Department of Microbiology and Immunology, College of Biological Sciences, China Agricultural University, Key Laboratory of Agro-Microbial Resource and Application, Ministry of Agriculture, Beijing, China
Correspondence: Lifu Yang, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Key Laboratory of Rubber Biology and Genetic Resource Utilization, Hainan Province, 571737, China. Tel.: +86 898 2330 0595; fax: +86 898 2330 0315; e-mail: email@example.com
In this study, metagenomic DNA was screened for the Na+/H+ antiporter gene from the halophilic bacteria from Daban Salt Lake using Escherichia coli KNabc lacking three major Na+/H+ antiporters, and two genes psmrAB predicted to encode paired small multidrug resistance (PSMR) family proteins, the homolog of YvdSR, were finally cloned. Only the simultaneous presence of psmrAB, but not the single gene alone, conferred the tolerance of E. coli KNabc to up to 0.6 M NaCl and at alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc cells carrying psmrAB, which had the highest activity at pH 9.0. However, a detailed test for antimicrobial drugs showed that E. coli DH5α with psmrAB only exhibited slight resistance to chloramphenicol, but not other representative antimicrobial drugs especially ethidium bromide. Protein sequence alignment showed that neither PsmrA nor PsmrB has homology with known single-gene or multiple-gene Na+/H+ antiporters, or such proteins as TetA(L) and MdfA with Na+/H+ antiport activity. Taken together, PsmrAB should function mainly as a novel two-component Na+/H+ antiporter. This is the first example of a PSMR family member that exhibits Na+/H+ antiporter activity.
In bacteria, Na+/H+ antiporters are ubiquitous secondary transporters that catalyze the efflux of intracellular alkali cations in exchange for external protons, which play a vital role in reducing the cytoplasmic concentration of toxic alkali cations and supporting Na+(Li+)/K+-dependent intracellular pH homeostasis under alkaline conditions (Ito et al., 1999; Padan et al., 2005). Na+/H+ antiporter genes or the genes with Na+/H+ antiporter activity have been increasingly cloned and functionally identified in Escherichia coli mutants KNabc or EP432 lacking major antiporters (Padan et al., 2004). So far, Na+/H+ antiporters are sorted into two main kinds based on the number of genes: One kind of Na+/H+ antiporters are encoded by a single gene including nhaA (Karpel et al., 1988), nhaB (Pinner et al., 1992), nhaC (Nakamura et al., 1996), nhaD (Ito et al., 1997), napA (Waser et al., 1992), nhaP (Utsugi et al., 1998), nhaG (Gouda et al., 2001) and nhaH (Yang et al., 2006c). The other kind of Na+/H+ antiporters containing multiple subunits are encoded by an operon or a gene cluster such as mrp operon from Bacillus subtilis (Ito et al., 1999), mnh gene cluster from Staphylococcus aureus (Hiramatsu et al., 1998) and pha2 gene cluster from Sinorhizobium fredii (Jiang et al., 2004; Yang et al., 2006a). Moreover, an unique tetracycline/H+ antiporter TetA(L) was reported to possess Na+/H+ antiporter activity (Cheng et al., 1994). Another E. coli multidrug resistance (MDR) protein MdfA with a broad-specificity MDR phenotype (Edgar & Bibi, 1997) was also characterized to exhibit Na+(K+)/H+ antiporter activity (Lewinson et al., 2004).
In our previous studies, a novel species Halobacillus dabanensis D-8T was isolated and characterized from Daban Salt Lake in Xinjiang Province, China (Liu et al., 2005), and two genes nhaH (Yang et al., 2006c) and nap (Yang et al., 2006b) were cloned from H. dabanensis and found to possess Na+/H+ antiporter activity. Our recent survey on the biodiversity of the culturable halophilic bacteria in the above area showed at least 10 potential novel species or genera (Wu et al., 2010). As almost all halophilic microorganisms have potential Na+ ion transport mechanisms to expel Na+ ions from the interior of the cells depending on Na+/H+ antiporters (Oren, 1999), it is very likely that some important (even novel) Na+/H+ antiporter genes exist in the above area containing such many potential novel species.
