JOURNAL OF BACTERIOLOGY, Feb. 1989, p. 1192-1195 Vol. 171, No. 20021-9193/89/021192-04$02.00/0Copyright © 1989, American Society for Microbiology

Wide Distribution of Homologs of Escherichia coli Kdp K+-ATPaseamong Gram-Negative Bacteria

MARK 0. WALDERHAUG, E. DAVID LITWACK, AND WOLFGANG EPSTEIN*

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637

Received 12 July 1988/Accepted 5 November 1988

We used Southern blotting to screen a variety of bacterial genes for homology to the kdp genes of Escherichiacoli, genes that encode an ATP-driven K+ transport system. We found that most enterobacteria have sequenceshomologous to those of the three kdp structural genes and the kdpD regulatory gene. A number of distantlyrelated species, including some cyanobacteria, have sequences homologous to those of the structural genes butnot the regulatory gene. In all cases only a single region of homology was found. These results suggest thatATP-driven transport systems similar to the Kdp system in structure and regulation are found in many entericorganisms. In other gram-negative organisms, the ATPase is more divergent, retaining good homology at theDNA level only to the highly conserved phosphorylated subunit of the ATPase.

Until a few years ago, it was widely believed that ATP-driven cation transport systems, apparently ubiquitous inhigher animals, were relatively infrequent in bacteria. Thisassumption has changed as a number of bacterial ATP-driven systems have been characterized. These includesystems for Ca2+ (11), Na' (7), and a K+-stimulated ATPaseof known DNA sequence in Streptococcus faecalis (19);' theKdp ATPase of Escherichia coli (4); and a K+-stimulatedATPase in Rhizobium sp. strain UMKL20 (12). There ishomology at the protein level of all El-E2 ATPases se-quenced to date (16). These ATPases, also referred to asP-ATPases (15), are characterized by an acyl-phosphateintermediate and sensitivity to vanadate. The homologsinclude examples from gram-negative (8) and gram-positive(19) bacteria, fungi (1, 6), yeasts (17), and higher eucaryotes(10, 13, 18). We decided to investigate whether transportsystems very similar to Kdp exist in other bacteria by usingSouthern blot analysis to search for DNA homology.Chromosomal DNA was prepared from 45-ml cultures

grown overnight at 370C in medium containing tryptone (10g/liter; Difco Laboratories), yeast extract (5 g/liter), and KCI(10 g/liter). The cells were chilled, collected by centrifuga-tion at 5,000 x g for 5 min, suspended in 1 ml of 100 mM Trishydrochloride (pH 8)-10 mM disodium EDTA (pH 8.0)-100mM glucose-lysozyme (2 mg/ml; Boehringer MannheimBiochemicals) and were incubated on ice for 30 min. Sodiumdodecyl sulfate was added to 5 mg/ml and proteinase K(Boehringer Mannheim) was added to 1 mg/ml, and themixture was incubated at 37°C for 1 h. The suspension wasthen extracted with phenol at room temperature, and theaqueous layer was removed. The phenol layer was reex-tracted with 10 mM Tris hydrochloride (pH 8.0)-0.1 mMdisodium EDTA, and the aqueous layers were combined.One-half volume of 7.5 M ammonium acetate was added,and the DNA was precipitated with 2 volumes of ethanolafter incubation for 1 h on ice. DNAs from Saccharomycescerevisiae, Bacillus coagulans, Bradyrhizobium japonicum,and the other cyanobacterial strains tested were provided byThomas Petes, Abdul Matin, Barry Chelm, and RobertHaselkorn, respectively. DNAs from Halobacterium halo-bium, Methanococcus ioltae, and Bacillus subtilis wereprovided by Alvin Markovitz. Synechococcus sp. strain

* Corresponding author.

