PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Microbiol. Author manuscript; available in PMC Oct 1, 2010.
Published in final edited form as:
PMCID: PMC2765581
NIHMSID: NIHMS145892
Cation/proton antiporter complements of bacteria: why so large and diverse?
Terry A. Krulwich,1* David B. Hicks,1 and Masahiro Ito2
1Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, 1 G. Levy Pl., New York, NY 10029 USA
2Graduate School of Life Sciences, Toyo University, Oura-gun, Gunma 374-0193, Japan
*For correspondence, terry.krulwich/at/mssm.edu; Tel. (+1) 212 241-7280; Fax (+1) 212 996-7214
Most bacterial genomes have 5–9 distinct genes predicted to encode transporters that exchange cytoplasmic Na+ and/or K+ for H+ from outside the cell, i.e. monovalent cation/proton antiporters. By contrast, pathogens that live primarily inside host cells usually possess 0–1 such antiporters while other stress-exposed bacteria exhibit even higher numbers. The monovalent cation/proton antiporters encoded by these diverse genes fall into at least eight different transporter protein families based on sequence similarity. They enable bacteria to meet challenges of high or fluctuating pH, salt, temperature or osmolarity, but we lack explanations for why so many antiporters are needed and for the value-added by specific antiporter types in specific settings. In this issue of Molecular Microbiology, analyses of the pH-dependence of cytoplasmic [Na+], [K+], pH and transmembrane electrical potential in the “poly-extremophile” Natranaerobius thermophilus are the context for assessment of the catalytic properties of 12 predicted monovalent cation/proton antiporters in the genome of this thermophilic haloalkaliphile. The results provide a profile of adaptations of the poly-extremophilic anaerobe, including a proposed role of cytoplasmic buffering capacity. They also provide new perspectives on two large monovalent cation/proton antiporter families, the NhaC and the Cation/Proton antiporter-3 (CPA3) antiporter families.
Keywords: alkaliphile, anaerobe, sodium-resistance, Mrp, NdhF, NhaC
Key physiological functions of bacteria are supported by monovalent cation/proton antiporters that catalyze active efflux of Na+ and/or K+ in exchange for H+ from outside the cell, i.e. Na+(K+)/H+ antiporters, These antiporters are secondary active transporters that catalyze active efflux of Na+ and K+ using the energy of the electrochemical gradient of protons that is generated across the cell membrane by distinct transporter events, e.g. ion-pumping ATPases or respiratory complexes (Padan et al., 2005, Slonczewski et al., 2009). Na+(K+)/H+ antiporters support cytoplasmic pH homeostasis, tolerance to alkali and to fluctuations in osmolarity, and concomitantly support resistance to toxic levels of their efflux substrates, especially Na+ (Padan et al., 2005, Slonczewski et al., 2009). They also enhance resistance to elevated temperatures that increase the cation permeability of the cell (Slonczewski et al., 2009).
Bacteria that live primarily in the cytoplasm of host cells and are sheltered by the homeostatic mechanisms of the host often lack genes predicted to encode a Na+(K+)/H+ antiporter, e.g. the aphid endosymbiont Buchnera aphidicola, or have only a single such gene, e.g., the human pathogen Rickettsia prowazekii (data from www.membranetransport.org). By contrast, most other bacterial genomes have multiple genes and operons predicted to encode Na+(K+)/H+ antiporters. Non-marine bacteria typically have 5–9 such antiporter genes and operons, while more ecologically stressed or versatile bacteria have 11–14 gene loci predicted to encode Na+(K+)/H+ antiporters (Table 1). Extensive information is available about single monovalent cation/proton antiporter proteins, especially NhaA from Escherichia coli, for which insights have been gained into the structure-function of the antiporter protein and how those properties relate to its physiological setting (Padan, 2008). Still, we do not understand why so many antiporter genes are stably maintained in bacterial genomes and the value added for the diverse mixes of antiporter types found among bacteria. The study of the “poly-extremophilic” anaerobe Natranaerobius thermophilus reported by Mesbah et al. in this issue of Molecular Microbiology moves us further towards building correlations between the physiology of particular lifestyles and properties of the monovalent cation/proton antiport complement (Mesbah, 2009).
Table 1
Table 1
Cation/proton antiporter candidates for Na+(K+)/H+ capacity as revealed by genomic analyses of selected bacteria.a
N. thermophilus grows well when confronted by the combined challenges of pH 9.5, a temperature of 53° C, and the presence of 3.3 M Na+. Under these conditions, the bacterium maintains a cytoplasmic pH of 8.3 (Mesbah, 2009). The external and cytoplasmic pH values at 53 C are equivalent to higher pH values at 25–30° C, so N. thermophilus tolerates a high cytoplasmic pH in the same way as do aerobic alkaliphiles when growing at pH 10.5 and above (Padan et al., 2005, Slonczewski et al., 2009). However, the growth rate of poly-extremophilic N. thermophilus drops as the internal pH rises further, consistent with evidence that cytoplasmic pH is a major determinant of the upper pH limit for growth of aerobic alkaliphiles (Padan et al., 2005). At pH values above the optimum for N. thermophilus, the cytoplasmic pH remains ~1 pH unit more acidic than the outside pH although growth and antiporter-based pH homeostasis come to a stop. Mesbah et al. attribute this to high cytoplasmic buffering capacity, which is highest as pH rises (Mesbah, 2009). Aerobic alkaliphiles also have high cytoplasmic buffering capacity at very alkaline pH but it does not replace the antiport role in alkaliphily (Slonczewski et al., 2009).
