Cloning And Identification Of A Novel CDG Family Transporter Gene CdffT From Planococcus Sp. NEAU-ST10-9

The moderate halophiles are a group of microorganisms that can grow optimally in the presence of3-15%NaCl, which were shown to possess high-efficency halo-alkaline tolerant capacity. Compared to non-halophilic bacteria, the moderately halophiles are believed to contain more unique types of halo-alkaline tolerant genes and more sophisticated molecular genetic mechnism to cope with saline and alkaline conditions. Planococcus sp. NEAU-ST10-9is a moderate halophile recently isolated by our lab from saline and alkaline soils in the Songnen Plain, which can tolerate10%NaCl and grow well at pH12.In this study, genomic DNA was screened for the Na+/H+antiporter gene from the moderate halophile Planococcus sp. NEAU-ST10-9by slection in Escherichia coli KNabc lacking three major Na+/H+antiporters. A2.7-kb DNA fragment consisting of three open reading frames （ORF1-3） proceeded by their respective promoter-like and Shine-Dalgarno （SD） sequences was finally cloned. ORF1designated Hyp consisting of200residues has the highest identity （49%） with a hypothetical protein from Planococcus donghaensis MPA1U2. ORF2designated TetR consisting of177residues has the highest identity （49%） with a TetR family transcriptional regulator from Bacillus pseudofirmus OF4. ORF3designated CdffT consisting of306residues has the highest identity （43%） with a cation diffusion facilitator （CDF） family transporter Bs_CdffT from Bacillus selenitireducens MLS10. Because the function of Bs CdffT has not been characterized experimentally, ORF3was also aligned with all the known CDF family transporters including Bs_CzcD （Accesion.version No. AAB53029.1） from Bacillus subtilis, Rm_CzcD （Accesion.version No. YP₁45596.1） from Ralastonia metallidurans, Sc_COTl （Accesion.version No. AAA74884.1） and Sc_ZRC1（Accesion.version No. NP₀13970.1） from Saccharomyces cerevisiae, and Ec ZitB （Accesion.version No. AAC73839.1） and Ec_FieF （Accesion.version No.NP418350.1） from E. coli that have been identified experimentally. ORF3showed very low identity with the above known CDF family transporters （17.2%for Ec_ZitB,15.2%for Rm_CzcD,14.3%for Sc ZRC1,13.0%for Bs_CzcD,12.9%for Sc_COT1and11.9%for Ec_FieF）. Based on the identity analysis, we proposed that CdffT should be a novel CDF family transporter.Although no ORFs were predicted to encode Na+/H+antiporter in the above mentioned2.7-kb DNA fragment, amino acid composition and topological analysis showed that CdffT is the sole tansmembrane trnasporter of low polarity with six hydrophobic transmembrane segments （TMSs）.In order to identify the exact ORF with Na+/H+antiport activity, cdffT gene encoding a putative CDF family transporter, together with its promoter-like and SD sequences, was separately subcloned by PCR amplification into a T-A cloning vector pEASY T3. The resulting plasmid designated pEASY T3-cdffT and the empty vector pEASY T3as a neagtive control were then transformed in the E. coli KNabc, respectively. To test the ability of CdffT to induce salt tolerance, E. coli KNabc/pEASY T3-cdffT and KNabc/pEASY T3were grown in LBK medium containing0-0.5M NaCl or0-20mM LiCI. As a result, E. coli KNabc/pEASY T3-cdffT could grow in the presence of0.4M NaCl, but E. coli KNabc/pEASY T3as a negative control could not survive in the presence of0.2M NaCl. Also, E. coli KNabc/pEASY T3-cdffT could grow in the presence of10mM LiCI, but E. coli KNabc/pEASY T3as a negative control could not survive