PCC 6301, Citrobacter freundii CF1 ATCC 8090, andAcholeplasma laidlawii were provided by WolfgangNitschmann, Eduardo Groisman, and Thomas Wilson, re-spectively; all other strains were from the collection of theUniversity of Chicago Hospital and were made available tous by Josephine Morello.The four DNA probes used are shown in Fig. 1. Screening

was done with probe K, which includes the kdpABC operonencoding the three structural proteins of Kdp, part of thepromoter of this operon, and part of the kdpD gene. Theother three probes represent smaller regions: probe A carriesonly the kdpA gene implicated in forming the K+-binding siteof Kdp (3); probe B includes the kdpB gene, which containsthe site of acyl-phosphorylation in these ATPases and in-cludes the smaller regions of homology at the protein levelfound in all other ATPases of this type sequenced to date(16); and probe D contains sequences from the middle of thekdpD gene that encode a positive regulator of expression ofthe kdpABC operon. Probes were prepared by cuttingpWE1001 or pWE1103 with the appropriate restriction en-zymes (Fig. 1), separating fragments by electrophoresis inlow-melting-point agarose (Bethesda Research Laborato-ries, Inc.), cutting out the desired bands, and labeling with[ax-32P]dCTP (3,000 Ci/mol; Amersham Corp.) by the randomoligonucleotide primer method of Feinberg and Vogelstein(5).

Restriction enzyme digestion, gel electrophoresis on 0.9%agarose, transfer to membranes, and hybridization wereperformed as described by Maniatis et al. (14) with twochanges: (i) DNA was transferred to nylon membranes(Biotrans; Schwartz/Mann) instead of nitrocellulose, and (ii)prehybridization and hybridization were done in the pres-ence of Blotto (9) instead of Denhardt solution. The blottedand hybridized membranes were washed in a Blotto solutionwith 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate) for 1 h at 23°C and then 1 h at 55°C. All gels includedthe size standards indicated in the legend to Fig. 1, anegative control of DNA from E. coli TK2281 [F- thi rhalacZ nagA trkA405 trkDl A(kdpAE)81], and a positive con-trol for labeling of probes A and D from strain TK2642,which is like TK2281 but A(kdpDE)54 instead ofA(kdpAE)81. The A(kdpDE)54 deletion retains most of thesequence of probe D and all sequences of the other probes.

Excellent homology was detected when DNAs from a

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NOTES 1193

EoRI

pmD

plasmid pWEl 001

EoRI Miul Mlul Mlul Avail

60600bp 41000 bp :2600 bp

C B

probe K

probe B

A

probe AI-

plasmid pWE 1103

probe D

FIG. 1. kdp genes of E. coli and the regions used as probes. Restriction sites used in constructing plasmid or probe are shown above thebase-pair scale of the region. The arrows represent the two transcripts of the region: that for the three structural genes, A through C, and thatfor the two regulatory genes, D and E. The limits of the five kdp genes are shown by open boxes. Small shaded boxes below kdpB representareas of good protein homology common to all sequenced E1-E, ATPases or P-ATPases. At the bottom are shown the two plasmids and theprobes obtained from them; vector (pBR322) sequences are shown as crosshatched broken boxes. The sizes of the probes are as follows: A,1,783 bp; B, two fragments of 1,719 and 583 bp; D, 1,700 bp; and K, 4,934 bp.

number of enterobacterial species were examined. For mostenterobacteria, a single band with homology was detectedwhen the EcoRI fragments were examined, except for Sal-monella typhimurium and Proteius mirabilis, for which twobands were detected (Fig. 2 and 3). In these cases, only a

single band was detected when the DNA was cut withHindIII, indicating that there is only a single cluster ofsequences homologous to kdp.

Examination of other organisms revealed much weakerhomology at best. DNA from Anacystis nidulans R2, Syn-echococcus sp. strain PCC 6301, Anabaena sp. strain 7120,Anabaena torulosa, Anabaena sp. strain L31, and B. japoni-cum species hybridized at the same level of stringency butnot to the same degree. Hybridization to DNA from a

number of other species (A. laidlawii, B. coagulans, B.subtilis, H. halobitim, M. voltae, Rhodobacter sphaeroides,S. faecalis, Streptococcus sanguis) and from the yeastSaccharomyces cerevisiae did not reveal homology afterwashing under conditions of weak stringency (2x SSC atroom temperature). Prolonged exposure of the labeled blotof EcoRI-cut H. halobium DNA showed a relatively darkband at 23 kilobase pairs (kbp) that stood out from a diffusebackground, and Saccharomyces cerevisiae showed a bandof about 3 kbp upon long exposure (data not shown). Theseresults are of dubious significance, because distinct bandswere seen in the negative control upon long exposure (Fig.3).The species exhibiting the best homology (Fig. 2) had good

