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Journal of Bacteriology, October 2007, p. 7511-7514, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00968-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Complex Formation by the mrpABCDEFG Gene Products, Which Constitute a Principal Na+/H+ Antiporter in Bacillus subtilis{triangledown}

Yusuke Kajiyama,1,2 Masato Otagiri,1 Junichi Sekiguchi,2 Saori Kosono,1* and Toshiaki Kudo1,3

Environmental Molecular Biology Laboratory, Wako, Saitama,1 Department of Applied Biology, Shinshu University, Ueda,2 Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan3

Received 19 June 2007/ Accepted 3 August 2007


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ABSTRACT
 
The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na+/H+ antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.


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TEXT
 
Mrp antiporters (also designated Sha [9], Pha [12], Mnh [3], or Sno [1]) are unique among monovalent cation/H+ antiporters in terms of their molecular features, distribution, and physiological function (reviewed in reference 15). They belong to the secondary cation-proton antiporter 3 (CPA-3) family (TC 2.A.63) in the sequence-based transporter classification (TC) system (13). This family represents a particular multigene structure consisting of either seven or six members, while all the other monovalent CPAs are encoded by a single gene. The feature of multigene structure as well as some genetic evidence has suggested that the Mrp antiporter is likely a multicomponent antiporter (3). In Bacillus subtilis, the Mrp (Sha) antiporter is encoded by an operon consisting of mrpABCDEFG (4). We showed that the B. subtilis Mrp plays a critical role in Na+ homeostasis during growth and also the transition to sporulation (7, 9, 10, 17). The B. subtilis mrp genes are reported to be essential because the mutants show drastic NaCl sensitivity and cannot grow on Luria-Bertani (LB) plates containing approximately 0.17 M NaCl (6). Deletion of any one of the mrpABCDEFG genes results in the NaCl sensitivity phenotype in culture (5, 17), suggesting that the mrpABCDEFG gene products are likely to form a functional complex involved in Na+ extrusion. However, no direct molecular evidence for complex formation has yet been presented. Here we addressed complex formation by the mrpABCDEFG gene products by pull-down and blue native (BN)-polyacrylamide gel electrophoresis (PAGE) analyses.

Construction of B. subtilis strains in which each Mrp is functionally replaced by its His-tagged form. We previously constructed B. subtilis strains harboring a deletion in each of the mrpABCDEFG genes (17). We introduced each of the mrpABCDEFG genes with a C-terminal His tag coding sequence into each of the corresponding mrp-deleted strains. For construction, an mrp gene was amplified by PCR so as to incorporate a SalI site, a ribosome binding site (5'-AAAGGAGGAT-3'), an SpeI site at the 5' end, and a sequence encoding a six-histidine (His6) tag and an EcoRI site at the 3' end. The amplified gene products were digested with SalI and EcoRI and cloned into the aprE-targeting pAPNC213 vector (11). The resulting plasmids were transformed into each corresponding B. subtilis mrp mutant (TY1 to TY7), and the transformants were selected on the basis of spectinomycin resistance. The integration of the gene encoding the His-tagged Mrp into the aprE site was confirmed by PCR using primers outside of the integration site. The genes coding for His-tagged Mrp are expressed under the control of the Pspac promoter, derived from pAPNC213, which is inducible by isopropyl-ß-D-thiogalactopyranoside(IPTG). The B. subtilis strains used in this study are shown in Table 1.


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  		TABLE 1. B. subtilis strains used in this study

The Mrp-deleted strains (TY1 to TY7) do not grow on LB agar plates containing 0.2 M NaCl (Fig. 1A) (17). The His-tagged form of Mrp, with the exception of MrpA, complemented the deletion of each mrp gene for growth in the presence of 1 M NaCl (Fig. 1B), indicating that the addition of the His tag did not affect its function. The MrpA-deleted strain was not, however, complemented by the original mrpA gene (Fig. 1B, {Delta}A/A-his). We found a DNA sequence that is predicted to form a secondary structure immediately downstream of the GTG start codon of mrpA (positions 3 to 40; {Delta}G = –9.7 kcal/mol, calculated by the Mfold Web server [18]). We introduced a synonymous mutation (G9 to C) to avoid the formation of the secondary structure, which decreased the calculated {Delta}G to –4.3 kcal/mol. The synonymous mutation allowed complementation of the growth of the mrpA mutant on 1 M NaCl (Fig. 1B, {Delta}A/A*-his).


