Sodium ion binding proteins

The present invention relates to the cloning and expression of a sodium ion binding protein. In particular, the invention relates to cloning and expression of a nhaS gene. The nhaS gene product, NhaS, is a protein characterized by binding to and sequestering sodium ion (Na.sup.+). The invention further relates to functional fragments of a sodium ion binding protein, which fragments are characterized by their ability to bind to sodium ion. In a specific embodiment, the fragment is a fragment of NhaS. The gene encoding the sodium binding protein can be introduced into cells to produce desalination bioreactors. The gene encoding the sodium binding protein can also be introduced into plants as a transgene to produce plants that are resistant to sodium. The sodium binding protein itself may be used for treatments involving Na.sup.+ /K.sup.+ ATPase disorders, e.g., in heart disease; the protein also may be introduced parenterally, preferably orally, to bind to and sequester dietary sodium.

TABLE OF CONTENTS Page 
1. FIELD OF THE INVENTION 
2. BACKGROUND OF THE INVENTION 
3. SUMMARY OF THE INVENTION 
4. BRIEF DESCRIPTION OF THE DRAWINGS 
5. DETAILED DESCRIPTION OF THE INVENTION 
5.1. THE NhaS CODING SEQUENCE 
5.2. EXPRESSION OF THE NhaS 
5.2.1. EXPRESSION SYSTEMS 
5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS THAT EXPRESS THE 
NhaS 
5.2.3. RECOVERY OF THE NhaS PROTEIN 
5.3. GENERATION OF ANTIBODIES THAT DEFINE THE NhaS 
5.4. USES OF THE NhaS: SALT TOLERANT PLANTS AND ENGINEERED CELL LINES 
6. EXAMPLE: CLONING AND EXPRESSION OF NhaS, A SODIUM ION BINDING PROTEIN 
6.1. MATERIALS AND METHODS 
6.1.1. BACTERIAL STRAINS AND PLASMIDS 
6.1.2. DNA SEQUENCING 
6.1.3. ASSAYS OF Na.sup.+ /H.sup.+ ANTIPORT AND Na.sup.+ BINDING 
6.2. RESULTS 
1. FIELD OF THE INVENTION 
The present invention relates to the cloning and expression of a sodium ion 
binding protein. In particular, the invention relates to cloning and 
expression of a nhaS gene. The nhaS gene product, NhaS, is a protein 
characterized by binding to and sequestering sodium ion (Na.sup.+). The 
invention further relates to functional fragments of a sodium ion binding 
protein, which fragments are characterized by their ability to bind to 
sodium ion. In a specific embodiment, the fragment is a fragment of NhaS. 
The gene encoding the sodium binding protein can be introduced into cells 
to produce desalination bioreactors. The gene encoding the sodium binding 
protein can also be introduced into plants as a transgene to produce 
plants that are resistant to sodium. The sodium binding protein itself may 
be used for treatments involving Na.sup.+ /K.sup.+ ATPase disorders, e.g., 
in heart disease; the protein also may be introduced parenterally, 
preferably orally, to bind to and sequester dietary sodium. 
2. BACKGROUND OF THE INVENTION 
Na.sup.+ /H.sup.+ antiporters are ubiquitous in living cells and have been 
assigned a large variety of important functions (Boron, W. F., & Boulpaep, 
E. L. (1983) J. Gen. Physiol. 81, 29-52; Krulwich, T. A. (1983) Biochim. 
Biophys. Acta 726, 245-264; Aronson, P. S. (1985) Annu. Rev. Physiol. 47, 
545-560; Grinstein, S., ed. (1988) Na.sup.+/H.sup.+ Exchange CRC Press, 
Boca Raton, Fla.), the most straight-forward being the regulation of the 
cytoplasmic level of Na.sup.+. The antiporters are integral membrane 
proteins that carry out either electroneutral or electrogenic exchange of 
Na.sup.+ for H.sup.+ that is driven by primary ion translocation events. 
Several genes encoding eukaryotic (Sardet, C. Franchi, A., & Pouyssegur, 
J. (1989) Cell 56, 271-280; Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. 
J., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouyssegur, J., & 
Donowitz, M. (1991) EMBO J. 10, 1957-1967); Hildebrandt, F., Pizzonia, J. 
H., Reilly, R. F., Reboucas, N. A., Sardet, C., Pouyssegur, J., Slayman, 
C. W., Aronson, P. S., & Igarashi, P. (1991) Biochim. Biophys. Acta 1129, 
105-108; Reilly, R. F., Hildebrandt, R., Biemesderfer, D., Sardet, C., 
Pouyessegur, J., Aronson, P. S., Slayman, C. W., & Igarashi, P. (1991) Am. 
J. Physiol. 261, F1088-94; Orlowski, J., Kandasamy, R. A., & Shull, G. E. 
(1992) J. Biol. Chem. 267, 9331-9339; Jia, Z. -P., McCullough, N., Martel, 
R., Hemmingsen, S., & Young, P. G. (1992) EMBO J. 11, 1631-1640) and 
prokaryotic (Goldberg, E. B., Arbel, T. Chen, J. Karpel, R., Mackie, G. 
A., Schuldiner, S., & Padan, E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 
2615-2619; Karpel, R., Olami, Y., Taglicht, D., Schuldiner, S., & Padan, 
E. (1988) J. Biol. Chem. 263, 10408-10414; Pinner, E., Padan, E., & 
Schuldiner, S. (1992) J. Biol. Chem. 267, 11064-11068; Waser, M., 
Hess-Bienz, D., Davies, K., & Solioz, M. (1992) J. Biol. Chem. 267, 
5396-5400) Na.sup.+ /H.sup.+ antiporters have been cloned, and the gene 
product of one of these genes, the nhaA gene from Escherichia coli, has 
been purified and studied in proteoliposomes (Taglicht, D., Padan, E., & 
Schuldiner, S. (1991) J. Biol. Chem. 266, 11289-11294). As with other 
secondary ion porters, no genetic or biochemical evidence has emerged for 
additional components of these transport systems. 
In bacteria, electrogenic Na.sup.+ /H.sup.+ antiporters may be part of the 
mechanism for cytoplasmic pH homeostasis in the alkaline range of pH for 
growth (Krulwich, T. A. (1983) Biochim. Biophys. Acta 726, 245-264; Booth, 
I. R. (1985) Microbiol. Rev. 49, 359-378). In particular, electrogenic 
Na.sup.+ /H.sup.+ antiport activity appears to be required in order for 
extremely alkaliphilic bacteria, such as Bacillus firmus and Bacillus 
alcalophilus, growing at pH 10.5 to maintain a cytoplasmic pH of 8.3 
(Krulwich, T. A., & Guffanti, A. A. (1989) Annu. Rev. Microbiol. 43, 
435-463). We have taken advantage of Na.sup.+ /H.sup.+ 
antiporter-deficient mutants of Escherichia coli that have been 
constructed during the extensive recent dissection of the molecular 
biology and genetics of the antiport complement in E. coli by Padan, 
Schuldiner and their colleagues (Goldberg, E. B., Arbel, T. Chen, J. 
Karpel, R., Mackie, G. A., Schuldiner, S., & Padan, E. (1987) Proc. Natl. 
