ABSTRACT The transcription of nhaA, encoding the main Na+/H+ antiporter of Escherichia coli, is induced by Na+, regulated by NhaR, and affected by H-NS. In this work the roles of the two nhaApromoters (P1 and P2) were studied by analysis of transcription both in vivo and in vitro and promoter mutations. We found that P1 is an NhaR-dependent, Na+-induced, and H-NS-affected promoter both in the exponential and stationary phases. An in vitro transcription assay demonstrated that P1 is activated by ς70-RNA polymerase and both NhaR and H-NS increase the specificity of P1. Remarkably, in marked contrast to P1, P2 exhibits very low activity during the exponential phase but is induced in the stationary phase to become the major promoter. Furthermore, P2 is activated by ςS and is neither induced by Na+nor dependent on NhaR or affected by H-NS. Hence, this work establishes that nhaA has a dual mode of regulation, each involving a different promoter, and reveals that P2 and ςS together are responsible for the survival of stationary-phase cells in the presence of high Na+, alkaline pH, and the combination of high Na+ and alkaline pH, the most stressful condition.

Na+/H+ antiporters are membrane proteins that play a major role in pH and Na+ homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na+/H+ antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na+/H+ antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 Å resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na+/H+ antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII–IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na+/H+ antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1–G11). The transcription of NhaA has been shown to be regulated by a novel Na+-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.

The NhaA Na+/H+ antiporter is the main system responsible for adaptation to Na+ and alkaline pH (in the presence of Na+) in Escherichia coli and many other enteric bacteria. It is under intricate control. At the protein level it is regulated directly by pH, one of its regulatory signals. A pH shift from 7 to 8.5 activates the antiporter and, in a fashion correlated with the activity change, confers a conformation change that, in isolated membrane vesicles, is reflected in the exposure of trypsin-cleavable sites. H225 and G338 are essential for the pH response of NhaA. nhaA transcription is dependent on NhaR, a positive regulator of the LysR family, and is regulated by Na+, the other environmental signal. Na+ affects the NhaR/nhaA interaction directly by changing the footprint of NhaR on nhaA in a pH-dependent fashion. The expression of nhaA is also under global regulation of H-NS. We suggest that the pattern of regulation of nhaA found in E. coli is a paradigm for the response of proteins and genes to H+ and Na+, the most common ions that challenge every cell.

We used partially purified NhaR and a highly purified His‐tagged NhaR derivative to identify the cis‐regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR‐binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G−92, G−60, G−29 and A−24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a ΔnhaR strain; accordingly, these bases were protected in vitro by the purified His‐tagged NhaR. 100 mM Na+, but not K+, removed the protection of G−60 conferred by His‐tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G−60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G−60, G−92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR–nhaA contact points.

H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has been shown to exhibit a binding preference for certain AT-rich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo, In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A K-d of 1-2 mu M was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.

nhaAencodes an Na 1 /H 1 antiporter inEscherichia coliwhich is essential for adaptation to high salinity and alkaline pH in the presence of Na 1 . We used Northern (RNA) analysis to measure directly the cellular levels ofnhaAmRNA.NhaRbelongstotheLysRfamilyofregulatoryproteins.Consistentwithourpreviousdatawith annhaA*-*lacZfusion, NhaR was found to be a positive regulator and Na 1 was found to be a specific inducer ofnhaAtranscription. In thenhaA*-*lacZfusion, maximal induction was observed at alkaline pH. In contrast, in thenhaA 1 strain both the level ofnhaAexpression and the induction ratio were lower at alkaline pH. This differencemaybeduetotheactivityofNhaAinthewild-typestrainasNhaAefficientlyexcretedNa 1 atalkaline pHandreducedtheintracellularconcentrationofNa 1 ,thesignalforinduction.Wealsoshowedthatalthough the global regulatorrpoSwas not involved innhaAregulation, the global regulatorhnsplayed a role. Thus, the expression of nhaA*-*lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carryinghnsrepressed expression and restored Na 1 induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns 1 background acted only as an Na 1 -dependent positive regulator, acted as a repressor in anhnsstrain in the absence of Na 1 but

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 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.