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A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake

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The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth
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  A tenth  atp   gene and the conserved  atpI   gene of a Bacillus atp   operon have a role in Mg 2  uptake David B. Hicks*, ZhenXiong Wang* † , Yi Wei, Rebecca Kent, Arthur A. Guffanti, Horia Banciu ‡ , David H. Bechhofer,and Terry A. Krulwich § Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029Edited by Paul D. Boyer, University of California, Los Angeles, CA, and approved July 8, 2003 (received for review May 18, 2003) The atp operonofalkaliphilic Bacilluspseudofirmus OF4,asinmostprokaryotes, contains the eight structural genes for the F-ATPase(ATP synthase), which are preceded by an  atpI   gene that encodesa membrane protein of unknown function. A tenth gene,  atpZ  , hasbeen found in this operon, which is upstream of and overlappingwith  atpI  . Most  Bacillus  species, and some other bacteria, possess atpZ  homologues.AtpZispredictedtobeamembraneproteinwitha hairpin topology, and was detected by Western analyses. Dele-tion of  atpZ  ,  atpI  , or  atpZI   from  B. pseudofirmus  OF4 led to arequirement for a greatly increased concentration of Mg 2  forgrowth at pH 7.5. Either  atpZ  ,  atpI  , or  atpZI   complemented thesimilar phenotype of a triple mutant of  Salmonella typhimurium (MM281), which is deficient in Mg 2  uptake.  atpZ   and  atpI  , sepa-ratelyandtogether,increasedtheMg 2  -sensitive 45 Ca 2  uptakebyvesicles of an  Escherichia coli   mutant that is defective in Ca 2  andNa  efflux.WehypothesizethatAtpZandAtpI,ashomooligomers,andperhapsasheterooligomers,areMg 2  transporter,Ca 2  trans-porter, or channel proteins. Such proteins could provide Mg 2  ,which is required by ATP synthase, and also support chargecompensation, when the enzyme is functioning in the hydrolyticdirection; e.g., during cytoplasmic pH regulation. P rokaryotic  atp  operons encode the cell membrane F-type ATPase (ATP synthase) that couples the energy of anelectrochemical H  gradient (or sometimes Na  ), to the syn-thesis of ATP, from ADP and P i . In the reverse reaction, the ATPase hydrolyzes ATP concomitant with H  (or Na  ) efflux,thereby contributing to cytoplasmic pH regulation and  or gen-eration of a transmembrane electrochemical gradient underfermentative conditions (1–4). Most  atp  operons, like that of   Escherichia coli , contain the eight structural genes for the ATPase,  atpBEFHAGDC , which are preceded by  atpI   (5). The  Escherichia coli atpI   is expressed, and its product associates withthe membrane, as predicted from its deduced sequence (6–9).Whereas there is no demonstrated effect of AtpI on expressionor assembly of the ATPase, an  atpI   deletion strain of   E. coli  hasbeen reported to have a reduced growth yield (7). There is nofunction established for this “mysterious ninth gene” (10) thataccounts for such an effect. We report here the finding of another gene, encoding a membrane protein, that is upstream of the  atpI   gene, and within the  atp  operon of alkaliphilic  Bacillus pseudofirmus  OF4. This gene, designated  atpZ,  was discoveredduring attempts to introduce site-directed changes in alkaliphile-specific motifs of the membrane-embedded F-ATPase subunitsof   B.pseudofirmus OF4(11).Acassetteintroducedjustupstreamof the putative  atp  promoter abolished  atp  expression. Thisfinding led us to reexamine the location of the  atp  operonpromoter, to the inclusion of   atpZ  in the extended operon, andthen to an exploration of the effects of deleting  atpI   as well as  atpZ . The results suggest a cation translocation function for AtpIand AtpZ, which could be widely relevant to the diverse pro-karyotes that possess homologues of one or both of theseproteins. Materials and Methods Bacterial Strains, Plasmids, and Growth Conditions.  