Acad. Sci. U.S.A. 84, 2615-2619; Karpel, R., Olami, Y., Taglicht, D., 
Schuldiner, S., & Padan, E. (1988) J. Biol. Chem. 263, 10408-10414; 
Pinner, E., Padan, E., & Schuldiner, S. (1992) J. Biol. Chem. 267, 
11064-11068). Using heterologous complementation of Na.sup.+ /H.sup.+ 
antiporter-deficient strains by cloned genes from alkaliphilic Bacillus 
firmus OF4, we have isolated and characterized one putative structural 
gene for a Na.sup.+ /H.sup.+ antiporter from the alkaliphile, designated 
nhaC (Ivey, D. M., Guffanti, A. A., Bossewitch, J. S., Padan, E., & 
Krulwich, T. A. (1991) J. Biol. Chem. 266, 23483-23489). It encodes a 
membrane protein whose deduced amino acid sequence is consistent with at 
least ten transmembrane regions and which markedly enhances the Na.sup.+ 
/H.sup.+ antiport activity of membranes from E. coli NM81; this mutant 
strain carries a deletion in one of the E. coli antiporter genes (nhaA). 
Another alkaliphile gene that restored partial Na.sup.+ resistance to E. 
coli NM81 was the product of the alkaliphile cadC gene, a probable cadmium 
binding protein (Ivey, D. M., Guffanti, A. A., Shen, A., Kudyan, N., & 
Krulwich, T. A. (1992) J. Bacteriol., in press). 
3. SUMMARY OF THE INVENTION 
The present invention relates to the cloning and expression of a sodium ion 
binding protein. In particular, the invention is based, in part, on the 
cloning and expression of a nhaS gene using heterologous complementation 
of Na.sup.+ /H.sup.+ antiporter-deficient strains by cloned genes from 
alkaliphilic Bacillus firmus OF4. A clone was isolated and characterized 
which encoded a putative structural protein for a Na.sup.+ /H.sup.+ 
antiporter (Ivey, D. M., Guffanti, A. A., Bossewitch, J. S., Padan, E., & 
Krulwich, T. A. (1991) J. Biol. Chem. 266, 23482-23489). Preliminary 
indications revealed the expression of a small protein product (&lt;10,000 
daltons) expressed from the clone carrying the nhaC gene that appeared to 
fractionate primarily with the membrane (Ivey, D. M., Guffanti, A. A., 
Bossewitch, J. S., Padan, E., & Krulwich, T. A. (1991) J. Biol. Chem. 266, 
23483-23489, FIG. 5). Coincident with these observations, further sequence 
determinations on that clone indicated the presence of a second orf (open 
reading frame) that could account for the small protein, and whose deduced 
sequence and location just downstream from an alkaliphile Na.sup.+ 
/H.sup.+ antiporter gene invited an investigation into its function. The 
gene product, referred to as NhaS, is a protein characterized by binding 
to and sequestering sodium ion (Na.sup.+). 
The invention further relates to functional fragment of a sodium ion 
binding protein, which fragments are characterized by their ability to 
bind to sodium ion. In a specific embodiment, the fragment is a fragment 
of NhaS. The NhaS protein enhances resistance of bacteria into which the 
nhaS gene has been introduced, to Na.sup.+ by sequestering Na.sup.+ and 
reducing its cytotoxicity. 
The protein product of the nhaS gene is very basic, with a calculated pI of 
12.12. The calculated molecular weight of the full length protein is 7100 
Daltons. The NhaS protein has some sequence similarity to genes encoding 
Na.sup.+ /K.sup.+ ATPases in a region of the N-terminal half of the much 
larger ATPases. The sequence similarity does not extend to the ATP binding 
and acylation regions of the ATPases. 
In one embodiment, the NhaS protein can be used for modeling drugs that 
bind to Na.sup.+ /K.sup.+ ATPase, based on the sequence similarity of the 
proteins. The Na.sup.+ binding properties of the NhaS protein make it 
useful in a variety of medical and agricultural applications, including 
but not limited to treatments involving Na.sup.+ /K.sup.+ ATPase 
disorders, e.g., in heart disease; and to bind to and sequester dietary 
sodium, especially for subjects on a reduced sodium diet or who are 
suffering from hypertension. 
In yet a further embodiment, the nhaS gene can be used to engineer cell 
lines, in particular plant cell lines, or to prepare transgenic plants. 
Engineered cell lines containing the nhaS gene, or a Na.sup.+ binding 
fragment thereof, would be useful as desalination bioreactors. Transgenic 
plants that contain the nhaS gene should be salt resistant. 
Thus it is a particular advantage of the present invention that it provides 
a protein characterized by binding to sodium ion. 
It is yet another advantage of the present invention that a gene encoding a 
protein, which protein is characterized by binding to sodium ion is 
provided, preferably for introduction into cells or to produce plants 
transgenic for the gene.

5. DETAILED DESCRIPTION OF THE INVENTION 
The gene identified in this study is immediately downstream from a putative 
structural gene for an alkaliphile Na.sup.+ /H.sup.+ antiporter, nhaC, and 
is likely to be transcribed together with nhaC. The gene product is 
apparently a small Na.sup.+ binding protein that functions on the 
cytoplasmic side of the membrane, as opposed to the classic larger 
periplasmic binding proteins that have been extensively characterized in 
prokaryotes (Quiocho, F. A. (1990) Phil. Trans. R. Soc. Lond. B 326, 
341-351). The Na.sup.+ binding assay indicates that crude extracts 
containing levels of cloned gene product that are insufficient to identify 
the protein on Coomassie blue-stained gels in comparisons with control 
extracts, result in binding activity up to 10-fold that of those in 
control extracts. The sequestration of Na.sup.+ probably plays a major 
role in restoring Na.sup.+ -resistance to antiporter-deficient mutants of 
E. coli. We thus propose to call the gene encoding the Na.sup.+ binding 
protein nhaS. 
The present invention relates to the cloning and expression of nhaS. In 
particular, the invention relates to cloning and expression of a nhaS 
gene. The nhaS gene product, NhaS, is a protein characterized by binding 
to and sequestering sodium ion (Na.sup.+). The invention further relates 
to functional fragment of a sodium ion binding protein, which fragments 
are characterized by their ability to bind to sodium ion. In a specific 
embodiment, the fragment is a fragment of NhaS. 
The NhaS protein is associated especially with alkaliphilic bacteria, which 
are highly dependent on Na+ regulation for pH homeostasis. In particular, 
these bacteria need to maintain a cytoplasmic pH that is much more acidic 
than the external medium when the latter is well above pH 10. The NhaS 
protein enhances resistance of bacteria or other cells into which the nhaS 
gene has been introduced to Na.sup.+ by sequestering Na+ and reducing its 
cytotoxicity. In bacteria, the protein appears to operate on the 
cytoplasmic side of the membrane. 
The protein product of the nhaS gene is very basic, with a calculated pI of 
12.12. The calculated molecular weight of the full length protein is 7100 
Daltons. The NhaS protein has some sequence similarity to genes encoding 
Na.sup.+ /K.sup.+ ATPases in a region of the N-terminal half of the much 
larger ATPases. The sequence similarity does not extend to the ATP binding 
and acylation regions of the ATPases. 
In one embodiment, the NhaS protein can be used for modeling drugs that 
bind to Na.sup.+ /K.sup.+ ATPase, based on the sequence similarity of the 
proteins. 
