for 15 min at 4 °C, yielding whole-cell suspension. The membrane fraction was harvested by centrifuging at 40 000 r.p.m. for 90 min in a Beckman 70Ti rotor at 4 °C and suspended in TSE buffer. A protease inhibitor cocktail (Sigma-Aldrich) was added. The protein concentrations of the whole-cell suspension and membrane fraction were measured by the Lowry method (Lowry et al., 1951) with BSA as a standard. The same volume of SDS loading buffer was added to each sample, which was boiled for 3 min at 100 °C, after which the proteins were separated by 12 % polyacrylamide SDS gels (Schagger & von Jagow, 1987). The gels were then transferred to nitrocellulose filters (Bio-Rad) electrophoretically overnight in Tris/glycine/methanol buffer (25 mM Tris, 192 mM glycine, 20 %, v/v, methanol, pH 8.3).
The following antibodies and reagents were used for detection in immunoblot analyses: for McpB (B. subtilis) and McpX (B. pseudofirmus OF4), 1 : 2000 rabbit anti-B. subtilis McpB antibody and 1 : 3000 goat anti-rabbit–horseradish peroxidase (HRP) conjugate (Bio-Rad); and for NaVBP–CFP and CFP, 1 : 250 (whole-cell analyses) or 1 : 1000 (membrane fraction analyses) rabbit anti-GFP polyclonal antibody (Clontech), 1 : 3000 goat anti-rabbit–HRP (Bio-Rad), and Can Get SignalM Immunoreaction Enhancer Solution (Toyobo). Detection and analysis of chemiluminescence images were conducted using the quantitative imaging system Fluor-S MAX (Bio-Rad) according to the protocol provided in the manufacturer's instructions (Amersham Biosciences). NaVBP could not be detected in immunoblot analyses of either whole cells or membranes using the anti-NaVBP mouse L30/34 mAb (data not shown).
IFM.
The method described by Hiraga et al. (1998) was adapted for B. pseudofirmus OF4 strains. Wild-type and mutant strains were grown as described above for immunoblot analyses. Final culture (1 ml) was added to 10 ml 70 % ethanol, mixed gently and left for 1 h at room temperature. The fixed cells were harvested by centrifugation (700 g for 15 min at 4 °C) and suspended in 1 ml 70 % ethanol. A glass slide, S-2215 (Matsunami Glass), was covered by 20 µl poly-L-lysine hydrobromide (1 mg ml–1), left for 5 min, washed with distilled water, and air-dried. The ethanol-fixed cell suspension (10 µl) was then dropped on the poly-L-lysine-coated slide and air-dried for 20 min. The slide was covered with 100 µl lysozyme solution (2 mg ml–1 in 25 mM Tris/HCl, pH 8.0, 50 mM glucose, 10 mM EDTA) and incubated for 5 min at room temperature. The slide was washed with 5 ml PBSTE (140 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 0.05 % Tween 20, 10 mM EDTA, pH 8) three times and then incubated with PBSTE-BSA (PBSTE containing 2 % BSA) for 15 min at room temperature.
The slide was treated for 1 h at room temperature with the primary antibody solution (1 : 1000 mouse anti-NaVBP L30/34 mAb or 1 : 2000 rabbit anti-B. subtilis McpB polyclonal antibody in PBSTE-BSA), and covered with a cover glass in a shading moisture chamber. The slide was washed with 5 ml PBSTE three times (the cover glass was removed by washing). After the slide had been washed with PBSTE, it was again incubated with PBSTE-BSA for 15 min at room temperature. It was then treated for 1 h at room temperature with a secondary antibody solution [1 : 1000 Alexa Fluor 488 rabbit anti-mouse IgG (Molecular Probes) for the NaVBP, and Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes) for the McpX in PBSTE-BSA]. The slide was then covered with a cover glass in a shading moisture chamber and again washed with 5 ml PBSTE three times. After washing with PBSTE, it was covered with a cover glass and sealed with enamel. The microscopic images were obtained by an FW4000 Fluorescence Image Analysis Workstation (Leica Geosystems), and processed with Photoshop CS software (Adobe Systems). In each experiment, over 30 cells were assessed with respect to the subcellular localization of NaVBP and McpX, and the length of the cells. All the results shown are from a total of three independent experiments. The localization of NaVBP and McpX was classified by two researchers (S. F. and M. I.) as: A, one pole; B, two poles; C, one pole and side(s); D, two poles and side(s); E, side(s); or diffuse (Tables 3 and 4).