Alkaliphilic  Bacillus pseudofirmus  OF4 strain 811M, a methionine auxotrophof   B. pseudofirmus  OF4 (12), was the parent strain for deletionmutants in  atpZ ,  atpI  , and  atpZI  . Routine cloning was performedin  Escherichia coli  DH5  , except for pG  host4 plasmids (Ap-pligene, Pleasanton, CA), which were cloned in  E. coli  XL-1 Blue(Promega). The phenotypic effects of   atpZ ,  atpI  , and  atpZI   wereexamined in a Na   H  and Ca 2   H  antiporter mutant of   E. coli , KNabc (  nhaA   nhaB  chaA) (13), and in the Mg 2  uptakemutant of   Salmonella typhimurium  MM281 (  corA45 ::MudJ  mgtA21 ::MudJ  mgtB10 ::MudJ) (14). Inducible gene expression was carried out in  E. coli  C43(DE3) (15). All plasmid inserts andPCR products were completely sequenced at the Utah StateBiotechnology Center (Logan, UT), or at the DNA Core at theMount Sinai School of Medicine. Ampicillin (Ap) was used at100   g  ml  1 in  E. coli  and  S. typhimurium , and erythromycin(Em) selection was conducted at 250   g  ml  1 in  E. coli . For  B. pseudofirmus  OF4811M in complex or defined media, Em wasused at 0.6   g  ml  1 , and in protoplast regeneration DM3 plates(16), 0.1–0.3   g  ml  1 Em was used.Complementation of the Mg 2  -uptake defect of   S. typhi- murium  MM281 was determined by passing 100-  l portions of overnight cultures (grown in LB   Ap medium plus 100 mMMgSO 4 )thatwerepelletedandresuspendedinLBmedium(withno MgSO 4  or Ap) into 2 ml of test medium (LB   Ap withdifferent concentrations of MgSO 4 ). The tubes were shaken at37°C for 6.5 h, and the absorbance at 600 nm was determined. Alkaliphile strains were grown in semidefined (16) or definedmedia at 30°C, with glucose or malate as the carbon source. Themedia were buffered with 0.1 M Mops-NaOH at pH 7.5, and 0.1M Na 2 CO 3  NaHCO 3  at pH 10.5. The [Na  ] of the pH 7.5 buffer was raised to 0.2 M Na  by the addition of NaCl. In the definedmedia, QA, the yeast extract was omitted, and 0.1% glutamineand 0.1% alanine, 1   g  ml thiamine and biotin, and 10   g  mlmethionine were added. For growth experiments with trans-formed alkaliphile strains, overnight cultures grown in a richmedium at pH 7.5 (16), containing 50   g  ml kanamycin (Km), were diluted 200-fold into defined medium to start the experi-ment. The richer medium overcame deleterious effects of theplasmids on growth. Carryover from this medium resulted in thefaster growth of transformed, but not of untransformed strains,in complementation experiments (Fig. 4  B  vs.  A ). Transcriptional Start Site Mapping.  RNA was isolated as described(17) from late-logarithmic cultures of   B. pseudofirmus  OF4grown in semidefined media at pH 10.5. Reverse transcriptasereactions were carried out by using the SUPERSCRIPT pre- This paper was submitted directly (Track II) to the PNAS office.Abbreviations: Ap, ampicillin; RT, reverse transcription.*D.B.H. and Z.W. contributed equally to this work. † Presentaddress:DepartmentofMedicalOncology,Dana–FarberCancerInstitute,HarvardMedical School, Boston, MA 02115. ‡ Present address: Department of Biology, Babes-Bolyai University, 5–7 Clinicilor Street,3400 Cluj-Napoca, Romania. § To whom correspondence should be addressed at: Department of Pharmacology andBiological Chemistry, P.O. Box 1603, Mount Sinai School of Medicine, 1 Gustave L. LevyPlace, New York, NY 10029. E-mail: terry.krulwich@mssm.edu. www.pnas.org  cgi  doi  10.1073  pnas.1832982100 PNAS    September 2, 2003    vol. 100    no. 18    10213–10218      B     I     O     C     H     E     M     I     S     T     R     Y  amplification system (GIBCO  BRL, Life Technologies, Carls-bad,CA),accordingtothemanufacturer ’ sinstructions.RNA(50  g) and 50 pmol of a primer that had been end-labeled with[ 32 P]ATP by using T4 polynucleotide kinase were used in eachreaction. Two primers were used: 2700R in  atpZ , and 3024R in  atpI   (see Fig. 1  B ). The latter primer corresponds to that used inthe earlier study (17) that mapped the start site to base 2925 (allnumbering is based on the deposited sequence in the GenBankdatabase, accession number AF330160). The reverse transcrip-tase reactions were resolved on an 8 M urea  6% polyacrylamidedenaturing gel (12    16 cm), along with  32 P-labeled 100-bpmarkers. They were further analyzed on DNA sequencing gels,by using a Bio-Rad apparatus, along with a sequencing laddergenerated by using primer 2700R or primer 3024R, and atemplate consisting of the region from 2389 to 3108, which wasobtained by PCR, and cloned into pGEM7Zf(  ). RT-PCRs were carried out by annealing primer 3378R, corresponding tothe 5   end of   atpB , to 1   g of RNA. The reverse transcription(RT) reaction (0.5 – 2   l of 20   l) was then used as the templatefor PCR, by using the HotStarTaq master mix kit (Qiagen,Valencia, CA). Construction of Mutants.  