The Na.sup.+ binding properties of the NhaS protein make it useful in a 
variety of medical and agricultural applications. For example, the protein 
may be useful for treatments involving Na.sup.+ /K.sup.+ ATPase disorders, 
e.g., in heart disease. In another embodiment, the NhaS protein can be 
introduced parenterally, preferably orally, to bind to and sequester 
dietary sodium, especially for subjects on a reduced sodium diet or who 
are suffering from hypertension. In a further embodiment, the protein is 
immobilized on a solid support such as a membrane or resin which can be 
used to remove Na.sup.+ from chemicals or during dialysis. 
In yet a further embodiment, the nhaS gene can be used to engineer cell 
lines, in particular plant cell lines, or to prepare transgenic plants. 
Engineered cell lines containing the nhaS gene, or a Na.sup.+ binding 
fragment thereof, would be useful as desalination bioreactors. Transgenic 
plants that contain the nhaS gene should be salt resistant. Such plants 
may resist drought conditions, or be suitable for cultivation in briny 
soil or salt marshes. 
For clarity of discussion, the invention is described in the subsections 
below by way of example for the Bacillus firmus sodium ion binding protein 
NhaS. However, the principles may be analogously applied to clone and 
express the NhaS or sodium ion binding proteins of other species, 
especially alkaliphilic bacteria. 
5.1. THE NhaS CODING SEQUENCE 
The invention relates to isolated nucleic acids encoding a sodium ion 
binding protein. The invention further relates to a cell line stably 
containing a recombinant nucleic acid encoding a sodium ion binding 
protein. In another embodiment, the invention relates to a nucleic acid 
encoding a fragment of the sodium ion binding protein, which fragment 
binds sodium ion. 
The nucleotide coding sequence (SEQ. ID NO: 1) and deduced amino acid 
sequence (SEQ. ID NO: 2) for the Bacillus firmus OF4 nhaS gene are 
depicted in FIG. 1. The open reading frame encodes a 67 amino acid protein 
of about 7100 Daltons. Hydrophobicity analysis of the deduced protein 
(FIG. 5) suggests the possibility that the gene product, NhaS, associates 
with the membrane. This is consistent with the retention of NhaS protein 
in everted membrane vesicle preparations, evidence of membrane binding 
ability. 
This unusual protein is further characterized by its extraordinary 
basicity. The overall predicted pI of NhaS is 12.1. This basicity may 
facilitate association with negatively charged phospholipids in the 
membrane, and it may also function as a pH sensor, rendering Na.sup.+ 
binding sensitive to pH. 
The NhaS protein is further characterized by sequence similarity to 
numerous cloned genes encoding the .alpha. chain of the Na.sup.+ /K.sup.+ 
ATPase from various species and tissues, e.g., from Drosophila 
melanogaster. This region of the Na.sup.+ /K.sup.+ ATPase has been 
proposed to encompass parts of two putative membrane-spanning stretches in 
the N-terminal half of the molecule, and the small intervening loop that 
contains the tryptophan shown in common with NhaS. The loop also shows 
similarity with a small region of sequence in the gene encoding the 
cardiac sarcolemmal Na.sup.+ /Ca.sup.++ exchanger. However, the NhaS 
protein is further characterized by having a dramatically basic deduced 
sequence--KYRYK--that has no analog in the ATPase sequences (and thus 
requires introduction of a gap in the ATPase sequences in order to 
maximize similarity). Such a region in the NhaS protein clearly 
contributes to overall basicity, and likely has a role in the 
pH-dependence of the Na.sup.+ -binding activity. 
The invention also relates to nhaS genes isolated from other bacterial 
species especially from alkaliphilic species, including but not limited to 
Bacillus, such as B. alcalophilus, B. firmus, and Exiguobacterium 
aurantiacum, in which is believed NhaS activity exists. Members of the 
NhaS family are defined herein as those small proteins that bind Na.sup.+. 
Such Na.sup.+ binding proteins may demonstrate about 80% homology at the 
nucleotide level, and even 90% homology at the amino acid level. The 
alkaliphilic B. firmus sequence can be used to design degenerate or fully 
degenerate oligonucleotide probes which can be used to screen genomic 
libraries derived from appropriate bacterial which express NhaS. The 
N-terminus of the sequence depicted in FIG. 1 may advantageously be used 
to design such oligonucleotide probes, as this region is relatively 
conserved with Na.sup.+ /K.sup.+ ATPases, and thus should be conserved 
within the NhaS family. 
Alternatively, a polymerase chain reaction (PCR) based strategy may be used 
to clone nhaS. Two pools of degenerate oligonucleotides, corresponding to 
conserved motifs between the NhaS and Na.sup.+ /K.sup.+ ATPases may be 
designed to serve as primers in a PCR reaction. Examples of such conserved 
motifs which may be used include but are not limited to those found at the 
N-terminus. The template for the reaction is chromosomal DNA prepared from 
a bacterial strain known or expected to express NhaS. The amplified PCR 
fragment may be used to isolate a full length nhaS clone by radioactively 
labeling the amplified fragment and screening a library. 
Alternatively, the methods detailed in Section 6.1.1 infra, for cloning the 
nhaS DNA, may be utilized to obtain the corresponding nhaS DNA sequence 
from other bacterial species. Specifically, a genomic library constructed 
from DNA prepared from a bacterial strain known or expected to express a 
Na.sup.+ binding protein may be used to transform the bacterial strain 
NM81 which is deficient in the putative Na.sup.+ /H.sup.+ antiporter. 
Transformation of a recombinant plasmid coding for a sodium binding 
protein into an antiporter-deficient NM81 bacterial strain would be 
expected to enhance the Na.sup.+ -resistance of the bacteria, and this 
phenotype may be selected for by adjusting the Na.sup.+ content and pH of 
the medium. 
Cloning of other proteins in the nhaS family may be carried out in a number 
of different ways. The PCR based strategy described above for cloning of 
nhaS, may also be used to isolate encoding NhaS related proteins. 
Alternatively, a bacteriophage library may be screened, under conditions 
of reduced stringency, using a radioactively labeled fragment of the B. 
firmus nhaS clone. For a review of cloning strategies which may be used, 
see e.g., Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Cold 
Springs Harbor Press, New York; and Ausubel et al., 1989, Current 
Protocols in Molecular Biology, Green Publishing Associates and Wiley 
Interscience, New York. 
In accordance with the invention, nucleotide sequences which encode a NhaS, 
fragments, fusion proteins or functional equivalents thereof, may be used 
to generate recombinant DNA molecules that direct the expression of the 
NhaS, or a functionally active peptide, fusion protein or functional 
equivalent thereof, in appropriate host cells. Alternatively, nucleotide 
sequences which hybridize to portions of the nhaS sequence may also be 
used in nucleic acid hybridization assays, Southern and Northern blot 
analyses, etc. 