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ncbA) were transformed with plasmid pSC-CFP (yielding the SC34/pSC-CFP transformant) or pCFP (yielding the SC34/pCFP transformant). The transformants were grown as described above for immunoblot analyses. Small aliquots of the culture were spotted onto glass slides coated with 0.5 % agarose. Microscopic images were obtained on an FW4000 Fluorescence Image Analysis Workstation. Photoshop CS software was used to manipulate images, e.g. to create overlays, without altering the images or patterns of the cells themselves. In each experiment, over 30 cells were classified with respect to the subcellular localization of NaVBP–CFP and CFP. All the results shown are from a total of three independent experiments.
Migration assays.
Migration in soft agar was assayed by the method described previously (Fujinami et al., 2007). B. pseudofirmus OF4 wild-type and its derivative mutant strains were grown in MYE medium overnight and then grown to exponential phase on MYE, pH 10, as described above. Migration assays of B. pseudofirmus OF4 wild-type and its derivative strains in soft agar were conducted on plates containing MYE medium (pH 10) (Sturr et al., 1994) and solidified by the addition of 0.3 % Noble agar. Liquid culture (1 µl) was spotted onto the centre of the plate. After incubation of the plates at 37 °C for 10 h, the diameter of the colony was measured. All results shown are the means of three independent experiments.
Assays of tumbling bias.
B. pseudofirmus OF4 wild-type and its derivative strains were grown as described above. Qualitative assessment of tumbling bias in liquid MYE medium (pH 10) was carried out by the hanging-drop method using a Leica DMLB100 dark-field microscope (x400), a Leica DC 300F camera, and Leica IM50 version 1.20 software (Leica Geosystems).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Before undertaking IFM, confirmation was sought that anti-B. subtilis McpB antibody could detect an MCP of B. pseudofirmus OF4 by assessing the heterogeneity of any cross-reacting proteins in the alkaliphile on immunoblot analyses (Fig. 3). The bands in expected positions of McpA and McpB were detected in whole cells of B. subtilis 168 (Kirby et al., 2000). The location of the band corresponding to McpB is indicated with an arrow in Fig. 3. In whole cells of wild-type B. pseudofirmus OF4 (811M), a single band was detected at almost the same location as McpB of B. subtilis 168. We designated this MCP of B. pseudofirmus OF4 as McpX. Bands corresponding to McpX were also detected in whole cells of the channel mutant SC34, and in a channel mutant strain (SC34-R) to which ncbA had been restored to the chromosome (Fig. 3). The expression level of McpX was similar in the three alkaliphile strains.
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). Quantification of the co-localization of NaVBP and McpX is shown in Table 3 (top rows of each section). ‘Total cells’ designates the total number of cells that were analysed. The subcellular localization of NaVBP was observed in 92 % of wild-type (811M) cells. In 82 % of 811M cells, NaVBP was localized at a cell pole, and in 90 % of such cells, polar co-localization of NaVBP and McpX was observed. In the channel mutant SC34, no localized signal was observed with the antibody against the channel, as expected, except for a small signal that reflects the difficulty of setting the parameters ‘counted’ as fluorescence with complete precision. Unexpectedly, the polar localization of McpX was also significantly decreased in the channel mutant, from 78 % in the wild-type to 15 % in SC34 (Fig. 4, Table 3, second row of each section). A channel mutant with a restored ncbA gene at the native location (SC34-R) exhibited fluorescence with 76 % polar localization, 89 % of which was co-localized with McpX. McpX fluorescence in the SC34-R strain that was localized at the poles rose from 15 % in SC34 to 71 % in the SC34-R strain and 96 % of that was co-localized with NaVBP (Fig. 4, Table 3, third row of each section). Measurements of 50 cells of each strain showed that there were no significant differences in the cell length or width of the different strains. The mean cell lengths (µm) of 811M, SC34, SC34-R, 811M-AW, SC34/pSC-CFP and SC34/pCFP were 1.95±0.44, 1.65±0.36, 2.43±0.55, 2.39±0.63, 2.25±0.45 and 2.13±0.55, respectively. The mean cell widths (µm) of 811M, SC34, SC34-R, 811M-AW, SC34/pSC-CFP and SC34/pCFP were 0.82±0.05, 0.82±0.05, 0.84±0.05, 0.84±0.06, 0.84±0.05 and 0.85±0.04, respectively.