in the presence of5mM LiCI. In order to analyze the resistance of CdffT to alkaline pH, E. coli KNabc/pEASY T3-cdffT and KNabc/pEASY T3were grown in the LBK medium at the pH values from7.0to8.5. As a result, the growth of E. coli KNabc/pEASY T3was greatly reduced under alkaline conditions at pH7.5-8.0, compared with that at pH7.0, whereas CdffT conferred E. coli KNabc cells the capability to grow under alkaline conditions. There results suggest that cdffT may encode a Na+/H+antiporter.To test the ability of CdffT to tolerate divalent metal ions, E. coli KNabc/pEASY T3-cdffT and KNabc/pEASY T3were grown in the LBK medium0-1.0mM ZnCl2,0-2.0mM CoCl2and0-0.4mM CdCl2, respectively. As a result, E. coli KNabc/pEASY T3-cdffT showed a signifcant or extremely signifcant resistance to0.2-1.0mM ZnCl2or0.2-0.3mM CdCl2, compared to KNabc/pEASY T3. In contrast, there is a certain （but not significant） resistance difference between E. coli KNabc/pEASY T3-cdffT and KNabc/pEASY T3in the presence of0.5-1.0mM CoCl2. In order to confirm the above results, we plotted the time curves of E. coli KNabc/pEASY T3-cdffT and KNabc/pEASY T3growing in the presence of1.0mM ZnC12,0.75mM CoC12or0.25mM CdC12. As a result, CdffT showed a significant resistance to Zn2+and Cd2+, but not to Co2+Cation/H+antiport activity with everted membrane vesicles prepared from cells of E. coli KNabc strains carrying pEASY T3-cdffT or pEASY T3was determined by measuring the dequenching of acridine orange fluorescence upon addition of NaCl, LiCI, Na-free KC1, ZnCl2, CoCl2or CdCl2. As a result, not only Na+/H+and Li+/H+antiport activity but also K+/H+antiport activity were detected in everted membrane vesicles from KNabc/pEASY T3-cdffT, while no Na+/H+, Li+/H+or K+/H+antiport activity was detected in those from KNabc/pEASY T3. In addition, higher Zn2+/H+and Cd2+/H+antiport activity were detected in everted membrane vesicles from KNabc/pEASY T3-cdffT, compared with those from KNabc/pEASY T3. However, no Co2+/H+antiport activity was detected in everted membrane vesicles from KNabc/pEASY T3-cdffT or those from KNabc/pEASY T3. The above results indicated that cdffT gene should encode a Na+（Li+, K+, Zn2+, Cd2+）/H+antiporter. The effect of pH on cation/H+antiport activity was also measured. As a result, CdffT exhibited Na+（Li+,K+）/H+antiport activity at a wide range of pH between6.5and9.5, which was with the highest Na+（Li+, K+）/H+antiport activity at pH9.0, pH8.5, pH9.5, respectively. In contrast, CdffT exhibited Zn2+（Cd2+）/H+antiport activity at a wide range of pH between6.5and8.5, which was with the highest antiport activity at pH8.0. In order to assess the affinity of CdffT for cations, Michaelis-Menten kinetics of CdffT for Na+, Li+, K+, Zn2+or Cd2+were analyzed by measuring cation/H+antiport activity in everted membrane vesicles from KNabc/pEASY T3-cdffT at pH9.0, pH8.5, pH9.5or pH8.0with final concentrations of added cations varying from0-20mM. The apparent Km values of Na+, Li+, K+, Zn2+and Cd2+are1.10,1.30,1.18,1.00and2.19, respectively, suggesting that the affinity of CdffT for cations is Zn2+>Na+>K+>Li+>Cd2+.Based on the above results, we proposed that cdffT gene should encode a novel CDF family transporter, which transports both divalent metal ions Zn2+, Cd2+and monovalent cations Na+, Li+, K+as a multi-funcational pH-dependent cation/H+antiporter. To the best of our knowledge, this is the first report that a CDF family transporter can simultaneously exhibit dicvalent cation/H+and monovalent cation/H+antiport activity.