Mesbah et al. then move from the physiological context to the catalytic and kinetic properties of 12 predicted monovalent cation/proton antiporters that they identified in a draft sequence of the N. thermophilus genome. Except for one novel antiporter, Nta-Nha, they are categorized according to the sequence-based Transporter Classification System (Saier et al., 2006, Ren et al., 2007). The two main clusters, in terms of nomenclature, that contain antiporter families with secondary Na+(K+)/H+ antiporters are: CPA (Cation:Proton Antiporter) families, CPA1, CPA2 and CPA3; and Nha (Na+-H+ antiporter) families, NhaA, NhaB, NhaC and NhaD. The CaCA (Calcium/Cation Antiporter) family also has members that are Ca2+(Na+)(K+)/H+ antiporters (Radchenko et al., 2006). Most cation/proton antiporters are single hydrophobic gene products some of which function as homo-oligomers. The most structurally complex antiporters are CPA3 (or Mrp-type) antiporters. They have 6–7 different hydrophobic proteins (Group 2 and 1, respectively, in Figure 1) that are encoded in operons and are all required for antiport activity of a multi-subunit complex (Kajiyama et al., 2007, Morino et al., 2008, Swartz et al., 2005). The N. thermophilus Nt-Nha antiporter is encoded in an apparent single gene locus whose product is a homologue of the two large subunits of Group 1 CPA3 antiporters, MrpA and MrpD. These proteins have been suggested to be the antiporters although they are not active when expressed alone (Mathiesen & Hagerhall, 2003, Swartz et al., 2005). They both have oxidoreductase domains and sequence similarity to membrane subunits of proton-pumping NADH dehydrogenases, e.g. NuoL, NuoM and NuoN proteins of E. coli and NdhF of plants and cyanobacteria (Hamamoto et al., 1994, Mathiesen & Hagerhall, 2003, Swartz et al., 2005). MrpA proteins usually also have a MrpB/MnhB domain (Mathiesen & Hagerhall, 2003, Swartz et al., 2005). This domain is absent in both Nt-Nha homologues and the MrpA of the CPA3 cluster of N. thermophilus (Figure 1). Since “stand-alone” Nt-Nha and its homologues are often annotated as NdhF, we designate the Nta-Nha antiporter type as NdhF-a (-a for antiporter). Cyanobacteria possess genes for NdhF-a homologues that are longer than Nt-Nha and also lack MrpB/MnhB domains. They may well be Nt-Nha-like antiporters, but are omitted from Fig. 1 because they could be dispersed respiratory ndh genes, as those are common in cyanobacteria.
Figure 1
Figure 1
Diagrammatic representations of the genes encoding MrpA/MrpD/NdhF like proteins encoded outside CPA3 operons and operons of different CPA3 groups, showing the oxidoreductase and MnhB (MrpB) domains
Table 1 catalogues the number and types of antiporters in genomes of a diverse group of bacteria, including N. thermophilus and a mix of aerobes, anaerobes, extremophiles and non-extremophiles. The 12 N. thermophilus antiporters characterized by Mesbah et al., 2 CPA1, 1 CPA2, 8 NhaC and 1 Nt-Nha antiporter are catalogued together with 5 more predicted monovalent cation/proton antiporters that we identified in the completed N. thermophilus genome sequence. These include 1 more CPA2 type, 3 more NhaC type and 1 CPA3 type antiporter. The CPA3 locus has a gene arrangement distinct from Group 1 and 2. Such CPA3 antiporters are referred to as Group 3 CPA3 or Mrp types (Swartz et al., 2005). Assays of the subset of 12 antiporters by Mesbah et al. (2009) indicated that 4 of the NhaC types support K+ uptake but exhibit no cation/proton antiport activity in vitro. They may couple K+ uptake to exchange of organic substrates, as another NhaC-type antiporter catalyzes Na-lactate/2H-Malic exchange (Padan et al., 2005). All the other antiporters exhibit Na+(K+)/H+ activity except for one K+-specific NhaC antiporter. The extensive K+/H+ antiport capacity of N. thermophilus antiporters differs from the exclusive use of Na+ as the efflux substrate for monovalent cation/proton antiporters of aerobic alkaliphiles (Padan et al. 2005). Mesbah et al. note that the presence of a Na+-pumping ATPase in N. thermophilus raises the need for an alternate substrate for the antiporters. Use of K+ for antiport necessitates enhanced K+ re-uptake capacity to maintain robust cytoplasmic concentrations of this important cation.
