Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus 0F4 encoding an ABC-type transporter with similarityof sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridizat ion to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revea led that, as in B. subtilis, the natA gene was part of a putative gene locu s encoding an ABC-type transporter. In the alkaliphile, the transporter inv olved three genes, designated natCAB, that are part of a larger operon of u nknown function. This is in contrast to the two-gene natAB operon and to an other homolog from B. subtilis, the yhaQP genes. Like natAB, however, the a lkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escher ichia coli that is deficient in Naf extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart f rom natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malk , an ATP-binding component that was unaccompanied by candidates for other m al transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporte r were found in the fragment, either from the sequence analysis or from ana lyses of complementation of E. coli mutants by subclones of the 14.1-kb pie ce. There were a total of 12 ORFs whose closest and significant homologs we re genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B . subtilis homologs.