Due to the degeneracy of the genetic code, other DNA sequences which encode 
substantially the NhaS amino acid sequence, e.g., such as the deduced 
sequence depicted in FIG. 1, or a functional equivalent may be used in the 
practice of the present invention for the cloning and expression of the 
nhaS. Such DNA sequences include those which are capable of hybridizing to 
the nhaS sequence under stringent conditions, or which would be capable of 
hybridizing under stringent conditions but for the degeneracy of the 
genetic code. The stringency conditions may be adjusted in a number of 
ways. For example, when performing polymerase chain reactions (PCR), the 
temperature at which annealing of primers to template takes place or the 
concentration of MgCl.sub.2 in the reaction buffer may be adjusted. When 
using radioactively labeled DNA fragments or oligonucleotides to probe 
filters, the stringency may be adjusted by changes in the ionic strength 
of the wash solutions or by careful control of the temperature at which 
the filter washes are carried out. 
Altered DNA sequences which may be used in accordance with the invention 
include deletions, additions or substitutions of different nucleotide 
residues resulting in a sequence that encodes the same or a functionally 
equivalent gene product. The gene product itself may contain deletions, 
additions or substitutions of amino acid residues within the NhaS 
sequence, which result in a silent change thus producing a functionally 
equivalent NhaS. Such amino acid substitutions may be made on the basis of 
similarity in polarity, charge, solubility, hydrophobicity, 
hydrophilicity, and/or the amphipatic nature of the residues involved. For 
example, negatively charged amino acids include aspartic acid and glutamic 
acid; positively charged amino acids include lysine and arginine; amino 
acids with uncharged polar head groups having similar hydrophilicity 
values include the following: leucine, isoleucine, valine; glycine, 
alanine; asparagine, glutamine; serine, theonine; phenylalanine, tyrosine. 
As used herein, a functionally equivalent NhaS refers to a protein which 
binds sodium ion (Na.sup.+), but not necessarily with the same binding 
affinity of its counterpart native NhaS. 
The DNA sequences of the invention may be engineered in order to alter the 
NhaS coding sequence for a variety of ends including but not limited to 
alterations which modify expression of the gene product. For example, 
mutations may be introduced using techniques which are well known in the 
art, e.g. site-directed mutagenesis, to insert new restriction sites that 
facilitate cloning into expression vectors. In an embodiment of the 
invention, the nhaS or a modified nhaS sequence may be ligated to a 
heterologous sequence to encode a fusion protein. The fusion protein may 
be engineered to contain a cleavage site located between the NhaS sequence 
and the heterologous protein sequence, so that the NhaS can be cleaved 
away from the heterologous moiety. 
In an alternate embodiment of the invention, the coding sequence of nhaS 
could be synthesized in whole or in part, using chemical methods well 
known in the art. See, for example, Caruthers, et al., 1980, Nuc. Acids 
Res. Symp. Ser. 7: 215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10): 
2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21: 719; and Chow 
and Kempe, 1981, Nuc. Acids Res. 9(12): 2807-2817. Alternatively, the 
protein itself could be produced using chemical methods to synthesize the 
NhaS amino acid sequence in whole or in part. For example, peptides can be 
synthesized by solid phase techniques, cleaved from the resin, and 
purified by preparative high performance liquid chromatography. (E.g., see 
Creighton, 1983, Proteins Structures And Molecular Principles, W. H. 
Freeman and Co., New York pp. 50-60). The composition of the synthetic 
peptides may be confirmed by amino acid analysis or sequencing (e.g., the 
Edman degradation procedure; see Creighton, 1983, Proteins, Structures and 
Molecular Principles, W. H. Freeman and Co., New York, pp. 34-49. 
5.2. EXPRESSION OF THE NhaS 
In order to express a biologically active NhaS, the nucleotide sequence 
coding for NhaS, or a functional equivalent as described in Section 5.1 
supra, is inserted into an appropriate expression vector, i.e., a vector 
which contains the necessary elements for the transcription and 
translation of the inserted coding sequence. The nhaS gene products as 
well as host cells or cell lines transfected or transformed with 
recombinant nhaS expression vectors can be used for a variety of purposes. 
These include but are not limited to generating antibodies (i.e., 
monoclonal or polyclonal) that bind to the receptor, including those that 
competitively inhibit Na.sup.+ binding and "neutralize" NhaS activity. 
5.2.1. EXPRESSION SYSTEMS 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing the nhaS coding sequence and 
appropriate transcriptional/translational control signals. These methods 
include in vitro recombinant DNA techniques, synthetic techniques and in 
vivo recombination/genetic recombination. See, for example, the techniques 
described in Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, 
Cold Spring Harbor Laboratory, New York and Ausubel et al., 1989, Current 
Protocols in Molecular Biology, Greene Publishing Associates and Wiley 
Interscience, New York. 
A variety of host-expression vector systems may be utilized to express the 
nhaS coding sequence. These include but are not limited to microorganisms 
such as bacteria transformed with recombinant bacteriophage DNA, plasmid 
DNA or cosmid DNA expression vectors containing the nhaS coding sequence; 
yeast transformed with recombinant yeast expression vectors containing the 
nhaS coding sequence; insect cell systems infected with recombinant virus 
expression vectors (e.g., baculovirus) containing the nhaS coding 
sequence; plant cell systems infected with recombinant virus expression 
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) 
or transformed with recombinant plasmid expression vectors (e.g., Ti 
plasmid) containing the nhaS coding sequence; or animal cell systems 
infected with recombinant virus expression vectors (e.g., adenovirus, 
vaccinia virus) including cell lines engineered to contain multiple copies 
of the nhaS DNA either stably amplified (e.g., CHO/dhfr) or unstably 
amplified in double-minute chromosomes (e.g., murine cell lines). 
The expression elements of these systems vary in their strength and 
specificities. Depending on the host/vector system utilized, any of a 
number of suitable transcription and translation elements, including 
constitutive and inducible promoters, may be used in the expression 
vector. For example, when cloning in bacterial systems, inducible 
promoters such as pL of bacteriophage .lambda., plac, ptrp, ptac (ptrp-lac 
hybrid promoter) and the like may be used; when cloning in insect cell 
systems, promoters such as the baculovirus polyhedrin promoter may be 
used; when cloning in plant cell systems, promoters derived from the 
genome of plant cells (e.g., heat shock promoters; the promoter for the 
small subunit of RUBISCO; the promoter for the chlorophyll a/b binding 
protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the 
coat protein promoter of TMV) may be used; when cloning in mammalian cell 
systems, promoters derived from the genome of mammalian cells (e.g., 
metallothionein promoter) or from mammalian viruses (e.g., the adenovirus 
late promoter; the vaccinia virus 7.5K promoter) may be used; when 
generating cell lines that contain multiple copies of the nhaS DNA SV40-, 
BPV- and EBV-based vectors may be used with an appropriate selectable 
marker. 
In bacterial systems a number of expression vectors may be advantageously 
selected depending upon the use intended for the NhaS expressed. For 
example, when large quantities of NhaS are to be produced for structural 
studies and protein modelling or for use in an immobilized form to 
sequester Na.sup.+ vectors which direct the expression of high levels of 
fusion protein products that are readily purified may be desirable. Such 
vectors include but are not limited to the E. coli expression vector 
pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the nhaS coding 
sequence may be ligated into the vector in frame with the lac Z coding 
region so that a hybrid NhaS-lac Z protein is produced; pIN vectors 
(Inouye & Inouye, 1985, Nucleic acids Res. 13: 3101-3109; Van Heeke & 
Schuster, 1989, J. Biol. Chem. 264: 5503-5509); and the like. pGEX vectors 
may also be used to express foreign polypeptides as fusion proteins with 
glutathione S-transferase (GST). In general, such fusion proteins are 
soluble and can easily be purified from lysed cells by adsorption to 
glutathione-agarose beads followed by elution in the presence of free 
glutathione. The pGEX vectors are designed to include thrombin or factor 
Xa protease cleavage sites so that the cloned NhaS polypeptide can be 
released from the GST moiety. 