Mutant constructs were made inpG  host4 carrying a temperature-sensitive replicon, and recom-binant plasmids were introduced into the alkaliphile by proto-plast transformation. The general strategy for making the mu-tant construction was the one described by Biswas  et al.  (18), asapplied to alkaliphiles (16, 19, 20).For construction of in-frame    atpZ ,    atpI  , and    atpZI   mu-tations, two sets of PCRs for each mutant were performed on wild-type chromosomal DNA to amplify and introduce appro-priate restriction sites into segments upstream and downstreamof the region to be deleted: nucleotides 2740 – 2889 for   atpZ,nucleotides 2924 – 3292 for  atpI, and nucleotides 2740 – 3292 for  atpZI. Digests of the upstream and downstream pair of PCRproducts were prepared, and then were ligated to the appropri-ately digested low-copy Gram-negative vector pMW118 (NipponGene, Toyama, Japan).  Eco RI digests of each of these plasmids were ligated with  Eco RI-digested pG  host4 to produce a chi-meric plasmid that was used to create each of the deletions in thealkaliphile. A growth temperature of 40 ° C was used duringmanipulations in  E. coli , so that maintenance of the plasmiddependedonthelow-copyrepliconofpMW118,thusminimizingpotential toxicity of the hydrophobic gene products. Cloning of  atpZ  ,  atpI  , and  atpZI  .  A PCR product generated from  B. pseudofirmus  OF4 chromosomal DNA with designed  Bam HIand  Eco RI sites was cloned into pMW118. The product con-tained  atpZ  and 1.3 kb of upstream region.  atpZI   was cloned ina similar way. For the cloning of   atpI  , the same strategy wasfollowed,exceptthatthetemplateforthePCRwaschromosomalDNA from the   atpZ mutant. Expression of   atpI   in this con-struct would be under the control of the native promoter of theoperon. For overexpression of   atpZ , the gene was cloned into the vector pET-3a (Novagen), by using designed  Nde I and  Xho I sitesat the 5   and 3   end of   atpZ , respectively. Everted membrane vesicles were isolated from IPTG-induced cultures of   E. coli C43(DE3) harboring the empty plasmid or pET-3a-  atpZ.  For theexpressionof   atpZ in  B.pseudofirmus OF4,aPCRproduct(usingVent DNA polymerase, New England Biolabs) that contained  atpZ  and 1.3 kb of upstream sequence was blunt-end-cloned intothe shuttle vector pYH56 (16). ATPase and Protein Assays.  Everted membrane vesicles wereprepared from strains grown to mid-logarithmic stage in definedmedia at pH 7.5 and 10.5 with malate as the carbon source, and were assayed for octyl glucoside-stimulated ATPase activity(21). Protein was measured by the Lowry method (22). Assays of  45 Ca 2  Accumulation and Uptake by  E. coli   Transformants.  E. coli  KNabc transformed with pMW118, or the recombinantplasmid with  atpZ ,  atpI  , or  atpZI   were grown overnight in LBK (LB medium containing KCl instead of NaCl) (23) and Ap. Thecells were washed once and were resuspended in 50 mM1,3-bis[tris(hydroxymethyl)methylamino]propane buffer, pH7.5. Three milliliters of cell suspension (  1 mg of protein per ml) were shaken in 15-ml conical tubes at 37 ° C with 100  M  45 CaCl 2 (0.1  Ci  mmol; 1 Ci  37 GBq). At the end of a 1-h incubation, with 1 mM glucose where indicated, 1 ml was removed and vacuum filtered by using 25-mm GF  F glass microfiber filters(Whatman). The air-dried filters were counted by liquid scintil-lation counting. Experimental values were corrected for binding.Binding was assessed in the presence of 5% butanol, whichreleased internalized solute. For  45 Ca 2  uptake assays, right-side-out membrane vesicles were prepared from the same trans-formants of   E. coli  KNabc by the method of Kaback (24) in 10mM Bis-Tris propane, pH 7.5.  45 Ca 2  uptake was measured byadding 100  M  45 CaCl 2  to a 1-ml suspension containing 0.25 mg vesicle protein. At 5-sec intervals during incubation at 37 ° C, the vesicle suspension was rapidly filtered through 0.45-  m HAWPfilters(Millipore)andwashedwith2.5mloftheBis-Trispropanebuffer. Radioactivity was measured by scintillation counting. Fig. 1.  atp  operon and upstream region with proposed promoter elementsof B.pseudofirmus OF4.