homology to all three of the smaller kdp probes. Theseinclude probe A for the kdpA gene involved in K+ binding,probe D for the gene required for expression of the Kdpsystem (Fig. 4), and probe B for the regions of homologyretained in all transport ATPases of this class (Fig. 1). Inother species the homology was weaker, requiring longerexposure of the blots to show bands when probe K or B (Fig.3) was used. In these species homology with probes A and Dwas weaker still (data not shown). This finding is consistentwith the much higher homology of distantly related ATPasesto the KdpB subunit, which forms the phosphorylated inter-mediate, than to other regions of kdp.

...~~~~~~~~~~~~~~~~~~~~........................0...........

.. .......

*:.~~~~~~~~~~~~~~~~~~~~~~~~.............:...~~~~~~~~~~~~~~~~~~~~...........

*~~~ ~ ~ ~ ~ ~:: : ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......~~~~~~~~~~~~~~.. ...

: 8~~~~22:

...~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~.............

*.: :.. :.:.:.:..~~~~~~~~~~~~~~~~~~~~~~~~~~~~. .

FIG. 2. Hybridization of probe K to enterobacterial species.

EcoRI-cut chromosomal DNA was separated on 0.9%t agarose for 14

h at 2 V/cm. Three micrograms of DNA was applied per lane, or less

when hybridization was especially efficient; 0.3 plg of DNA was run

in the positive control and standard lanes. Hybridization of blots

was done by adding 1 x 106 cpm of boiled probe (1.7 x 107 cpm/tgof DNA). The incorporation of label into probe was measured by

Cerenkov counting in water, with no correction for efficiency.

Washed blots were exposed to Kodak X-AR film at -70°C for 18 h.

The autoradiogram shows the following DNAs: lane 1, positive

control; lane 2, negative control; lane 3, C. f,-eundii CF1; lane 4,

Salmonella tvphimulriumn SL4; lane 5, Shligella flexneri; lane 6,

Yersinia pestis; lane 7, SerratiaJm.arc escens; lane 8, K.pne.moniae;

lane 9, Enterobacicer cloacae; lane 10, P. a~eruginosaJ; and lane 11,

standards.

BamHI

E

EcoRI

V=. I I I

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1194 NOTES

1 2 3 4 5 6 7 8 9 10 11

9300 bp7100

.:_0i:.:.'

__NWM

..

-.. 622

FIG. 3. Hybridization of probe B with cyanobacterial and Proteus DNA. The conditions of electrophoresis, blotting, and labeling were thesame as those described in the legend to Fig. 2. Exposure of the blot was for 7 days at -700C. The autoradiogram shows the following DNAs:lane 1, positive control; lane 2, negative control; lane 3, Anacystis nidulans R2; lane 4, Synechococcus sp. strain PCC 6301; lane 5, Anabaenasp. strain 7120; lane 6, Anabaena sp. strain L31; lane 7, Anabaena torulosa; lane 8, B. japonicum; lane 9, Proteus mirabilis; lane 10, Proteusvulgaris; and lane 11, standards. No band is visible in lane 5, but in other experiments a single band of approximately 9 kbp was observed.

In control experiments we detected sequences homolo-gous to pBR322 in Shigella flexneri and Klebsiella pneumo-niae. For the former there was only a single band in DNA

1 2 3 4 5 6 7 8 9 1 0 digested with HindIII; for the latter there were four bands,whether cut by HindIII or EcoRI. The same bands were alsoseen when these species were examined with probes A andB, indicating pBR322 contamination of those probes.

4-+ 8500 bp These results demonstrate the presence of sequenceshomologous to kdp in a number of gram-negative species.