In yeast, a number of vectors containing constitutive or inducible 
promoters may be used. For a review see, Current Protocols in Molecular 
Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley 
Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors 
for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. 
Press, New York, Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. 
II, IRL Press, Washington, D.C., Ch. 3; and Bitter, 1987, Heterologous 
Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, 
Acad. Press, New York, Vol. 152, pp. 673-684; and The Molecular Biology of 
the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor 
Press, Vols. I and II. 
In cases where plant expression vectors are used, the expression of the 
nhaS coding sequence may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV 
(Brisson et al., 1984, Nature 310: 511-514), or the coat protein promoter 
of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used; 
alternatively, plant promoters such as the small subunit of RUBISCO 
(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science 
224: 838-843); or heat shock promoters, e.g., soybean hsp17.5-E or 
hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6: 559-565) may be used. 
These constructs can be introduced into plant cells using Ti plasmids, Ri 
plasmids, plant virus vectors, direct DNA transformation, microinjection, 
electroporation, etc. For reviews of such techniques see, for example, 
Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic 
Press, New York, Section VIII, pp. 421-463; and Grierson & Corey, 1988, 
Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. 
An alternative expression system which could be used to express NhaS is an 
insect system. In one such system, Autographa californica nuclear 
polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. 
The virus grows in Spodoptera frugiperda cells. The nhaS coding sequence 
may be cloned into non-essential regions (for example the polyhedrin gene) 
of the virus and placed under control of an AcNPV promoter (for example 
the polyhedrin promoter). Successful insertion of the nhaS coding sequence 
will result in inactivation of the polyhedrin gene and production of 
non-occluded recombinant virus (.i.e., virus lacking the proteinaceous 
coat coded for by the polyhedrin gene). These recombinant viruses are then 
used to infect Spodoptera frugiperda cells in which the inserted gene is 
expressed. (E.g., see Smith et al., 1983, J. Viol. 46: 584; Smith, U.S. 
Pat. No. 4,215,051). 
In mammalian host cells, a number of viral based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, 
the nhaS coding sequence may be ligated to an adenovirus 
transcription/translation control complex, e.g., the late promoter and 
tripartite leader sequence. This chimeric gene may then be inserted in the 
adenovirus genome by in vitro or in vivo recombination. Insertion in a 
non-essential region of the viral genome (e.g., region E1 or E3) will 
result in a recombinant virus that is viable and capable of expressing 
NhaS in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. 
Sci. (USA) 81: 3655-3659). Alternatively, the vaccinia 7.5K promoter may 
be used. (E.g., see Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79: 
7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et al., 
1982, Proc. Natl. Acad. Sci. 79: 4927-4931). 
Specific initiation signals may also be required for efficient translation 
of inserted nhaS coding sequences. These signals include the ATG 
initiation codon and adjacent sequences, referred to as Kosak sequences, 
which differ from those utilized by prokaryotes. In cases where only a 
portion of the nhaS coding sequence is inserted, exogenous translational 
control signals, including the ATG initiation codon, must be provided. 
Furthermore, the initiation codon must be in phase with the reading frame 
of the nhaS coding sequence to ensure translation of the entire insert. 
These exogenous translational control signals and initiation codons can be 
of a variety of origins, both natural and synthetic. In addition, 
eukaryotic expression vectors should contain the signals necessary for 
transcriptional termination and polyadenylation. The efficiency of 
expression may be enhanced by the inclusion of appropriate transcription 
enhancer elements, transcription terminators, etc. (see Bitter et al., 
1987, Methods in Enzymol. 153: 516-544). 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express the 
NhaS may be engineered. Rather than using expression vectors which contain 
viral origins of replication, host cells can be transformed with the nhaS 
DNA controlled by appropriate expression control elements (e.g., promoter, 
enhancer, sequences, transcription terminators, polyadenylation sites, 
etc.), and a selectable marker. Following the introduction of foreign DNA, 
engineered cells may be allowed to grow for 1-2 days in an enriched media, 
and then are switched to a selective media. The selectable marker in the 
recombinant plasmid confers resistance to the selection and allows cells 
to stably integrate the plasmid into their chromosomes and grow to form 
foci which in turn can be cloned and expanded into cell lines. This method 
may advantageously be used to engineer cell lines which express the NhaS 
on the cytoplasmic surface of the cell membrane, and which bind Na.sup.+. 
A number of selection systems may be used, including but not limited to the 
herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), 
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 
1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine 
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be 
employed in tk, hgprt or aprt cells, respectively. Also, antimetabolite 
resistance can be used as the basis of selection for dhfr, which confers 
resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 
77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, 
which confers resistance to mycophenolic acid (Mulligan & Berg, 1981), 
Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the 
aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 
150:1); and hygro, which confers resistance to hygromycin (Santerre, et 
al., 1984, Gene 30:147) genes. Recently, additional selectable genes have 
been described, namely trpB, which allows cells to utilize indole in place 
of tryptophan; hisD, which allows cells to utilize histinol in place of 
histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); 
and ODC (ornithine decarboxylase) which confers resistance to the 
ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO 
(McConlogue L., 1987, In: Current Communications in Molecular Biology, 
Cold Spring Harbor Laboratory ed.). 
5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS THAT EXPRESS THE 
NhaS 
The host cells which contain the coding sequence and which express the 
biologically active gene product may be identified by at least four 
general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence 
or absence of "marker" gene functions; (c) assessing the level of 
transcription as measured by the expression of nhaS mRNA transcripts in 
the host cell; and (d) detection of the gene product as measured by 
immunoassay or by its biological activity. 
In the first approach, the presence of the nhaS coding sequence inserted in 
the expression vector can be detected by DNA-DNA or DNA-RNA hybridization 
using probes comprising nucleotide sequences that are homologous to the 
nhaS coding sequence, respectively, or portions or derivatives thereof. 
In the second approach, the recombinant expression vector/host system can 
be identified and selected based upon the presence or absence of certain 
"marker" gene functions (e.g., thymidine kinase activity, resistance to 
antibiotics, resistance to methotrexate, transformation phenotype, 
occlusion body formation in baculovirus, etc.). For example, if the nhaS 
coding sequence is inserted within a marker gene sequence of the vector, 
recombinants containing the nhaS coding sequence can be identified by the 
absence of the marker gene function. Alternatively, a marker gene can be 
placed in tandem with the nhaS sequence under the control of the same or 
different promoter used to control the expression of the nhaS coding 
sequence. Expression of the marker in response to induction or selection 
indicates expression of the nhaS coding sequence. 
In the third approach, transcriptional activity for the nhaS coding region 
can be assessed by hybridization assays. For example, RNA can be isolated 
and analyzed by Northern blot using a probe homologous to the nhaS coding 
sequence or particular portions thereof. Alternatively, total nucleic 
acids of the host cell may be extracted and assayed for hybridization to 
such probes. 