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ncbA strain could result entirely from secondary delocalization of MCPs rather than from a channel function of NaVBP itself. Evidence against this line of reasoning was the earlier observation that the NaVBP channel inhibitor nifedipine caused inverse chemotaxis behaviour of wild-type B. pseudofirmus OF4 toward the chemoattractant aspartate when the inhibitor was added (at 50 µM) to the chemotaxis assay buffer (Ito et al., 2004b). IFM experiments were conducted to test whether comparable exposure of the wild-type strain to nifedipine caused delocalization of NaVBP or McpX. These experiments revealed no such delocalization (Fig. 4b).
NaVBP–CFP expressed from a plasmid localizes at cell poles in the ncbA deletion strain
Transformants of B. pseudofirmus OF4-SC34 (ncbA) cells with plasmid pSC-CFP (SC34/pSC-CFP), in which CFP is fused to the channel, or control pCFP (SC34/pCFP) were grown in MYE, pH 10, and subjected to immunoblot analyses using antibodies against GFP. As shown in Fig. 5, bands corresponding to the expected location for NaVBP–CFP fusion were detected in whole-cell and membrane fractions of the SC34/pSC-CFP transformant (white arrows). The band for CFP was detected in whole cells of SC34/pSC-CFP but not in membrane fractions (filled arrows). Anti-NaVBP mouse mAb was also used for this detection. However, the antibody was not able to detect denatured NaVBP for immunoblot analyses. This mAb can probably recognize an antigen only when NaVBP maintains 3D structure.
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). NaVBP–CFP was localized at cell poles in SC34/pSC-CFP (Fig. 6a), and CFP was not localized in SC34/pCFP (Fig. 6b). Polar localization of NaVBP–CFP was observed in 88 % of SC34/pSC-CFP cells (Table 4).
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). IFM studies of the subcellular localization of McpX and NaVBP showed a decrease in the polar localization of both McpX and NaVBP in 811M-cheAW in comparison with the wild-type parent (Fig. 4a, Table 3, fourth rows of both sections). The polar localization of McpX decreased from 78 % in the wild-type to 33 % in 811M-cheAW, with only 51 % co-localized with NaVBP in the mutant. The polar localization of NaVBP decreased from 82 % in the wild-type to 33 % in 811M-cheAW, with only 50 % co-localized with McpX in the mutant.
Migration assays and tumbling bias assessments of B. pseudofirmus OF4 strains with different NaVBP status
Migration assays in soft agar and observations of the tumbling bias in liquid medium were conducted on B. pseudofirmus OF4 wild-type and its derivative mutant strains to ascertain whether the effects of ncbA status on polar localization of McpX correlated with swarming capability in soft agar and tumbliness (Fig. 7, Table 5). As shown in earlier studies of an up-motile alkaliphile variant and its mutant derivative (Ito et al., 2004b), deletion of ncbA (in SC34) from the wild-type strain led to a smaller colony diameter and a more tumbly phenotype relative to wild-type. Restoration of ncbA (in SC34-R) restored wild-type motility on soft agar and reduced the tumbliness. Transformation of the channel mutant SC34 with a multicopy plasmid encoding ncbA–ecfp (SC34/pSC-CFP) led to almost wild-type motility and tumbliness. Control plasmid pCFP, encoding ECFP, did not restore motility or reverse the tumbly phenotype of strain SC34.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The role in pH homeostasis was presumed to reflect a contribution of NaVBP to entry of Na+ in support of the Na+/H+ antiport that maintains pH homeostasis during alkalinization of the medium, e.g. during a pH 8.5
10.5 shift of the outside pH. The upward pH change would potentiate opening of the channel (Ito et al., 2004b). The role of NaVBP observed in the pH-shift experiments was especially notable in the presence of low [Na+] and when the medium contained low concentrations of solutes that enter the cell together with Na+. No deficit was observed in the growth yield of the channel mutant at pH 10.5 in MYE medium containing optimal [Na+] and solutes that are co-transported with Na+, so no role for NaVBP in pH homeostasis under optimal conditions was indicated in the earlier studies. Here, we found that the channel mutant SC34 exhibited a defect in growth to the mid-exponential phase in the Na+-replete, malate-containing medium at pH 10.5. These results indicated that NaVBP does have a role in alkaline pH homeostasis, even when adequate Na+ and solutes are present. This raises the possibility that the lag in motility in the channel mutant is a result of suboptimal pH homeostasis, since the pH range for optimal swimming of B. pseudofirmus OF4 is below the highest values for optimal non-fermentative growth (Fujinami et al., 2007). However, the role of NaVBP in pH homeostasis cannot explain the dramatic effect of channel loss on chemotaxis. Most of the original assays of chemotaxis were conducted at pH 8.5, at which the pronounced inverse chemotaxis phenotype was consistently observed in the mutant, although no pH homeostasis problem would exist (Ito et al., 2004b). Rather, we hypothesize that the effect of NaVBP status on the chemotaxis behaviour of B. pseudofirmus OF4 is related to the interaction between NaVBP and chemotaxis proteins.