Figure 1
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  		FIG. 1. Complementation of mrp-deleted strains by each His-tagged form of Mrp. (A) Cells of B. subtilis strains TY1 ({Delta}A), TY2 ({Delta}B), TY3 ({Delta}C), TY4 ({Delta}D). TY5 ({Delta}E), TY6 ({Delta}F), and TY7 ({Delta}G) and wild-type strain UOT1285 (WT) were streaked onto an LB plate containing 0.2 M NaCl. (B) Strains SK702 ({Delta}A/A*-his), KY8 ({Delta}B/B-his), KY9 ({Delta}C/C-his), KY10 ({Delta}D/D-his), KY11 ({Delta}E/E-his), KY12 ({Delta}F/F-his), and KY13 ({Delta}G/G-his) grew on an LB plate containing 1 M NaCl and 1 mM IPTG, but SK701 ({Delta}A/A-his) did not. (C) Western blots to detect His-tagged MrpA. The membrane samples (20 µg of protein) of TY1 ({Delta}A), SK701 ({Delta}A/A-his), and SK702 ({Delta}A/A*-his) were loaded onto an SDS-12.5% polyacrylamide gel without boiling. His-tagged MrpA was immunodetected using an anti-His tag antibody (arrowhead).

We examined the levels of His-tagged MrpA proteins in the membrane fraction of TY1 ({Delta}A), SK701 ({Delta}A/A-his), and SK702 ({Delta}A/A*-his) by Western blot analysis. Membrane samples were prepared from cells grown at 37°C in LBK1/2 medium (10 g of tryptone, 5 g of yeast extract, and 5 g of KCl per liter) (10) containing 1 mM IPTG to the late exponential phase of growth. The cell pellets were suspended in TMKN buffer (30 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 mM KCl, and 500 mM NaCl) containing 10 µg/ml DNase I, 10 µg/ml RNase A, and 1 mM phenylmethylsulfonyl fluoride and subjected to cell lysis by French press (1,000 kg/cm2, twice). Following removal of cell debris (6,000 x g for 10 min), the membrane fraction was sedimented by centrifugation at 100,000 x g for 1 h and washed once with TMKN buffer. The membrane samples (20 µg of protein) were loaded onto a sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel without boiling. His-tagged MrpA was immunodetected using an anti-His tag antibody. We detected His-tagged MrpA proteins in the membrane fraction of SK702, but not SK701 (Fig. 1C). It may be considered that the secondary structure of DNA prevented the expression of mrpA.

All His-tagged Mrp proteins associate with MrpE protein. Using the above-mentioned strains, we performed pull-down assays to examine the association of each His-tagged Mrp protein with MrpE, the antibody of which was the only one available. Cells of SK702 and KY8 to KY13 were grown in LBK1/2 containing 0.1 mM (KY12), 0.05 mM (SK702, KY8, and KY10), 0.025 mM (KY11 and KY13), or no IPTG (KY9) (the IPTG concentrations were also used in the experiment of Fig. 3). Membrane samples were prepared as described above and incubated in TMKN buffer containing 1% N-dodecyl-ß-D-maltoside (DDM) for 1 h at 4°C for solubilization. After centrifugation (100,000 x g for 1 h), the supernatant containing the solubilized membrane proteins was incubated with a 0.5-ml bed volume of equilibrated TALON metal affinity resin (Clontech) for 1 h at 4°C. Unbound proteins were washed away with 25 ml of TMKN buffer containing 0.05% DDM and 10 mM imidazole and then eluted with 5 ml of TMKN buffer containing 0.05% DDM and 250 mM imidazole. The eluted sample was concentrated by ultrafiltration using the Amicon Ultra-4 filter (molecular weight cutoff, 30,000; Millipore). The concentrated sample containing 5 µg of proteins was subjected to SDS-PAGE and Western blotting using an anti-His tag (MBL, Japan) or anti-MrpE antibody (8).


Figure 3
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  		FIG. 3. BN/SDS-PAGE. The solubilized membrane samples of SK702 and KY8 to KY13 were loaded onto the first-dimension (1D) BN-PAGE gel and then the second-dimension (2D) Tricine SDS-PAGE gel (4% stacking-10% running gel) (top). His-tagged Mrp proteins were probed with an anti-His tag antibody. The range in which the signals are detected in all lanes in the 1D is shown with dotted lines, and the molecular size of the center is estimated to be 410 kDa by comparison with a standard curve of a NativeMark unstained protein standard (Invitrogen). No signals were detected with the membrane sample of UOT1285 (the negative control) in the region between the dotted lines (bottom). The molecular sizes of His-tagged Mrp proteins estimated from the molecular standard (Precision Plus protein standards [Bio-Rad]) in the 2D are indicated to the right of the top panel. The migration pattern of the Precision molecular standard is shown to the left of the bottom panel.

In Fig. 2, all His-tagged Mrp proteins were shown to bind to the metal affinity resin by probing with an anti-His tag antibody. Immobilized His-tagged MrpA, MrpB, MrpC, MrpD, MrpF, and MrpG successfully pulled down MrpE. The shift of an MrpE-corresponding band was observed in strain KY11 because of the additional His tag (Fig. 2, lane 6 from the left). These results indicated that MrpE was in contact with all the other Mrp proteins, directly or indirectly, in the membrane and suggested that all the Mrp proteins are likely to form a multicomponent complex.