In the fourth approach, the expression of the NhaS protein product can be 
assessed immunologically, for example by Western blots, immunoassays such 
as radioimmuno-precipitation, enzyme-linked immunoassays and the like. The 
ultimate test of the success of the expression system, however, involves 
the detection of the biologically active NhaS gene product. A number of 
assays can be used to detect receptor activity including but not limited 
to Na.sup.+ binding assays. 
5.2.3. RECOVERY OF THE NhaS PROTEIN 
Once a clone that produces high levels of biologically active NhaS is 
identified, the clone may be expanded and used to produce large amounts of 
the protein, which may be purified using techniques well-known in the art 
including, but not limited to immunoaffinity purification, chromatographic 
methods including high performance liquid chromatography or more 
preferably cation exchange chromatography, "affinity" chromatography based 
on the affinity of NhaS for Na.sup.+, immunoaffinity purification using 
antibodies and the like. 
Where the nhaS coding sequence is engineered to encode a cleavable fusion 
protein, purification may be readily accomplished using affinity 
purification techniques. For example, a collagenase cleavage recognition 
consensus sequence may be engineered between the carboxy terminus of NhaS 
and protein A. The resulting fusion protein may be readily purified using 
an IgG column that binds the protein A moiety. Unfused NhaS may be readily 
released from the column by treatment with collagenase. Another example 
would be the use of pGEX vectors that express foreign polypeptides as 
fusion proteins with glutathionine S-transferase (GST). The fusion protein 
may be engineered with either thrombin or factor Xa cleavage sites between 
the cloned gene and the GST moiety. The fusion protein may be easily 
purified from cell extracts by adsorption to glutathione agarose beads 
followed by elution in the presence of glutathione. In this aspect of the 
invention, any cleavage site or enzyme cleavage substrate may be 
engineered between the NhaS sequence and a second peptide or protein that 
has a binding partner which could be used for purification, e.g., any 
antigen for which an immunoaffinity column can be prepared. 
5.3. GENERATION OF ANTIBODIES THAT DEFINE THE NhaS PROTEIN 
Various procedures known in the art may be used for the production of 
antibodies to epitopes of the recombinantly produced NhaS. Neutralizing 
antibodies i.e., those which compete for the Na.sup.+ binding site of the 
receptor are especially preferred for diagnostics and therapeutics. Such 
antibodies include but are not limited to polyclonal, monoclonal, 
chimeric, single chain, Fab fragments and fragments produced by an Fab 
expression library. 
For the production of antibodies, various host animals may be immunized by 
injection with the NhaS including but not limited to rabbits, mice, rats, 
etc. Various adjuvants may be used to increase the immunological response, 
depending on the host species, including but not limited to Freund's 
(complete and incomplete), mineral gels such as aluminum hydroxide, 
surface active substances such as lysolecithin, pluronic polyols, 
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, 
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille 
Calmette-Guerin) and corynebacterium parvum. 
Monoclonal antibodies to NhaS may be prepared by using any technique which 
provides for the production of antibody molecules by continuous cell lines 
in culture. These include but are not limited to the hybridoma technique 
originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), 
the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology 
Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and 
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques 
developed for the production of "chimeric antibodies" (Morrison et al., 
1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, 
Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing 
the genes from a mouse antibody molecule of appropriate antigen 
specificity together with genes from a human antibody molecule of 
appropriate biological activity can be used. Alternatively, techniques 
described for the production of single chain antibodies (U.S. Pat. No. 
4,946,778) can be adapted to produce NhaS-specific single chain 
antibodies. 
Antibody fragments which contain specific binding sites of NhaS may be 
generated by known techniques. For example, such fragments include but are 
not limited to: the F(ab').sub.2 fragments which can be produced by pepsin 
digestion of the antibody molecule and the Fab fragments which can be 
generated by reducing the disulfide bridges of the F(ab').sub.2 fragments. 
Alternatively, Fab expression libraries may be constructed (Huse et al., 
1989, Science, 246:1275-1281) to allow rapid and easy identification of 
monoclonal Fab fragments with the desired specificity to NhaS. 
5.4. USES OF THE NhaS: SALT TOLERANT PLANTS AND ENGINEERED CELL LINES 
The nhaS DNA, NhaS expression products, antibodies and engineered cell 
lines described above have a number of uses as a dietary therapy where 
control of sodium intake is desired, or for preparing recombinant plants 
capable of growth in briney water or under drought conditions. 
In another embodiment of the invention, the NhaS itself, or a fragment 
containing its Na.sup.+ binding site, could be administered in vivo. 
Preferably a NhaS fragment lacks the membrane-binding portion of the 
protein. The free NhaS or the soluble peptide fragment could bind 
Na.sup.+, thus reducing side effects in vivo. 
Recently, computer generated models for interactions have been developed 
and in a specific embodiment of the invention information derived from 
computer modeling of NhaS may be used for design of inhibitors of the 
Na.sup.+ /K.sup.+ ATPase. Changes made to NhaS sequences, using for 
example techniques for site directed mutagenesis and expression of mutant 
proteins in cell lines, may be used to further define the functional role 
of particular protein regions and residues. 
Nucleic acids, in particular vectors, are introduced into the desired host 
cells by methods known in the art, e.g., transfection, electroporation, 
microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate 
precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA 
vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 
267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et 
al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990). 
6. EXAMPLE: CLONING AND EXPRESSION OF NhaS, A SODIUM ION BINDING PROTEIN 
A gene, designated nhaS, has been identified downstream from the putative 
Na.sup.+ /H.sup.+ antiporter-encoding nhaC gene on the chromosome of 
alkaliphilic Bacillus firmus OF4. The deduced nhaS gene product would be a 
protein with an Mr of 7,000, a pI of 12.12, and shared sequence similarity 
to a region in the N-terminal half of the Na.sup.+ /K.sup.+ ATPase. An 
Escherichia coli strain with a deletion in one Na.sup.+ /H.sup.+ 
antiporter gene (nhaA) exhibited enhanced activity of its residual, 
nhaB-dependent membrane antiport activity when transformed with a plasmid 
containing nhaS. An E. coli mutant that was deleted in both nhaA and nhaB 
showed no significant increase in Na.sup.+ /H.sup.+ antiport when 
transformed with the same plasmid, but nonetheless showed greatly enhanced 
resistance to high concentrations of NaCl relative to a control 
transformant. Direct evidence for a Na.sup.+ -binding activity of the nhaS 
product in extracts of the E. coli transformant was obtained using a 
sodium sensitive fluorescent probe. NhaS is proposed to be a sodium 
binding protein that can enhance the Na.sup.+ -resistance of 
antiporter-deficient strains by increasing the availability of Na.sup.+ to 
the integral membrane antiporters on the cytoplasmic side of the membrane 
and by sequestering Na.sup.+ while rate-limiting efflux mechanisms 
catalyze extrusion of the cation. 