In summary, the major findings on the localization of alkaliphile NaVBP and an MCP that emerge from this work are as follows. (i) The putative alkaliphile MCP designated McpX localized at cell poles, as anticipated from similar findings in other systems (Kirby et al., 2000). Some McpX appeared to localize at the sides of the cell (Fig. 4, Table 3). MCPs are translocated by the helically distributed Sec machinery (Shiomi et al., 2006). It is possible that the experiments captured McpX that was in transit or that was localized to a central region destined to be a new pole. (ii) NaVBP also exhibited localization to the cell poles, as shown by both IFM (Fig. 4, Table 3) and fluorescence microscopy on cells expressing CFP fused to the channel (Fig. 6, Table 4). The expression of the fused channel restored wild-type motility properties, as assessed in soft agar plates, and reversed tumbly properties, as assessed qualitatively by microscopic examination (Table 5). (iii) A significant percentage of the polarly localized McpX and NaVBP exhibited co-localization (Fig. 4, Table 3). On the other hand, the pattern of NaVBP localization did not correlate well with the pattern of peritrichous flagellum location in wild-type B. pseudofirmus OF4. The wild-type strain typically has one to two flagella per cell, and these are located at various locations around the cell body (Fujinami et al., 2007). (iv) The expression level of McpX was not markedly changed in the ncbA mutant that lacks NaVBP, but McpX was significantly delocalized. This effect was reversed appreciably in a strain in which ncbA was restored to the chromosome (Fig. 4, Table 3). (v) Significant delocalization of both NaVBP and McpX was observed when cheAW genes of the wild-type were disrupted (Table 3). This delocalization was not as complete as in E. coli, in which CheAW has been shown to form a complex with MCPs that is required for polar localization as well as clustering (Irieda et al., 2006; Kentner et al., 2006). Perhaps association of NaVBP and McpX is sufficient to allow a low level of polar co-localization in B. pseudofirmus OF4.
The finding that mutational loss of NaVBP leads to delocalization of McpX raises the possibility that such delocalization is in part responsible for the altered chemotactic responses, rather than loss of channel function itself being completely responsible. However, the functional capacity of NaVBP is also essential for normal chemotaxis. Nifedipine addition to chemotaxis assay buffers results in inverse chemotaxis (Ito et al., 2004b), but does not result in delocalization of NaVBP and McpX. This supports the possibility that NaVBP-mediated fluctuations in membrane potential play a role in the signalling pathway for chemotaxis of B. pseudofirmus OF4, as suggested for not yet identified channels in earlier studies of chemotaxis in Spirochaeta aurantia (Goulbourne & Greenberg, 1983) and E. coli (Tisa et al., 1993, 2000). The co-localization of NaVBP and MCPs further raises the possibility that modulation of NavBP function occurs via dynamic changes in NavBP phosphorylation, as mediated by chemoreceptor activation of the CheA kinase and CheZ phosphatase system, which are presumably also present at the cell poles (Baker et al., 2006). This could lead to changes in NavBP gating, as occurs in many eukaryotic voltage-gated ion channels (Levitan, 1999). The cytoplasmic C terminus of NavBP contains four aspartate residues that could serve as phosphoacceptors for the CheA kinase; changes in C-terminal phosphorylation of eukaryotic channels can profoundly affect channel gating (Park et al., 2006). Further work will be needed to define the nature of MCP and channel interactions in the alkaliphile, and how such interactions affect channel function and chemotaxis. Finally, these studies raise the question of whether, as inferred by the earlier investigators cited above, voltage-gated ion channels are involved in signalling events or other features of bacterial chemotaxis in bacteria beyond a subset of extremophiles.
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ACKNOWLEDGEMENTS |
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Edited by: C. Edwards
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 26 July 2007;
revised 4 September 2007;
accepted 6 September 2007.
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