Figure 2
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  		FIG. 2. Pull-down assays. His-tagged MrpA, MrpB, MrpC, MrpD, MrpE, MrpF, and MrpG were purified using TALON metal affinity resin from the membrane samples of SK702 and KY8 to KY13, respectively. The eluted samples (5 µg of protein) were separated by SDS-PAGE using a 10-to-20%-gradient acrylamide gel, and immunodetected by an anti-His tag antibody (top, arrowheads). Eluted samples of the same amount were also separated by Tricine SDS-PAGE using a 4% stacking-10% running acrylamide gel and immunodetected by an anti-MrpE antibody (bottom, star). The membrane sample of UOT1285 was used as the negative control (NC).

All the mrpABCDEFG gene products form a multicomponent complex. We next performed BN-PAGE experiments to detect a putative Mrp antiporter complex. The membrane fractions of SK702 and KY8 to KY13 were prepared and solubilized as described above in a concentration of 100 µg of protein in 100 µl of TMKN buffer. After centrifugation (100,000 x g for 15 min at 4°C), the supernatant containing solubilized proteins was mixed with 5 µl of Coomassie brilliant blue solution (5% Serva blue G, 500 mM aminocaproic acid). Twenty microliters of the samples was loaded onto NativePAGE Novex Bis-Tris gels (4 to 16%) (Invitrogen). Buffers and electrophoresis conditions were in accordance with the manufacturer's manual. After electrophoresis, the gels were destained in a solution of 10% acetate-20% ethanol, washed with distilled water, and soaked for 30 min in a solution containing 50 mM dithiothreitol and 1% SDS. After being washed with distilled water, the gels were cut into lanes for use in two-dimensional Tricine SDS-PAGE using a 4% stacking-10% running gel, according to the protocol described by Schägger (14). Gels were then subjected to Western blotting using the anti-His tag antibody.

As shown in Fig. 3, all the His-tagged Mrp proteins were detected at the same position in the first-dimension BN-PAGE. The size of this complex was estimated to be approximately 410 kDa from the molecular standard, which is probably overestimated because of lipids and detergents bound to the complex. Heuberger et al. showed that there is a correlation between the molecular masses of membrane proteins on BN-PAGE (MBNP) and the intrinsic molecular masses (MAA), that is, MBNP = 1.8 x MAA (2). If the Mrp complex consists of only each monomeric Mrp subunit, the molecular mass of 410 kDa from BN-PAGE is converted to 228 kDa using the above correlation, which is close to the molecular mass of 210 kDa calculated on the basis of amino acid sequence. We cannot exclude possibilities that more than one molecule of some Mrp proteins (particularly small subunits such as MrpB, -C, -E, -F, and -G) is included in the Mrp complex and that unknown components other than the Mrp proteins are associated with the complex. It will be necessary to determine the molecular weight of the Mrp complex more precisely in order to clarify its subunit composition.

In this study, we showed that the mrpABCDEFG gene products actually form a multicomponent complex. This is the first molecular evidence to show that the Mrp antiporter is a multicomponent transporter in the CPA-3 family. We previously reported the functional involvement of conserved transmembrane acidic residues of the MrpA (ShaA) subunit (8) and are currently doing a similar study for the other Mrp proteins. Based on the evidence in this study, the determination of residues important for Mrp function will provide more precisely the molecular architecture of the Mrp antiporter, including ion translocation site(s).


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ACKNOWLEDGMENTS
 
This work was partially supported by grants for the Bioarchitect Research, Eco-Molecular Research, and DRI Research programs from RIKEN. This work was also supported by a grant-in-aid for scientific research (C) to S.K.


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FOOTNOTES
 
* Corresponding author. Mailing address: Environmental Molecular Biology Lab., RIKEN, Wako, Saitama 351-0198, Japan. Phone: 81-48-467-9545. Fax: 81-48-462-4672. E-mail: kosono{at}riken.jp Back

{triangledown} Published ahead of print on 10 August 2007. Back


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Journal of Bacteriology, October 2007, p. 7511-7514, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00968-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kajiyama, Y., Otagiri, M., Sekiguchi, J., Kudo, T., Kosono, S. (2009). The MrpA, MrpB and MrpD subunits of the Mrp antiporter complex in Bacillus subtilis contain membrane-embedded and essential acidic residues. Microbiology 155: 2137-2147 [Abstract] [Full Text]  
  • Morino, M., Natsui, S., Swartz, T. H., Krulwich, T. A., Ito, M. (2008). Single Gene Deletions of mrpA to mrpG and mrpE Point Mutations Affect Activity of the Mrp Na+/H+ Antiporter of Alkaliphilic Bacillus and Formation of Hetero-Oligomeric Mrp Complexes. J. Bacteriol. 190: 4162-4172 [Abstract] [Full Text]  

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