6.1. MATERIALS AND METHODS 
6.1.1. BACTERIAL STRAINS AND PLASMIDS 
The cloned DNA fragment from alkaliphilic B. firmus OF4 that was the 
starting point for this investigation was in pGJX5 is a derivative of 
pM4.10, which had been selected from a library of MboI-digested B. firmus 
OF4 DNA. Chromosomal DNA was prepared from B. firmus OF4 by the method of 
Marmur (1961, J. Mol. Biol. 3:208-218). The chromosomal DNA was partially 
digested with MboI (at 0.04 unit/.mu.g chromosomal DNA) and ligated into 
BamHI-digested and dephosphorylated pSPT18 ("library 1") or pGEM3Zf(+) 
("library 2"). Recombinant plasmids were transformed into E. coli JM109, 
and approximately 105 colonies were pooled, inoculated into 50 ml of LB 
containing ampicillin, and grown overnight at 37.degree. C. Plasmid DNA 
was isolated (Del Sal et al., 1989, BioTechniques 7:514-519) and used for 
transformation of NM81. Analyses of the subsequent clones were conducted 
in part by Southern analysis and by other standard restriction mapping and 
subcloning techniques (Ausubel et al. (eds.), 1987, Current Protocols in 
Molecular Biology, John Wiley & Sons, New York; Sambrook et al., 1989, 
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y.). Probe DNA used for Southern 
analyses was radioactively labeled using the Prime-a-Gene kit (Promega). 
Subcloning study was carried out by standard molecular biological 
manipulations (Ausubel et al. (eds.), 1987, Current Protocols in Molecular 
Biology, John Wiley & Sons, New York). E. coli strain NM81 (.sub..DELTA. 
nhaA, kan.sup.R) (Padan, E., Maisler, N., Taglicht, D., Karpel, R., & 
Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302) and E. coli strain 
EP432 (.sub..DELTA. nhaA, .sub..DELTA. nhaB, kan.sup.R, cam.sup.R) (EP432) 
were obtained from Dr. Etana Padan (Institute for Life Sciences, Hebrew 
University, Jerusalem, Israel). These strains were routinely grown on LBK 
medium (Goldberg, E. B., Arbel, T., Chen, J., Karpel, R., Mackie, G. A., 
Schuldiner, S., & Padan, E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 
2615-1619) at pH 7.5, containing 50 .mu.g/ml kanamycin and, when 
transformed with plasmids, 100 .mu.g/ml ampicillin. For specific 
determinations of Na.sup.+ resistance, the Na.sup.+ content and pH of the 
medium were adjusted as indicated. 
6.1.2. DNA SEQUENCING 
CsCl gradient-purified plasmid DNA was prepared by a large scale alkaline 
lysis procedure for DNA sequencing (Ausubel, F. M., Brent, R., Kingston, 
R. E., Moore, D. D., Smith, J. A., Seidman, J. G., & Struhl, K. (eds.) 
(1987) Current Protocols in Molecular Biology., John Wiley & Sons, Inc., 
New York). Both strands of the regions of interest were sequenced using an 
Applied Biosystems 373A DNA sequencer in the DNA Core Laboratory of the 
Brookdale Center for Molecular Biology at the Mount Sinai School of 
Medicine. Oligonucleotide primers were synthesized in the same facility 
using the Applied Biosystems 380B DNA synthesizer. The Genetics Computer 
Group Sequence Analysis Software Package (Devereux, J., Haeberli, P., & 
Smithies, O. (1984) Nucl. Acids Res. 12, 387-395) was used for sequence 
analyses on a VAX 4000-300 computer. 
6.1.3. ASSAYS OF Na.sup.+ /H.sup.+ ANTIPORT AND Na.sup.+ BINDING 
Assays of antiport were carried out on everted membrane preparations from 
transformed E. coli strains. The vesicles were prepared according to the 
method of Rosen and colleagues (Ambudkar, S. V., Zlotnick, G. W., & Rosen, 
B. P. (1984) J. Biol. Chem. 259, 6142-6146; Rosen, B. P. (1986) Methods 
Enzymol. 125, 328-336), with a few modifications as described by Goldberg 
et al. (Goldberg, E. B., Arbel, T., Chen, J., Karpel, R., Mackie, G. A., 
Schuldiner, S., & Padan, E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 
2615-1619) and Ivey et al. (Ivey, D. M., Guffanti, A. A., Bossewitch, J. 
S., Padan, E., & Krulwich, T. A. (1991) J. Biol. Chem. 266, 23483-23489). 
Several accommodations were made to the apparent lability of the 
alkaliphile gene product(s) in this heterologous system. Specifically, 
cells were grown from small inocula for about 8 h, harvested, and 
immediately used for membrane preparations, without freezing or storage in 
the cold. The membranes were assayed immediately after preparation. 
Protein concentrations were determined by the method of Lowry (1951 , J. 
Biol. Chem., 193:256-275) using egg white lysozyme as a standard. The 
membranes were assayed immediately after preparation. For assays of 
Na.sup.+ binding, crude extracts were prepared by passing cells through a 
French Press (10,000 lb/in2). Unbroken cells and cell wall debris were 
separated by centrifugation at 12,000.times.g for ten minutes. The 
supernatant was then dialyzed (in tubing) with a molecular weight cut-off 
of 3,500 daltons) for five hours against 20 mM Tris-Hepes, pH 7.5, with 
two changes of buffer. Protein was determined by the method of Lowry 
(Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J. 
Biol. Chem. 193,265-275) using egg white lysozyme as the standard. 
The antiport assays of everted vesicles involved the establishment of a 
.sub..DELTA. pH (transmembrane pH gradient) by the addition of D-lactate 
to the membrane suspension, followed by the partial abolition of that 
gradient by the addition of NaCl or LiCl . The establishment and abolition 
of the .sub..DELTA. was monitored by the quenching of acridine orange 
fluorescence and its reversal (Goldberg, E. B., Arbel, T., Chen, J., 
Karpel, R., Mackie, G. A., Schuldiner, S., & Padan, E. (1987) Proc. Natl. 
Acad. Sci. U.S.A. 84, 2615-1619; Ambudkar, S. V., Zlotnick, G. W., & 
Rosen, B. P. (1984) J. Biol. Chem. 259, 6142-6146). A Perkin-Elmer Cetus 
model 650-10S spectrofluorometer was used, with an excitation wavelength 
of 430 nm and emission wavelength of 570 nm. 
The Na.sup.+ binding assay was conducted on cell extracts using SBFI 
(Harootunian, A. R., Kao, J. P. Y., Eckert, B. K., & Tsien, R. Y. (1989) 
J. Biol. Chem. 264, 19458-19467). Crude extract was preincubated for five 
minutes in 20 mM Tris-Hepes, pH 7.5, with 5 .mu.M SBFI in the presence of 
various concentrations of NaCl. Fluorescence was monitored at a fixed 
emission wavelength of 505 nm, while the excitation wavelength was scanned 
between 300 and 400 nm. 
6.2. RESULTS 
Further sequence analysis of the cloned alkaliphile DNA fragment in pGJX5, 
downstream from nhaC, indicated the presence of an open reading frame 
(orf2) starting 50 bp downstream from nhaC. The next closest downstream 
open reading frame was an additional 400 bp beyond orf2. The nucleotide 
sequence and the deduced amino acid sequence of a product that orf2 might 
encode are shown in FIG. 1. The putative product would be a very basic 
protein, with a pI of 12.12 and a molecular weight of 7,100 daltons. The 
databases indicated that this gene was novel and that it had some sequence 
similarity to genes encoding Na.sup.+ /K.sup.+ ATPases in a region of the 
N-terminal half of these much larger proteins, distinct from the ATP 
binding and acylation regions. Given this similarity and the proximity to 
nhaC on the alkaliphile chromosome, we sought to examine the activity of 
the orf2 gene product in E. coli strains with deletions in one or both of 
the Na.sup.+ /H.sup.+ antiporter-encoding genes. 