To obtain as many (especially novel) Na+/H+ antiporter genes as possible, metagenomic DNA was screened from the culturable halophilic bacteria in the above area and several genes with Na+/H+ antiport activity were finally cloned. Of these, psmrAB were found to encode a pair of putative PSMR family proteins, the homolog of YvdSR in B. subtilis. In this study, we reported the cloning and characterization of psmrAB and finally proposed that PsmrAB should function mainly as a novel two-component Na+/H+ antiporter.
Materials and methods
Bacterial strains and growth conditions
Halophilic bacteria were enriched in the Sehgal–Gibbons medium containing 0.5% casein, 1% yeast extract, 0.5% tryptone, 0.2% KCl, 0.3% trisodium citrate, 2% MgSO4. 7H2O plus 7.5% NaCl at pH 7.5. Escherichia coli strain KNabc, lacking three major Na+/H+ antiporters (NhaA, NhaB and ChaA) and its transformant cells were grown in the LBK medium consisting of 1.0% tryptone, 0.5% yeast extract and 87 mM KCl, to which NaCl or LiCl was added at indicated concentrations when necessary. Ampicillin was added to a final concentration of 50 μg mL−1 for the selection of transformant cells. To test the resistance of PsmrAB to the antimicrobial drugs, E. coli DH5α/pEASY T3-psmrAB and DH5α/pEASY T3 (as a negative control) were grown overnight on the LB medium plates and then continued to be grown on the fresh LB medium plates containing the different concentrations of drugs for 48 h. Cell growth was monitored turbidimetrically at 600 nm.
Preparation and electroporation of electro-competent E. coli KNabc or DH5α cells
A 5-mL overnight culture of E. coli KNabc or DH5α cells was inoculated into 100 mL of LBK or LB medium and grown to an optimal density of 0.4 at 600 nm. Cells were harvested by centrifugation at 4000 g for 10 min at 4 °C and washed for three times in 10 mL of ice-cold sterile 10% glycerol solution before electro-competent preparation. Recombinant plasmids (20–200 ng) were added to 50–200 μL of cell suspension and mixed thoroughly. Electroporation was carried out at a field strength of 16 kV cm−1 in combination with an electric resistance of 300 Ω at 25 mF in a 0.1-cm electroporation cuvette.
Cloning and subcloning of DNA fragment containing Na+/H+ antiporter genes
Soil samples (20-cm deep) were widely collected around Daban Salt Lake in Xinjiang Province, P.R. China, and then the culturable halophilic bacteria were enriched in the Sehgal–Gibbons medium containing 7.5% NaCl. The metagenomic DNA was extracted from the enriched cultures and partially digested with Sau3AI. The DNA fragments with 4–10 kb were separated by agarose electrophoresis and ligated into pUC18, which had been digested with BamHI and dephosphorylated with bacterial alkaline phosphatase, using T4 DNA ligase. Competent cells of E. coli KNabc were transformed with the ligated reaction mixture and spread on LBK medium plates containing 0.2 M NaCl, 1.5% agar and 50 mg mL−1 of ampicillin. The plates were incubated at 37 °C for 20 h and colonies picked for further studies. Subcloning of one or more ORFs including their respective promoter-like and SD sequences was carried out by PCR amplification, purification and re-ligation into a T-A cloning vector pEASY T3 (Beijing TransGen Biotech Co., Ltd). The forward primer for psmrAB is 5′-TAATGGTGGAAGATTGTATG-3′ and the reverse primer is 5′-GTCGGTGTCGAAAGTTGTA-3′.
Preparation of everted membrane vesicles
Escherichia coli KNabc cells carrying pEASY T3-psmrAB and pEASY T3 (as a negative control) were grown in LBK medium up to the mid-exponential phase and harvested by centrifugation at 5000 g, 4 °C for 10 min. Everted membrane vesicles were prepared from transformant cells of E. coli KNabc/pEASY T3-psmrAB and KNabc/pEASY T3 by the French Pressure cell method at 2000 psi and collected by ultracentrifugation at 100 000 g for 1 h as described by Rosen (1986). The vesicles were resuspended in a buffer containing 10 mM Hepes-Tris (pH 7.0), 140 mM choline chloride, 0.5 mM dithiothreitol and 250 mM sucrose and stored at −70 °C before use.