4- 6900 There was extensive and good homology to the sequences inmany enterobacteria and Pseudomonas aeruginosa, butmore limited and weaker homology in cyanobacteria. In all

4- 2800 cases only a single homologous band was detected with atleast one restriction enzyme, implying that the sequenceshomologous to kdp are clustered in all of the species tested.The sequence in S. faecalis, known from sequence anal-

ysis of cloned genes to be moderately homologous to kdp atthe protein level (141 of 583 amino acids identical [19]), was

_I _not detected here, as expected from the extremely poor4 1700 homology at the DNA level. A Kdp homolog appears to exist

in the thermophilic gram-positive acidophile Bacillus aci-docaldarius because there is cross-reactivity with antibodiesto Kdp of a 70,000-dalton protein seen only in cells grown atlow K+ concentration (2). Our hybridization data and infer-ences from the sequences of distantly related organismssuggest that transport ATPases of the E1-E2 class will befound in many, probably most, gram-negative and gram-

FIG. 4. Hybridization of probe D with enterobacterial DNA. The positive bacteria.conditions of electrophoresis, blotting, and labeling and the samples Our data for homologs of kdp genes differ somewhat fromused were the same as those described in the legend to Fig. 2, except predictions of the bacterial phylogeny presented by Woesethat probe D was used instead of probe K. Exposure was for 18 h, (20). We did not detect homology to sequences of gram-as for Fig. 2. positive bacteria but did observe homology to sequences of

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NOTES 1195

some cyanobacteria that are supposed to be more distantlyrelated to enteric organisms. The enteric organisms belongto the y-3 branch of the purple bacteria in that classification.We did not detect homology in R. sphaeroides, a member ofthe ot-3 group of purple bacteria, but did detect homology inB. japonicum, presumably an a-2 purple bacterium. Theimperfect correlation with the phylogeny could be explainedif kdp genes were lost from some branches. It is also possiblethat in some instances kdp genes were obtained from transferacross group boundaries rather than inheritance from acommon ancestor. Less plausible, but to be considered, isthe possibility that genes encoding nonessential proteinshave a different evolutionary flexibility than those encodingrRNA.

We thank the colleagues mentioned who provided the bacterialstrains or DNA samples used in this work.

This work was supported by a Chicago Heart Association fellow-ship to M.O.W. and by Public Health Service grant GM22323 fromthe National Institute of General Medical Sciences.

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2. Bakker, E. P., A. Borchard, M. Michels, K. Altendorf, and A.Siebers. 1987. High-affinity potassium uptake system in Bacillusacidocaldarius showing immunological cross-reactivity with theKdp system from Escherichia coli. J. Bacteriol. 169:4342-4348.

3. Dorus, E., J. E. Hesse, and W. Epstein. 1985. The cation-bindingsite of Kdp, a bacterial transport ATPase, p. 743-746. In 1.Glynn and C. Ellory (ed.), The sodium pump. The Company ofBiologists, Cambridge.

4. Epstein, W., V. Whitelaw, and J. Hesse. 1978. A K' transportATPase in Escherichia coli. J. Biol. Chem. 253:6666-6668.

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12. Lim, S. T. 1985. K+-ATPase from Rhizobium sp. UMKL 20.Arch. Microbiol. 142:393-396.

13. MacLennan, D. H., C. J. Brandl, B. Korczak, and N. M. Green.1985. Amino-acid sequence of a Ca2+ + Mg2+-dependent AT-Pase from rabbit muscle sarcoplasmic reticulum, deduced fromits complementary DNA sequence. Nature (London) 316:696-700.

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15. Pedersen, P. L., and E. Carafoli. 1987. Ion motive ATPases. I.Ubiquity, properties, and significance to cell function. TrendsBiochem. Sci. 12:146-150.

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17. Serrano, R., M. C. Kielland-Brandt, and G. R. Fink. 1986. Yeastplasma membrane ATPase is essential for growth and hashomology with (Na+ + K+)-, K'-, and Ca2+-ATPase. Nature(London) 319:689-693.

18. Shull, G. E., A. Schwartz, and J. B. Lingrel. 1985. Amino-acidsequence of the catalytic subunit of the (Na+ + K+)ATPasededuced from a complementary DNA. Nature (London) 316:691-695.

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