(  A ) atp operonstructureshowinganupstreamgene, vpr   (an incomplete ORF), and the  atpBEFHAGDC   structural genes, as well asthe  Z  and I  genes that are proposed to be part of the atp transcriptional unit.Thesolidarrowindicatestheproposedpromoterfromthisarticle,thedottedarrow is the previously described promoter (17), and the upward arrowheadindicates the site of insertion of a spectinomycin-resistance cassette in apreliminary experiment that led to a malate-negative phenotype, and henceto this article. ( Upper  ) The bar is marked in kb. Below the operon is anexpansionoftheregionfrom  2to5kbthatshowstheregionsdeletedinthemutants constructed for this article. ( B ) The sequence from bases 2541 – 3052.The primers used in the transcriptional start mapping (3024R and 2700R) areshown.Thestartsitefromtheearlierstudyisshownwithadownwarddottedarrow (base 2925), and the new start site is indicated with a downward solidarrow (base 2613). The proposed  35 promoter and  10 promoter elementsare bold. The primers used in conjunction with primer 3378R in the RT-PCR ofFig. 2 B  are also shown (2568F and 2619F). The one-letter amino acid desig-nations are shown above ( atpZ  ) or below ( atpI  ) their codons. 10214    www.pnas.org  cgi  doi  10.1073  pnas.1832982100 Hicks  et al.  Western Analysis. Everted membrane vesicles were prepared from  E. coli  transformants grown on LB   Ap medium, and the indi-cated alkaliphile strains grown on pH 7.5 QA media, with malateand 50  g  ml kanamycin. The vesicles, denatured in SDS samplebuffer, were resolved on mini-SDS  12% polyacrylamide gels(25). After transfer to nitrocellulose membranes, Western blots were developed by a chemiluminescence protocol (AmershamPharmacia Biosciences, Piscataway, NJ), according to manufac-turer ’ s recommendations. A synthetic peptide was prepared thatcorresponded to the last 15 amino acids of AtpZ, with anadditional cysteine at the N terminus. The peptide, conjugatedto keyhole limpet hemocyanin, was injected in rabbits to raisepolyclonal antisera (Covance Research Products, Denver, PA),from which the IgG fraction was purified (26), and used for theseanalyses. Results Reexamination of the Transcriptional Start and Probable Promoter ofthe  atp   Operon.  Introduction of a cassette in the chromosomalregion just upstream of   atpI  , in the position within  atpZ indicatedby the arrowhead in Fig. 1  A , resulted in a malate-minus growthphenotype, and loss of ATPase expression in  B. pseudofirmus OF4, even though the promoter had earlier been predicted to bedownstream of the cassette-insertion site, just upstream of   atpI  (17). To reexamine that promoter identification, primer exten-sion was carried out with the same primer, 3024R, that was usedin the earlier work (17). This primer sequence is in  atpI  , as shownin Fig. 1  B . The product of the RT reaction was electrophoresedon a polyacrylamide  urea gel that enabled approximate sizing of the product from  50 to 500 – 600 nucleotides, a broader rangethan would have been detected in the earlier experiments (17).Two products of the reaction were visualized: one that corre-sponded approximately to the size srcinally determined, which was 85 nucleotides, and a second product of   400 nucleotides.To precisely size the larger product by primer extension, it wasdesirable to use a primer upstream of 3024R, i.e., 2700R. Theresulting smaller reverse transcriptase product was determinedto be 67 or 68 nucleotides on a 6% polyacrylamide  ureasequencing gel (data not shown). This result indicated that thetranscriptional start site is base 2613 or 2614 of the depositedsequence, which is 293 (292) nucleotides upstream of the pre-dicted start codon of   atpI  . The smaller product observed with the3024R primer presumably resulted from either premature ter-mination of the RT reaction, because of RNA secondary struc-ture, or from a processed  atp  transcript fragment. Additional support for an  atp  transcriptional start near base2613 was obtained by RT-PCR. The reverse transcriptase reac-tion was carried out by using a primer corresponding to asequence from the 5   end of   atpB  (3378R), which was the first ATPase structural gene in the operon. The RT product was thensubjected to PCR, by using two sets of primers. For the first set,3378R was used with 2619F, whose 5   end is six nucleotidesdownstream of the proposed start site. In the second set, 3378R was used with 2568F, whose 3  end is 25 nucleotides upstream of the start site (Fig. 1  B ). As shown