Assays of Na+(Li+)/H+ and chloramphenicol/H+ antiport activity
The Na+(Li+)/H+ and chloramphenicol/H+ antiport activity of everted membrane vesicles was estimated according to the extent of the collapse of a performed proton gradient, with acridine orange as the pH indicator, as described by Rosen (1986). The assay mixture contained 10 mM Hepes-Tris (at the indicated pH from 6 to 9) or 10 mM Ches-KOH (pH 9.5), 140 mM choline chloride, 10 mM MgCl2, 2 μM acridine orange and 20–40 μg mL−1 protein of membrane vesicles. Potassium lactate (5 mM) was added to initiate respiration. Fluorescence was monitored with a Hitachi F-4500 fluorescence spectrophotometer (Hitachi Ltd, Tokyo, Japan) at excitation and emission wavelength of 495 and 530 nm, respectively.
DNA manipulation and sequence analysis
Preparation of plasmid DNA, extraction of metagenomic DNA, restriction enzyme digestion and ligation were carried out as described by Sambrook et al. (1989). DNA sequencing was performed by Beijing Genomics Institute (Beijing, China). The analyses for ORF, hydrophobicity and topology were carried out with the dnaman 6.0 software. Protein sequence alignment was performed through the National Center for Biotechnology Information (NCBI) using the website http://www.ncbi.nlm.nih.gov/blastp. Promoter prediction was performed using the website http://www.fruitfly.org/seq_tools/promoter.html.
Protein content determination
Protein content in everted membrane vesicles was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Nucleotide sequence accession number
The 5.2-kb nucleotide sequence reported in this study has been submitted to GenBank database with Accession number JQ350846.
Cloning and sequence analysis of Na+/H+ antiporter genes
A 5.2-kb DNA fragment was first obtained from Sau3AI-digested metagenomic DNA from the enriched halophilic bacteria in soil samples around Daban Salt Lake using E. coli KNabc. The plasmid pUC18 carrying this DNA fragment enabled E. coli KNabc cells to grow in medium containing 0.2 M NaCl or 5 mM LiCl. Sequence analysis showed that eight open reading frames (ORFs) are included in this DNA fragment and each ORF is preceded by a promoter-like sequence and a SD sequence. Of these eight ORFs, ORF3 has the highest identity with a TetR family transcriptional regulator (38%) (GenBank Accession No. YP_001114342) in Desulfotomaculum reducens, and also has higher identity (32%) with a TetR family transcriptional regulator (GenBank Accession No. YP_003561463) in Bacillus megaterium QM B1551. ORF4-5 have the highest identity with one pair of putative PSMR family proteins YP_003561462/YP_003561461 (55%, 58%) in B. megaterium QM B1551, respectively (Fig. 1b and c). Because that the functions of proteins YP_003561462 and YP_003561461 have not been characterized experimentally, ORF4-5 was also aligned with all four PSMR family protein pairs including YvdSR, YkkCD, EbrAB and YvaDE that have been identified experimentally in B. subtilis. ORF4-5 showed the highest identity (35%, 42%) with YvdSR pair among these four pairs (Fig. 1b and c). ORF4- and ORF5-encoded genes were designated as psmrA and psmrB, respectively, based on the identities with paired small multidrug resistance (PSMR) family protein genes.