Mesbah et al. (2009) also made notable findings for two antiporter families. The first of these was the finding of high antiport activity and evident host expression of 4 NhaC antiporters, suggesting that this antiporter type has a sizeable role in N. thermophilus whereas these antiporters thus far have been shown to have minor roles in aerobic Bacillus alkaliphiles (Padan et al., 2005). Perhaps NhaC antiporters have a greater role in the alkali-response of anaerobes than aerobes. NhaC also supports alkali-tolerance of anaerobic Desulfovibrio vulgaris (Stolyar et al., 2007). The second notable finding by Mesbah et al. is the demonstration of Na+(K+)/H+ activity for the Nt-Nha antiporter. To our knowledge, this is the first demonstration of antiport activity for a stand-alone type of MrpA/MrpD/NdhF protein. The results support the idea that MrpA and MrpD are the antiporter proteins of CPA3 complexes.
The study of N. thermophilus (Mesbah, 2009) supports the idea that the total number of antiporters is heavily influenced by the diversity of challenges rather than just intensity of one challenge. Poly-extremophilic N. thermophilus has 17 predicted antiporters, extremely alkaliphilic B. halodurans C-125 has only 5 predicted monovalent cation/proton antiporters, and neutralophilic Bacillus subtilis has 7 (Table 1). Perhaps extremophiles that face a particular environmental challenge have a specific, adapted antiporter that has a dominant role in confronting that central challenge whereas a bacterium with multiple challenges uses a larger group of antiporters with overlapping roles that cover all the contingencies. In B. halodurans C-125, the multi-subunit Mrp-type antiporter is required for alkaliphily and Na+-resistance and in alkaliphilic B. pseudofirmus OF4, Mrp is required for growth throughout its pH range of 7.5 to >11 (Hamamoto et al., 1994, Swartz et al., 2005). In B. subtilis, Mrp has a major role in Na+-resistance but is not essential, and alkaline pH homeostasis and osmo-regulation apparently rely on other antiporters (Padan et al., 2005). The roles of the 11 antiporters of alkaliphilic Bacillus clausii (Table 1), including 2 CPA3 antiporters, have not been studied. The large number may serve the multiple lifestyles of the bacterium as a host-associated probiotic bacterium as well as a freeliving alkaliphile just as the 17 antiporters of N. thermophilus presumably enable the poly-extremophile to meet its multiple challenges. Genetic studies will be required to assess whether any particular antiporter in these bacteria is dominant or essential under specific conditions.
  • Hamamoto T, Hashimoto M, Hino M, Kitada M, Seto Y, Kudo T, Horikoshi K. Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C-125. Mol. Microbiol. 1994;14:939–946. [PubMed]
  • Kajiyama Y, Otagiri M, Sekiguchi J, Kosono S, Kudo T. Complex formation by the mrpABCDEFG gene products, which constitute a principal Na+/H+ antiporter in Bacillus subtilis. J Bacteriol. 2007;189:7511–7514. [PMC free article] [PubMed]
  • Mathiesen C, Hagerhall C. The 'antiporter module' of respiratory chain Complex I includes the MrpC/NuoK subunit - a revision of the modular evolution scheme. FEBS Lett. 2003;5459:7–13. [PubMed]
  • Mesbah N, Gregory Cook, Juergen Wiegel. The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters. Molecular Microbiology. 2009;xxx:xxx. [PMC free article] [PubMed]
  • Morino M, Natsui S, Swartz TH, Krulwich TA, Ito M. 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. 2008;190:4162–4172. [PMC free article] [PubMed]
  • Padan E. The enlightening encounter between structure and function in the NhaA Na+-H+ antiporter. Trends Biochem Sci. 2008;33:435–443. [PubMed]
  • Padan E, Bibi E, Ito M, Krulwich TA. Alkaline pH homeostasis in bacteria: new insights. Biochim Biophys Acta. 2005;1717:67–88. [PMC free article] [PubMed]
  • Radchenko MV, Tanaka K, Waditee R, Oshimi S, Matsuzaki Y, Fukuhara M, Kobayashi H, Takabe T, Nakamura T. Potassium/proton antiport system of Escherichia coli. J. Biol. Chem. 2006;281:19822–19829. [PubMed]
  • Ren Q, Chen K, Paulsen IT. TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels. Nucleic Acids Res. 2007;35:D274–D279. [PubMed]
  • Saier MH, Jr, Tran CV, Barabote RD. TCDB: the Transporter Classification Database for membrane transport protein analyses and information. Nucleic Acids Res. 2006;34:D181–D186. [PMC free article] [PubMed]
  • Slonczewski JL, Fujisawa M, Dopson M, Krulwich TA. Cytoplasmic pH Measurement and Homeostasis in Bacteria and Archaea. Adv Microb Physiol. 2009;55:1–317. [PubMed]
  • Stolyar S, He Q, Joachimiak MP, He Z, Yang ZK, Borglin SE, Joyner DC, Huang K, Alm E, Hazen TC, Zhou J, Wall JD, Arkin AP, Stahl DA. Response of Desulfovibrio vulgaris to alkaline stress. J Bacteriol. 2007;189:8944–8952. [PMC free article] [PubMed]
  • Swartz TH, Ikewada S, Ishikawa O, Ito M, Krulwich TA. The Mrp system: a giant among monovalent cation/proton antiporters? Extremophiles. 2005;9:345–354. [PubMed]