in Fig. 2  A , a product of the rightsize, 760 base pairs, was observed for the wild-type with the set1 primers, whereas no product was generated from primer set 2.The set 1 product was absent when the RT enzyme was omittedfrom the RT reaction, indicating that the observed product wasnot due to DNA contamination of the RNA preparation.RT-PCR was also carried out on RNA from the   atpI   mutant,and similar results were obtained, except that the size of the set1 product was approximately the predicted size of 392 base pairs,taking into account the deleted region.The suggested promoter sequence shown in Fig. 1  B  has theconsensus sequence for the  Bacillus subtilis   35 element, TT-GACA, and a  10 element, TACGAT, which is an infrequentlyused element in  B. subtilis  (27). The transcriptional start site isfour nucleotides upstream of the predicted start codon of   atpZ (National Center for Biotechnology Information ORF finder), aputative ORF upstream of   atpI   that had not earlier beenconsidered part of the operon. A more likely start for AtpZ isthat shown in Fig. 1  B , which is based on the presence of apotential ribosome-binding site, and the size of the productdetected in Western blots by antibodies raised against a syntheticpeptide corresponding to the C-terminal sequence of AtpZ (seeFig. 2  B ). An alignment of several possible homologues of AtpZfrom  Bacillus  species is shown in Fig. 2 C , and additional homo- Fig. 2.  Demonstration of a promoter upstream of  atpZ   and of an AtpZprotein.(  A )RT-PCRwascarriedoutbyusingprimer3378RfortheRTreaction.The reaction was subjected to PCR by using 3378R, with either primer 2568F(set 1) or 2619F (set 2; see Fig. 1 B  for precise locations of these primers). Thereversetranscriptaseenzymewasleftout(  )orincluded(  )intheRTreactionasindicated.ThecontrollanesshowtheproductgeneratedfromPCRwiththeappropriate primer pairs, with chromosomal DNA as the template. The stan-dards are labeled in base pairs. Below the gel is a diagram showing thelocation of the primers, with the reverse primer in  atpB , and the forwardprimers either just after the proposed start site (set 1) or before the start site(set 2), as indicated by the numbered arrows. ( B ) Western blot of  atpZ  expression in  E. coli   and  B. pseudo fi  rmus  OF4. In  E. coli  , everted membranevesicles were isolated from induced cultures of strains with the empty vector,or with the cloned  atpZ  . In  B. pseudo fi  rmus  OF4, everted membrane vesicleswere prepared from the wild-type carrying pYH56 (empty vector), or  atpZ  carrying the empty vector, or with cloned  atpZ  . Lane 1,  E. coli   with emptyvector(0.25  g);lane2, E.coli  with atpZ  (0.25  g);lane3, B.pseudo fi  rmus OF4wild-type with empty vector (100   g); lane 4,  atpZ   with empty vector (100  g);lane5,  atpZ  with atpZ  (100  g).TheAtpZbandismarkedontheleft,andthe other bands represent nonspeci fi c reactions. Molecular weight markersare indicated on the left. ( C  ) Alignment of AtpZ homologues. Boxed residuesare charged amino acids shared by three of four of the candidate proteins.Otherresiduessharedbythreeorfourproteinsareshaded.Thetwopredictedtransmembrane helices (TMS) are overlined. Bps,  B. pseudo fi  rmus  OF4; Bh, Bacillus halodurans  C-125; Bm,  Bacillus megaterium ; Ba,  Bacillus anthracis .According to the National Center for Biotechnology Information ORF fi nder,the  B. anthracis  candidate has an additional 10 residues at the N terminus forwhichnostrongribosomebindingsite(RBS)canbeidenti fi ed;thereisagoodRBS for the leucine codon that lines up with the  fi rst amino acid of  B. pseudo fi  rmus OF4AtpZ.The B.megaterium candidateshownisanincompleteORF with at least 14 more amino acids at the N terminus. In this alignment,these extended segments have not been included. Hicks  et al.  PNAS    September 2, 2003    vol. 100    no. 18    10215      B     I     O     C     H     E     M     I     S     T     R     Y  logues are found in the database entries for genomes of   Clos-tridium difficile  and  Desulfitobacterium hafniense . Detection of AtpZ.  