The deduced amino sequence of PsmrA consists of 114 residues (Fig. 1a) with a calculated molecular weight of 12, 210 Dalton and a pI of 4.56. The most abundant residues of PsmrA were Gly (18/114), Ile (17/114), Phe (12/114), Leu (11/114) and Thr (11/114). The least abundant residues of PsmrA were His (1/114), Pro (1/114), Gln (1/114) and Arg (1/114). Among the 114 residues of PsmrA, 87 residues were hydrophobic, indicating that PsmrA is of low polarity. By contrast, the deduced amino sequence of PsmrB consists of 104 residues (Fig. 1a) with a calculated molecular weight of 11, 117 Dalton and a pI of 10.32. The most abundant residues of PsmrB were Gly (13/104), Ala (13/104), Leu (13/104), Phe (11/104) and Ile (11/104). The least abundant residues of PsmrB were Cys (1/104), Asp (1/104), Glu (1/104) and Gln (1/104). Among the 104 residues of PsmrB, 82 residues were hydrophobic, indicating that PsmrB is also of low polarity. Topological analysis showed that both PsmrA and PsmrB are composed of three transmembrane segments, respectively.
Identification of ORFs with Na+/H+ antiport activity
To identify the exact ORF(s) with Na+/H+ antiport activity, each ORF with its respective promoter-like and SD sequence was subcloned by PCR into a T-A cloning vector pEASY T3 and then transformed into E. coli KNabc to test whether it could restore the growth of E. coli KNabc in the presence of 0.2 M NaCl. No single ORF could enable E. coli KNabc to grow in the presence of 0.2 M NaCl, even if each one was separately inserted just downstream from the lac promoter of pEASY T3 in the forward orientation. Considering that ORF4-5 were predicted to encode a pair of putative PSMR family proteins that belong to transmembrane proteins, both of them were subcloned together into pEASY T3 and found to succeed in restoring the growth of E. coli KNabc in the presence of 0.2 M NaCl. It should be stressed that the psmrAB genes with their respective predicted promoters can also restore the growth of E. coli KNabc in the presence of 0.2 M NaCl when they were inserted just downstream from the lac promoter of pEASY T3 in the opposite orientation. Therefore, it is concluded that the original promoters of psmrAB genes should be functional in the E. coli cells. The strategy of subcloning of all ORFs was carried out as that of ORF4-5 shown in Fig. 2.
Resistance of PsmrAB to NaCl and pH
To test the salt tolerance of PsmrAB, E. coli KNabc/pEASY T3-psmrAB and KNabc/pEASY T3 were grown in the LBK medium containing 0–0.6 M NaCl or 5–50 mM LiCl. As shown in Fig. 3a, E. coli KNabc/pEASY T3-psmrAB could grow in the presence of up to 0.6 M NaCl, but E. coli KNabc/pEASY T3 as a negative control could not survive in the presence of 0.2 M NaCl. In contrast, E. coli KNabc/pEASY T3-psmrAB could grow only in the presence of 5 mM LiCl (data not shown). To analyze the resistance of PsmrAB to pH, E. coli KNabc/pEASY T3-psmrAB and KNabc/pEASY T3 were grown in the LBK medium at the pH values from 7 to 9. As shown in Fig. 3b, the growth of E. coli KNabc/pEASY T3 was greatly reduced under alkaline conditions, especially at pH 8.0, compared with that below neutral pH, whereas the coexpression of PsmrAB conferred E. coli KNabc cells with the ability to grow under alkaline conditions.
Determination of the resistance of PsmrAB to antimicrobial drugs
To determine whether PsmrAB exhibit a broad-specificity MDR phenotype, E. coli DH5α/pEASY T3-psmrAB and DH5α/pEASY T3 were grown on the LB medium plates containing the different concentrations of such representative antimicrobial drugs as ethidium bromide, which are usually used for the determination of the function of PSMR family proteins. Escherichia coli DH5α/pEASY T3-psmrAB only showed a slight resistance to chloramphenicol, but not any other representative antimicrobial drugs especially ethidium bromide (Table 1).