A subclone (pGRVH) in pGEM7f(+) containing orf2 as the sole complete open 
reading frame was constructed from pGJX5 by digesting this plasmid with 
HindIII, which cut 77 bp upstream from the 3' end of nhaC, and with EcoRV, 
which cut 270 bp downstream from orf2, well before the start of the next 
downstream open reading frame (FIG. 1). As shown in FIG. 2, a pGRVH 
transformant of the nhaA-deletion strain, E. coli NM81, exhibited enhanced 
membrane Na.sup.+ /H.sup.+ antiport activity with Na.sup.+ but not with 
Li+ as the substrate, relative to a control transformed with plasmid 
alone. By contrast, a pGRVH transformant of E. coli strain EP432, that 
carries deletions in both the nhaA and nhaB genes, did not show such 
enhancement. E. coli EP432 exhibited only a slight Na.sup.+ /H.sup.+ 
antiport activity that was not significantly enhanced in the membranes of 
the pGRVH transformant. In the presence of excess KCl, the Na.sup.+ 
/H.sup.+ antiport was no longer observed, consistent with the conclusion 
that this small residual activity is attributable to a K.sup.+ /H.sup.+ 
antiporter in the membrane (EP432). Under these conditions, the presence 
of pGRVH had no effect on the antiport activity (FIG. 2). 
Although transformation of the double nha deletion of E. coli with pGRVH 
did not enhance the Na.sup.+ /H.sup.+ activity of the membrane, it 
markedly enhanced the resistance of this strain to elevated concentrations 
of NaCl in the medium. In FIG. 3, growth of the following transformants of 
E. coli are compared on plating media of three different NaCl 
concentrations (0.2-0.4M) and 0.2M NaCl-containing medium at either pH 7.5 
or pH 8.6: pGEM, the plasmid control; pGM36, a plasmid encoding the E. 
coli nhaA gene (Padan, E., Maisler, N., Taglicht, D., Karpel, R., & 
Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302); pBE22, a plasmid 
containing the alkaliphile cadC gene (Ivey, D. M., Guffanti, A. A., Shen, 
A., Kudyan, N., & Krulwich, T. A. (1992) J. Bacteriol., in press); and 
pGRVH. The pGRVH transformant grew almost as well as the double deletion 
strain that was transformed with the nhaA-encoding plasmid, at pH 7.5 and 
various NaCl concentrations; both of these transformants grew better than 
the transformant carrying the alkaliphile cadC gene and the transformant 
with the plasmid control did not grow at all. At 0.2M NaCl and elevated 
pH, only the pGRVH transformant of E. coli strain EP432 grew. Since this 
transformant had not shown enhanced antiport activity, and the deduced 
orf2 product suggested that it was unlikely to be an integral membrane 
antiporter, we considered the possibility that the orf2 product might be a 
Na.sup.+ -binding protein. Such a protein might enhance Na.sup.+ /H.sup.+ 
antiport activity by somehow making the cytoplasmic substrate more 
available to the integral membrane antiporters; in the absence of Na.sup.+ 
/H.sup.+ antiporters, enhanced NaCl resistance might be conferred by 
sequestration of the cytoplasmic NaCl while sluggish residual mechanisms, 
e.g. modest Na.sup.+ /H.sup.+ antiport by other antiporters such as the 
K.sup.+ /H.sup.+ antiporter, extrude the cation. As shown in FIG. 4, 
extracts of the EP432 transformant with pGRVH had significantly enhanced 
Na.sup.+ binding activity relative to extracts from a control 
transformant. There was some variability from experiment to experiment in 
these crude extracts, but significant differences between control and 
transformant extracts were consistently observed in this concentration 
range of Na.sup.+ and protein, and increased extract protein gave 
increased binding. The primary data shown in FIG. 4A were in the lower 
range of NaCl examined, where the differences were most pronounced. In 
order to better assess the binding capacity of the extract from the 
transformant vs. the control, several experiments were conducted over a 
broader range of NaCl concentrations such that saturation of the probe was 
achieved, and the saturation fraction could be calculated and plotted. 
Such a plot is shown in FIG. 4B. 
The present invention is not to be limited in scope by the microorganisms 
deposited since the deposited embodiments are intended as illustrations of 
single aspects of the invention and any microorganisms which are 
functionally equivalent are within the scope of the invention. 
The present invention is not to be limited in scope by the exemplified 
embodiments which are intended as illustrations of single aspects of the 
invention, and any clones, DNA or amino acid sequences which are 
functionally equivalent are within the scope of the invention. Indeed, 
various modifications of the invention in addition to those skilled in the 
art from the foregoing description and accompanying drawings. Such 
modifications are intended to fall within the scope of the appended 
claims. 
It is also to be understood that all base pair sizes given for nucleotides 
are approximate and are used for purposes of description. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 572 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A ) NAME/KEY: CDS 
(B) LOCATION: 56..253 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AAACGTGGATAAAGCAAGTGGATTTGATGCAAAGTAAGGATAAGGAGGGATGACCATG58 
Met 
1 
TTTAGCCGTCTTATCTCAATTGTTAGTTTAATCTTGTCTTTTTACTTT106 
PheSerArgLeuIleSerIleValSerLeuIleLeuSerPheTyrPhe 
51015 
GCTTACAAATATCGCTACCGTGTTATTAATGCAGTGCTCGGACGCCGT154 
AlaTyrLysTyrArgTyrArgValIleAsnAlaValLeuGlyArgArg 
20 2530 
TGGCTGCGGAAAGTGATTATTGGTTTTGCGATGCAGATCCCGATGATC202 
TrpLeuArgLysValIleIleGlyPheAlaMetGlnIleProMetIle 
35 4045 
AGAGACCGTATGCTAGGATCCGTTCTGCAGTCTAATCGACCTCAAAAT250 
ArgAspArgMetLeuGlySerValLeuGlnSerAsnArgProGlnAsn 
5055 6065 
GTGTAACAAACAAGAAAGTCTGATGTTCACTTTAATGAAAGTTAAAGTATGGC303 
Val 
ATCAGACTTTTTCGTATATATACATACTATAAGGCTTATTAATGTCCTAACCTTTAGAGC363 
CCTTGTTATACT ATAAGGGATAGGAGGATTGTATCTTGAAAGTGAAGTGGAGTACGATAT423 
GTTTTGATTTAGATAATACGTTATATAACCATGAGTATGCTTTTAAGCGTGCGATTAAAC483 
AATGTTACTATACAAAACTTCAGCAATGGAAGATATCCGTTGATCACGCTCCTCCATT TG543 
AAGCATGGTTTACTACATTTAAATATTAT572 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
Met PheSerArgLeuIleSerIleValSerLeuIleLeuSerPheTyr 
151015 
PheAlaTyrLysTyrArgTyrArgValIleAsnAlaValLeuGlyArg 
202530 
ArgTrpLeuArgLysValIleIleGlyPheAlaMetGlnIleProMet 
354045 
IleArgAspArgMetLeuGlySerVal LeuGlnSerAsnArgProGln 
505560 
AsnVal 
65