Initial assessments of whether  atpZ  actuallyproduces a protein product were conducted by Western blotanalysis of membranes from wild-type  B. pseudofirmus  OF4grown in semidefined malate medium at pH 7.5 and 10.5, but no AtpZwasdetected.Apparentlytheantibodycouldnotdetectthelow levels of AtpZ found in such cells. Therefore,  atpZ  wascloned into the plasmid pET-3a behind an IPTG-induciblepromoter, by using the start codon deduced from this article. Asshown in Fig. 2  B , AtpZ was expressed at high levels in  E. coli C43(DE3) (lane 2); the negative control lacked a reactiveproduct of the same size.  atpZ  was also expressed in thealkaliphile   atpZ  mutant transformed with recombinant pYH56containing the  atpZ  coding sequence, and 1 kb of upstreamsequence. A band was observed that was the same size as thatseen in  E. coli  (lane 5), supporting the conclusion that the actualstart site corresponds to that shown in Fig. 1  B . No AtpZ signal wasdetectedfromeitherthe   atpZ ,orfromthewild-typestrainsharboring the empty plasmid (lanes 3 and 4). Phenotype of Deletion Mutants.  The    atpZ  and    atpI   mutantstrains of   B. pseudofirmus  OF4 grew like the wild-type strain onsemidefined media, with either malate or glucose as the carbonsource. The specific ATPase activities in membrane vesiclesfrom    atpZ  and    atpI   were similar to the wild-type (data notshown), which was consistent with the nonfermentative growthon malate. This result confirmed the nonpolar nature of thedeletions. Little growth deficit was observed on defined mediumat pH 10.5, ruling out a specific role of   atpZ  or  atpI   in alkaliphily.However, the mutant strains showed a pronounced growthdeficit when grown in a defined medium at pH 7.5. Recentstudies (28 – 31) suggest a role in Mg 2  transport for severalputative 2 TMS proteins. This finding led us to consider thepossibility that one or both of the first  atp  operon genes wasinvolved in Mg 2  acquisition. Because of the poor solubility of Mg 2  in high-pH settings, the alkaliphile is likely to requirespecial Mg 2  acquisition mechanisms for growth at pH 10.5, butmight have a lower overall capacity for this important functionatpH7.5.AsshowninFig.3  A ,allthreedeletionstrainsexhibiteda greatly increased requirement for Mg 2  in malate-containingQA-MopsmediaatpH7.5.Althoughnotshown,agrowthdeficit was also observed when glucose was the primary carbon sourceinstead of malate. As shown in Fig. 3  B , transformation of the   atpZ  strain with a plasmid-bearing  atpZ  resulted in nearly wild-type levels of growth over a broad range of MgSO 4  con-centrations. There were differences in pregrowth conditions andMg 2  carryover (see  Materials and Methods ), which were neces-sitated by effects of plasmids on growth. This finding led to theapparent ability of the wild-type transformant and comple-mented mutant to grow at lower Mg 2  concentrations than theuntransformed wild-type shown in Fig. 3  A . Effects of  atpZ  ,  atpI  , or  atpZI   Expression on the Phenotypes ofHeterologous Transport Mutants.  S. typhimurium  MM281 is defi-cient in Mg 2  uptake by virtue of disruptions in the genes forthree different transport systems, CorA, MgtA, and MgtB (14);complementation of this strain has proved useful in identifyingand developing information about new, heterologous Mg 2  transporters (32, 33). As shown in Fig. 4, low-copy plasmidsexpressing  atpZ ,  atpI  , or  atpZI   complemented the growth phe-notype of   S. typhimurium  MM281. The AtpZI combinationsupported significantly greater growth of the mutant on con-taminating and low added concentrations of Mg 2  than dideither AtpI or AtpZ alone.Because of the lack of availability of radioactive isotopes of Mg 2  , assays of Mg 2  transport are usually performed as theMg 2  -dependent inhibition of transport of other divalent cat-ions. This method was used to characterize the MgtE Mg 2  transporter of   B. pseudofirmus  OF4 (32), which exhibitedMg 2  -inhibitable uptake of   57 Co 2  in  S. typhimurium  MM281transformants. However, no evidence for AtpZ-, AtpI-, or AtpZI-dependent Co 2  uptake was obtained, although suchactivity was conferred by the positive control plasmid in whichthe alkaliphile  mgtE  gene was cloned (data not shown). Becausecrossinhibition occurs between Mg 2  and Ca 2   with sometransporters (34), the possibility that AtpZ and AtpI mightconfer Mg 2  -inhibitable  45 Ca 2  accumulation was assessed in  E. coli  KNabc, which is deficient in Ca 2  efflux (13). As shown inTable 1 for a 1-h accumulation experiment, the control trans-formant (cells harboring the empty vector) exhibited littleMg 2  -inhibitable  45 Ca 2  accumulation. The transformant ex-pressing  atpI  exhibitedasignificantincreaseinactivityonlywhen Fig. 3.  