Table 1. Minimum inhibitory concentrations of several representative antimicrobial drugs for Escherichia coli DH5α/pEASY T3 and DH5α/pEASY T3-psmrAB
Minimum inhibitory concentrations (μg mL−1)
Na+(Li+)/H+ antiport activity in everted membrane vesicles
Na+(Li+)/H+ antiport activity with everted membrane vesicles prepared from cells of E. coli KNabc strains carrying pEASY T3-psmrAB or pEASY T3 was determined by measuring the dequenching of acridine orange fluorescence upon addition of NaCl or LiCl. As shown in Fig. 4, both Na+/H+ and Li+/H+ antiport activity were detected in membrane vesicles from KNabc/pEASY T3-psmrAB, while no Na+/H+ or Li+/H+ antiport activity was detected in those from KNabc/pEASY T3. The effect of pH on Na+/H+ as well as Li+/H+ antiport activity was also measured. PsmrAB exhibited Na+/H+ antiport activity at a wide range of pH between 6.5 and 9.5, whereas no Li+/H+ antiport activity was measured below pH 8.0 (Fig. 5). Optimal pH for the Na+/H+ and Li+/H+ antiport activity was 9.0 (Fig. 5).
Due to the slight resistance of PsmrAB to chloramphenicol, chloramphenicol/H+ antiport activity was also determined based on the above method of the measurements of Na+(Li+)/H+ antiport activity. However, no chloramphenicol/H+ antiport activity was detected in membrane vesicles from KNabc/pEASY T3-psmrAB or KNabc/pEASY T3 at a wide range of pH between 6.5 and 9.5 (data not shown).
This study reports for the first time PSMR family protein genes psmrAB encoding a novel two-component Na+/H+ antiporter. PsmrAB could confer the E. coli KNabc the with capability of growing under alkaline conditions (Fig. 3), and both Na+/H+ and Li+/H+ antiport activity was detected in everted membrane vesicles from KNabc/pEASY T3-psmrAB, but not from KNabc/pEASY T3 (Fig. 4), which was with the highest Na+/H+ antiport and Li+/H+ antiport activity at pH 9.0 (Fig. 5). These confirm that psmrAB genes should encode a Na+/H+ antiporter. Known Na+/H+ antiporters include two main sorts: single-gene Na+/H+ antiporters such as NhaA, NhaB, etc. (Karpel et al., 1988; Pinner et al., 1992; Waser et al., 1992; Nakamura et al., 1996; Ito et al., 1997; Utsugi et al., 1998; Gouda et al., 2001; Yang et al., 2006c) and multigene Na+/H+ antiporters such as Mhn, Mrp or Pha2 (Hiramatsu et al., 1998; Ito et al., 1999; Jiang et al., 2004; Yang et al., 2006a). However, a careful protein alignment at the NCBI website showed that there is no identity between either of PsmrA or PsmrB and any known single-gene Na+/H+ antiporters or any subunit of multiple-gene Na+/H+ antiporters. Therefore, PsmrAB should encode a novel Na+/H+ antiporter, which is significantly different from these two kinds of Na+/H+ antiporters.
A unique tetracycline/H+ transporter TetA(L) displays Na+/H+ antiporter activity (Cheng et al., 1994). Another E. coli MDR protein MdfA with a broad-specificity MDR phenotype (Edgar & Bibi, 1997) possesses Na+(K+)/H+ antiporter activity (Lewinson et al., 2004). Both TetA(L) and MdfA are MDR-type transporters belonging to the major facilitator family (MF) with 12 transmembrane segments (Cheng et al., 1994; Lewinson et al., 2004). So far, known drug extrusion systems are sorted into four major groups: MF family; the small multidrug resistance (SMR) family; the resistance nodulation cell division family (RND) family; and the ATP binding cassette (ABC) family (Mine et al., 1998). SMR family transporters with usually three to four transmembrane helices are much smaller than MF family MDR-type transporters and therefore significantly different from the latter, although they exhibit a similar broad-specificity MDR phenotype (Bay et al., 2008). Therefore, this is the first example of a PSMR family member that exhibits Na+/H+ antiporter activity.
PsmrAB (ORF4-5) have the highest identity (55%, 58%) with a pair of putative PSMR family proteins YP_003561462/YP_003561461 in B. megaterium (Fig. 1b and c). So far, known PSMR family protein pairs were only identified in B. subtilis and sorted into four distinct members: YvdSR, YkkCD, EbrAB and YvaDE (Bay et al., 2008). PsmrAB have the highest identity with YvdSR pair among the above four PSMR family protein pairs (Fig. 1b and c). Also, only YvdSR pair is adjacent to a gene encoding a TetR family transcription regulator (GenBank Accession No. CAB15453) (Eppinger et al., 2011), the gene order of which is identical to that of TetR and PsmrAB. Our recent study showed that Bacillus species amount for 48% of culturable halophilic bacteria from soil samples around Daban Salt Lake (Wu et al., 2010). Therefore, it is the most possible that PsmrAB are the homolog of YvdSR pair in B. subtilis.
