Alkalophilic Bacillus firmus RAB generates variants which can grow at lower Na+ concentrations than the parental strain.

Obligately alkalophilic Bacillus firmus RAB cannot grow well on mediacontaining less than 5 mM Na+. However, variant strains canbeisolated on plates containing 2to3mM Na+.Thesevariants areobserved only rarely incultures that areplated before being subjected torepeated transfers inliquid medium.Cultures which havebeentransferred several times produce variants atan apparent frequency of2 x 10-4. Mostofthese variants areunstable, generating parental types atthehighfrequency of10%;however, stable variants can be isolated. These strains grow better thantheparental strain atveryhighpHvalues inthepresenceof5mM Na+ andhaveenhanced activity oftheNa+-H+antiporter that hasbeenimplicated inpHhomeostasis. Bycontrast, Na+-coupled solute uptake isindistinguishable fromthat oftheparental strain, andnoobvious changes inthe respiratory chain components areapparent inreduced versusoxidized difference spectra. Themembranes of thevariants showamarkedenhancement, onsodium dodecyl sulfate-polyacrylamide gradient electrophoresis, inonepolypeptide bandwith amolecular weight intherangeof90,000. Thefindings arediscussed fromthe point ofviewofgenetic mechanisms thatmight confer adaptability toevenmore extreme environments than usual andinviewofearlier models relating theNa+-translocating activities ofthealkalophiles. Thealkalophilic bacilli areofspecial bioenergetic interest bothbecause oftheir primary problem ofmaintaining a cytoplasmic pHthat ismuchlower thanthat oftheexternal milieu andbecause ofsecondary energetic problems that result fromthis necessity (11, 12). Clarification oftheproperties thatrelate toextremealkalophily willincreasingly require genetic approaches sothat thenumberofgenes and their products canbedefinitively assessed. Genesfrom alkalophilic bacilli havebeensuccessfully cloned andexpressed inEscherichia coli byHorikoshi andhiscolleagues (14, 20), andthere isareport, that awaits follow-up, ofthe transformation ofBacillus subtilis toa morealkalophilic phenotype withDNA fromanalkalophile (24). As yet, however, nogenetic transformations ofthealkalophiles themselves havebeenreported. Amongourattempts toachieve suchtransformations, we havetried touseDNA fromonealkalophilic species, Bacillusalcalophilus, totransform another alkalophilic species, Bacillus firmus RAB.Initial attempts werefocused upona difference intheNa+requirements ofthetwospecies. B. alcalophilus growswellwithout addedNa+aslong asno special precautions aretaken toavoid normal contaminating Na+levels. B.firmus RAB,bycontrast, isamoretypical alkalophile inrequiring substantial Na+,intherange of25 mM,foroptimal growth (5). Bothspecies apparently depend ontheactivity ofaNa+-H+antiporter tocatalyze acidificationofthecytoplasm relative totheexterior, butthe antiporter fromB.alcalophilus hasamuchhigher affinity for Na+thanthatfromB.firmus RAB (4,13). Attempts were accordingly madetotransform thelatter species toa"higher-Na+-affinity" phenotype withDNA fromB.alcalophilus. Screening wascarried outonplates containing mediumthat contained 2to3mM Na+,levels that wouldnotpermit the formation ofdiscrete colonies ofB.firmus RABwithin 48h. During thecourse oftheexperiments, however, itbecame clear thatoccasional cells ofB.firmus RAB cangrowata