Effect of [MgSO 4 ] on the growth of deletion strains of  B. pseudo fi  r-mus OF4.(  A )TheMgSO 4 concentrationwasvariedforovernightgrowthinthepH 7.5 QA – Mops-de fi ned media described in  Materials and Methods . ( B )Growthoftheindicatedtransformantsofwild-type B.pseudo fi  rmus OF4andthe  atpZ   mutant was monitored as in  A . The enhanced response of thewild-type with the control vector to low [MgSO 4 ] was due to a carryover ofMg 2  from the richer medium used for the pregrowth of transformed alka-liphilestrains,asdescribedin MaterialsandMethods .Theresultsarefromtwoindependent experiments, each carried out in duplicate. Fig.4.  ComplementationoftheMg 2  phenotypeof S.typhimurium MM281by a tpZ  ,  atpI  , and  atpZI  .  S. typhimurium  MM281 transformants were grownfor 6.5 h in LB  Ap medium, supplemented with the indicated concentrationsofMgSO 4 asdescribedin MaterialsandMethods .TheaveragesandSDof fi veindependent growth experiments, each done in duplicate, are shown. 10216    www.pnas.org  cgi  doi  10.1073  pnas.1832982100 Hicks  et al.  glucose was also added, but not as much as the transformantsexpressing  atpZ  and  atpZI  , both of which exhibited enhancedaccumulation even in the absence of glucose. In followupexperiments, right-side-out membrane vesicles from the sametransformants were assayed for  45 Ca 2  uptake in the absence of any intravesicular solute. Initial experiments showed that uptake was extremely rapid, such that the highest uptake was observedat the shortest time points that could be taken (5 sec). Subse-quent time points indicated a loss of   45 Ca 2  from the vesicles, as would be expected under unenergized conditions. Addition of anelectron donor, D-lactate, did not cause more sustained uptake,but instead inhibited uptake overall. This result was presumed toreflect energization of the remaining Ca 2  efflux pathway(s), which were expected to be present in the  chaA  deletion back-ground of   E. coli  KNabc (35). Subsequent vesicle experiments were conducted in the absence of added electron donors at 5-secpoints. As shown in Table 2, AtpZ-, AtpI- and AtpZI-dependent 45 Ca 2  uptake was observed. This uptake, after subtracting thebackground binding and uptake in the control transformant, wascompletely inhibited by addition of either 1 mM MgCl 2  or 200  M of the general cation channel inhibitor GdCl 3  (36) to theextravesicular space, just before the addition of   45 Ca 2  to startthe reaction. Discussion The results indicate that a tenth gene, designated  atpZ , is part of the  B. pseudofirmus  OF4  atp  operon. Because  atpZ  homologuesare found in a comparable position in  atp  operons of severalnonalkaliphilic  Bacillus  species, as well as  C. difficile  and  D. hafniense , the indications that AtpZ and AtpI have a role indivalent cation translocation may have broad applicability. Inaddition to the phenotypes of the  atpZ, atpI  , and  atpZI   mutantsof the alkaliphile, the enhancement of Mg 2  acquisition in theMg 2  uptake-deficient mutant strain,  S. typhimurium  MM281,and of Mg 2  -inhibitable  45 Ca 2  uptake in  E. coli  KNabc, arefurther evidence that AtpZ and AtpI support inward Mg 2  transport. In the whole-cell experiments in the heterologoussystems, the relative effectiveness of the alkaliphile genes was  atpZI     atpZ    atpI   in enhancing both growth of the Mg 2  uptake mutant of   S. typhimurium  and the Mg 2  -sensitive accu-mulation of Ca 2  in  E. coli . Only in the vesicle uptake experi-ments did  atpI   seem to confer higher activity than the othergenes, with  atpZI   now conferring the lowest. The likely expla-nation for this deviation from the whole-cell experiments is thatthe measurements in vesicles were too slow to capture the peakpoints of the fastest transporters. The faster the initial transport,the further the 5-sec assay point was down the slope of thesubsequent loss of internalized  45 Ca 2  . Such a loss would reflectan expected reequilibration of the Ca 2  in the absence of animposed potential.Different properties conferred by AtpZ, AtpI, and AtpZIsuggest that these molecules constitute the translocation path- way themselves, rather than acting through an indirect mecha-nism, by affecting some independent