nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+. We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA. NhaR belongs to the LysR family of regulatory proteins. Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription. In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH. In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH. This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction. We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role. Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion. Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested.

The Na ⍣ -specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na ⍣ /H ⍣ antiporter of Escherichia coli et al., 1994) Northern analysis of nhaA mRNA (Dover et al., 1996)

Na+/H+ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A delta nhaA strain, which is sensitive to Na+ and Li+, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na+/H+ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activated by alkaline pH and recognizes Li+ with high affinity.

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The mutation nhaAup (antup) has now been identified as a Glu134 to Ala substitution in NhaR and designated nhaR1. This was demonstrated by sequence analysis showing that the mutant contains a wild‐type nhaA but nhaR1 instead of nhaR and by the finding that nhaR1 cloned in a plasmid confers the NhaAup phenotype. Na+ (107 mM) increases by 5‐ to 10‐fold the level of nhaA transcripts, similar to the effect on the NhaR‐mediated expression of a nhaA‘‐’lacZ fusion. These results are in agreement with the notion that nhaR is a positive regulator which controls Na(+)‐dependent transcription of nhaA. The promoter region of nhaR and nhaR1 was found to reside within the BglII‐BamHI fragment of the C‐terminal sequences of nhaA. The mutation nhaR1, while increasing dramatically the level of transcription, reduces the requirement for Na+ by 3‐ to 5‐fold both for nhaA transcription and for the nhaR1‐mediated expression of nhaA‘‐’lacZ fusion. NhaR1, like NhaR, binds specifically to the promoter region of nhaA. However, at equal protein concentration NhaR1 binds more DNA and the NhaR1‐DNA complex shows higher mobility than that of NhaR‐DNA, suggesting the existence of two different binding complexes. Yet in this assay the DNA binding pattern of neither NhaR nor NhaR1 was affected by the addition of Na+. The possible relevance of these two DNA‐binding complexes to the Na(+)‐induced NhaR‐mediated expression is discussed.