Promoter analysis of the sodium-responsive V-ATPase (ntp) operon in Enterococcus hirae

Abstract.

The Enterococcus hirae ntp operon encodes all subunits of the vacuolar-type ATPase (V-ATPase), which transports Na⁺ or Li⁺. This operon is expressed preferentially in response to Na⁺, but not to Li⁺. Deletion analysis of the ntp promoter region in plasmids indicated that the AT-tract between –198 and –132 is required for Na⁺-specific transcriptional regulation. In addition, lithium-tolerant (LTR) mutants were isolated in which functional V-ATPase levels were high even in Na⁺-depleted medium. Western blot and Northern blot experiments revealed an increase in basal Na⁺-independent transcription in one of the mutants (LTR1). The nucleotide sequences of the ntp promoter region of the LTR mutants showed mutational conversion of single base-pairs between positions –23 and +1. Na⁺-independent expression of a reporter gene linked to the ntp promoter in plasmids was elevated by base substitutions at –23 to +1, and promoter activity induced by these base substitutions was lost by deletion of the region between –198 and –132. These results suggest that the AT-tract between –198 and –132 is indispensable for transcription of the ntp operon.