transporter in the cells. AtpZ and AtpI would be likely to function as homooligomersand, perhaps, AtpZ and AtpI can also form heterooligomers.Homooligomeric assemblies of CorA (37), and the 2 TMS ALR1and Mrs2 transporters of yeast (30, 38), have been reported, andmay function as channels. Although no conclusion can yet bedrawn about the transport mechanism of AtpZ and AtpI, theproperties observed thus far raise the possibility of a cationchannel.The unusual and diverse nature of the proteins to which aMg 2  transport function has been ascribed is notable (39). Thecurrent findings add to that diversity. Neither AtpZ nor AtpIhas the conserved motif of the CorA family of Mg 2  trans-porters (39) and the 2-TMS Mrs2-type transporters (28 – 31),nor do they show sequence similarity to other putative Mg 2  transporters such as MgtE, which was first isolated from analkaliphilic  B. pseudofirmus  DNA library (32). The CorA family possesses a putative magnesium transporter signature,Y  FGMNF, which is critical for activity (30, 31, 37). There isno evident counterpart in AtpZ or AtpI. We note, however,that the AtpZ proteins of two alkaliphilic  Bacillus  speciespossess three contiguous acidic residues at their C terminus. A discontinuous DDE motif has been shown to function indivalent metal binding (40), and contiguous acidic residueshave been associated with Ca 2  binding and translocatingproteins (34, 41).Coexpression of a cation translocation pathway with theF-ATPase from a single operon is a functionally satisfyingcombination. The nucleotide substrates for the enzyme areusually complexed with Mg 2  , and the cation has been sug-gested to play a role in establishing the asymmetry of thecatalytic sites (42). In addition, when the ATPase is function-ing hydrolytically to lower the cytoplasmic pH (43), a capacityof AtpZI, AtpZ, or AtpI to support cation flux could allowcharge-compensating, inward movement of cations that wouldmaximize the proton efflux. AtpI gene products from differentorganisms do not exhibit strong sequence conservation, andare less conserved than are deduced AtpZ sequences. Perhapsinactivation, loss, or modifications in AtpZ and AtpI function will ultimately be found to relate to different physiologicalneeds of diverse organisms.It is expected that extremely alkaliphilic bacteria require well adapted mechanisms for acquiring needed concentrationsof ions such as Fe 2  and Mg 2  , when growing at pH values ashigh as 10.5. The transporter complement encoded in thegenome of alkaliphilic  Bacillus haloduran s C-125 is predictedto include four distinct MgtE-like proteins (www.membrane- Table 1. AtpZ-, AtpI-, and AtpZI-enhanced  45 Ca 2  accumulationby transformants of  Escherichia coli   KNabc Transformedwith plasmid 45 Ca 2  accumulation, nmol  mg of cell protein*  Glucose   GlucoseNo add   MgCl 2  No add   MgCl 2 pMW118 2.1  0.3 2.4  0.4 2.8  0.1 1.9  0.2pMW- atpZ   4.5  0.8 2.3  0.4 11.8  0.1 1.9  0.1pMW- atpI   2.1  0.2 2.2  0.2 7.0  0.1 2.7  0.2pMW- atpZI   11.0  0.1 2.2  0.2 16.3  0.2 2.2  0.1 *Transformant cells were incubated with 100   M  45 Ca 2  for 1 h, with orwithout1mMMgCl 2 ,afterwhichtheaccumulationof 45 Ca 2  wasassayedasdescribed in  Materials and Methods . All values are corrected for bindingcontrols, which were carried out by butanol treatment. The results are theaverages of six independent experiments conducted in duplicate and areshown  SD. No add, no MgCl 2  added. Table 2. AtpZ-, AtpI-, and AtpZI-dependent  45 Ca 2  uptake inright-side-out membrane vesicles of  Escherichia coli   KNabc Vesicles fromtransformantswith plasmid 45 Ca 2  uptake, nmol  5 sec per mg of vesicle protein*No add   1 mM MgCl 2   200   M GdCl 3 pMW- atpZ   8.5  1.3 0.5  0.1 0.8  0.2pMW- atpI   9.5  1.5 0  0.2 0.8  0.2pMW- atpZI   6.6  1.1 0.1  0.2 0.6  0.2 *The values shown were corrected for the  45 Ca 2  bound and taken up by thevesicles from the control vector (pMW118) transformant. The values sub-tracted were 18.8  1.6, No add; 12.8  1.2,  1 mM MgCl 2 ; and 6.0  0.6,  GdCl 3 . The results, shown with SD, are the averages from six independentvesicle experiments assayed in duplicate. No add, no MgCl 2  added. Hicks  et al.  PNAS    September 2, 2003    vol. 100    no. 18    10217      B     I     O     C     H     E     M     I     S     T     R     Y
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