The SMR protein family is a bacterial multidrug transporter family mainly including three subclasses: the single-gene small multidrug pump, suppressor of GroEL mutation proteins (SUG) and PSMR family proteins (Bay et al., 2008). PSMR proteins are distinct from the other two subclasses of SMR proteins due to the requirement for simultaneous expression of both SMR homologs to confer a drug resistance phenotype (Bay et al., 2008). As shown in Fig. 3a, only the simultaneous presence of PsmrAB could confer E. coli KNabc NaCl resistance, indicating that PsmrAB should function as a heterodimer. The deduced amino sequence of PsmrA consists of 114 residues and that of PsmrB consists of 104 residues, which is consistent with the report that PSMR protein pairs generally consist of one protein with typical SMR protein length and a remaining protein that is longer (Bay et al., 2008). Topology analysis also showed that both PsmrA and PsmrB are composed of three transmembrane segments, respectively, which is also consistent with the report that PSMR family proteins are usually integral membrane proteins containing three to four transmembrane segments (Bay et al., 2008). Therefore, PsmrAB should belong to PSMR protein family.
Escherichia coli KAM3 lacking a restriction system and a main drug transporter AcrAB or E. coli DH5α and ethidium bromide, a representative of antimicrobial drugs, are usually used for the determination of PSMR family proteins (Jack et al., 2000; Masaoka et al., 2000). In this study, when pEASY T3-psmrAB were introduced into E. coli DH5α, PsmrAB was found to only be able to slightly enhance the resistance of E. coli DH5α to chloramphenicol but not any other antimicrobials especially ethidium bromide (Table 1). However, no chaloramphenicol/H+ antiport activity was detected in everted membrane vesicles from KNabc/pEASY T3-psmrAB and KNabc/pEASY T3 (data not shown). In B. subtilis, both of EbrAB (Bay et al., 2008), YkkCD (Masaoka et al., 2000) and YvaE of the YvaDE pair were characterized to be able to confer host drug resistance phenotype (Jack et al., 2000). However, neither protein of YvdSR pair could confer a drug resistance phenotype when expressed as single genes or in tandem (Chung & Sair, 2001). As it is most possible that PsmrAB are the homolog of YvdSR pair, PsmrAB cannot function as a MDR-type drug transporter just like YvdSR pair. Therefore, future studies must confirm whether YvdSR pair can also exactly exhibit Na+/H+ antiporter activity. Moreover, it is possible that the drug resistance conferred by PsmrAB was masked by the other MDR transporters in this strain, if PsmrAB were not sufficiently expressed in E. coli DH5α, which was suggested by the fact that E. coli DH5α by itself displayed very high resistance to such antimicrobial drugs as ethidium bromide (Table 1). Therefore, it would be more proper that the drug resistance of PsmrAB should be tested in the MDR-type transporter deficient E. coli KAM3, Based on our current data, we proposed that PsmrAB, as the homolog of YvdSR pair, should function mainly as a novel two-component Na+/H+ antiporter.
We are so grateful to Dr Terry A. Krulwich (Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of the City University, New York) for the kind gift of E. coli strain KNabc. This manuscript was supported by National Natural Science Foundation of China (Grant No. 30960009 and 31000055), Key Project of Returned Overseas Chinese Scholars of Heilongjiang Province of China (Grant No. 1251HZ001), Special Financial Grant from China Postdoctoral Science Foundation (Grant No. 201104408), Doctor Start-up Fund of Northeast Agricultural University (Grant No. 2009RC23) and Key Laboratory Open Fund of Soybean Biology of Ministry of Education (Grant No. SB11A05).
J.J., L.W. and H.Z. contributed equally to this work.