Halotolerant Cyanobacterium Aphanothece halophytica Contains an Na+-dependent F1F0-ATP Synthase with a Potential Role in Salt-stress Tolerance

Abstract

Aphanothecehalophyticaisahalotolerantalkaliphiliccyanobacteriumthatcangrowinmediaofupto3.0MNaClandpH11. Here,weshowthatinadditiontoatypicalH -ATPsynthase, Aphanothece halophytica contains a putative F1F0-type Na ATP synthase (ApNa+-ATPase) operon (ApNa+-atp). The operonconsistsofninegenesorganizedintheorderofputative subunits , ,I,hypotheticalprotein,a,c,b, ,and .Homologousoperonscouldalsobefoundinsomecyanobacteriasuch as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa -atp operon was isolated from the A.halophyticagenomeandtransferredintoanEscherichiacoli mutant DK8 ( atp) deficient in ATP synthase. The inverted membrane vesicles of E.coli DK8 expressing ApNa -ATPase exhibited Na -dependent ATP hydrolysis activity, which was inhibitedbymonensinandtributyltinchloride,butnotbythe protonophore,carbonylcyanidem-chlorophenylhydrazone (CCCP). The Na ion protected the inhibition of ApNa ATPasebyN,N -dicyclohexylcarbodiimide.TheATPsynthesis activitywasalsoobservedusingtheNa -loadedinvertedmembranevesicles.ExpressionoftheApNa -atpoperonintheheterologouscyanobacteriumSynechococcussp.PCC7942showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A.halophytica has additional Na -dependent F1F0-ATPase in thecytoplasmicmembraneplayingapotentialroleinsalt-stress tolerance.