©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Construction and Function of Chimeric Subunits Containing Regions from the Subunits of the FF ATPases of Escherichia coli and Bacillus megaterium(*)

(Received for publication, November 1, 1994)

Sharlene R. Matten (§) Randy A. Schemidt William S. A. Brusilow (¶)

From the Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The highly conserved beta subunit of the Escherichia coli F(1)F(0) ATPase was divided into three sections, each of which was exchanged with the homologous section of the beta subunit of the obligate aerobe Bacillus megaterium. Plasmids coding for the resultant six chimeric beta subunits varied in their abilities to complement two E. coli beta mutants as measured by testing transformed cells for aerobic growth on a nonfermentable carbon source or anaerobic growth on rich medium containing glucose. Two chimeras were able to restore both growth on succinate and anaerobic growth on rich medium. The genetic results corresponded to increased levels of membrane-bound ATPase and ATP synthase activities. These chimeric subunits were therefore capable of being assembled into functional E. coli ATPase complexes. The results indicate that chimeric beta subunits can be used to analyze assembly of the beta subunit and that the final 181 amino acids of the beta subunit might contain a region involved in functional energy coupling.


INTRODUCTION

ATP is synthesized by a class of ubiquitous enzymes referred to as F-type ATPases, or F(1)F(0) ATPases. In the cytoplasmic membranes of bacteria, the thylakoid membranes of chloroplasts, or the inner membranes of mitochondria, these enzymes utilize the energy of a transmembrane proton gradient to synthesize ATP from ADP and P(i). They consist of two sectors, an F(0) and an F(1). The F(0) is an intrinsic membrane protein complex, which forms a transmembrane proton channel. The F(1) sector is an extrinsic membrane protein complex, which is bound to the F(0) and which contains the catalytic sites for ATP synthesis or hydrolysis. The F(1) of Escherichia coli consists of five subunits: alpha, beta, , , and (for review, see (1) ). Of these, the beta subunit contains nucleotide binding sites involved in catalysis. The beta subunit has been strongly conserved evolutionarily. beta subunits isolated from sources as diverse as E. coli and cows contain over 60% identity of amino acid sequence(2) .

E. coli is a facultative anaerobe, able to grow both aerobically and anaerobically. During aerobic growth, the ATPase can act as an ATP synthase, using the energy of the proton gradient generated by the electron transport chain to synthesize ATP. Anaerobically, the ATPase can hydrolyze ATP generated from glycolysis to pump protons out of the cell, restoring the proton gradient that is required for transport of nutrients. During an analysis of the ATPase genes of the obligate aerobe Bacillus megaterium, we demonstrated that the beta subunit of this organism can complement E. coli mutants defective in uncD, the gene for beta(3) . Therefore, the B. megaterium beta is capable of being assembled into a hybrid E. coli-B. megaterium F(1) complex. We subsequently demonstrated that this cross-species assembly did not require any other B. megaterium subunits. We also demonstrated that an E. coli beta mutant carrying the B. megaterium beta gene had become an obligate aerobe as a result of carrying hybrid ATPases containing the B. megaterium beta subunit(4) .

A common approach to dissecting the function of a polypeptide or enzyme involves site-directed mutagenesis of individual amino acids suspected of being involved in determining the structure and function of the protein. The ability of homologous subunits from different organisms to be assembled into functional hybrid complexes provides a system for analyzing the effects of large numbers of evolution-driven amino acid changes on subunit assembly, structure, and function. A careful comparison of the differences between homologous subunits and the effects of those differences on subunit assembly and activity represents an alternative approach to site-directed mutagenesis for analyzing the significance of amino acid substitutions on the function of the beta subunit.

This paper describes a more detailed analysis of this cross-species subunit assembly process by dissecting the beta subunits of the two organisms. To determine which regions or residues of the beta subunit are involved in assembly, energy coupling, or both, we constructed chimeric beta subunits consisting of regions from both E. coli and B. megaterium and analyzed the abilities of those subunits to complement an E. coli beta mutant and to restore ATPase-dependent energy coupling to that mutant.


MATERIALS AND METHODS

Plasmids

The E. coli beta-only plasmid, pWSB39, was constructed by digesting the beta plasmid pRPG32 (5) with Tth3 and religating to delete the gene.

Construction of Hybrid beta Subunits

The beta gene of E. coli contains a SalI site and a PstI site, which divide the gene into three sections of 22%, 39%, and 39%. The beta gene of B. megaterium does not contain these sites. Both were added by site-directed mutagenesis of the B. megaterium beta gene in plasmid pSRM100, consisting of an SphI-BamHI fragment cloned from pMAS112 (4) into M13mp19. This insert carries most of the B. megaterium beta gene, including the two sites to be mutagenized. Single-stranded DNA was annealed to mutagenic primers, and the mutated DNA was constructed and isolated as described for the Amersham mutagenesis kit. The sequences of the mutagenic primers were: 1) construction of SalI site, 5`-GGGGCGTCTAAGTCGACTTTTTCACCTAATACG-3`; 2) construction of PstI site, 5`-CGTGATACGCTCCTGCAGCTGACCCATTTCCG-3`. The resultant mutant constructions, pSRM100.1 and pSRM100.2, were identical to pSRM100 except that pSRM100.1 contained a PstI site and pSRM100.2 contained both the PstI site and the SalI site in the identical locations of the PstI and SalI sites in the E. coli beta gene. The only change in the B. megaterium sequence was that isoleucine 103 was changed to valine by the construction of the SalI site.

Having divided both beta subunits into three interchangeable sections, we constructed chimeric beta genes in pSRM103, pSRM104, and pRAS009, by modification of pMAS112, which consists of the entire B. megaterium beta gene cloned into pUC19(6) . The appropriate section or sections of the B. megaterium beta gene were replaced with the homologous section from the E. coli beta gene by standard recombinant techniques. In like fashion, chimeric constructs pSRM107, pRAS007, and pRAS008 were constructed by modification of pWSB39, consisting of the entire E. coli beta gene cloned in pACYC184 (7) .

Other Methods

Complementation assays testing aerobic growth on succinate and anerobic growth on rich medium were conducted as described previously(4) . The strain in which the plasmids were tested were AN818 (FuncD409 argH pyrE recA nalA), described by Cox et al.(8) , and JP17 (FuncD argH pyrE recA::Tn10 entA), described by Lee et al.(9) . Membrane preparation and assay of membranes for ATPase and ATP synthase activities were performed as described previously(4) . LB medium consisted of 10 g/liter Difco Tryptone, 10 g/liter NaCl, and 5 g/liter Difco yeast extract. For anaerobic growth, 0.2% glucose was added to the LB medium.


RESULTS

Fig. 1shows a schematic of the E. coli beta subunit (M(r) = 50286), indicating the locations of SalI and PstI restriction enzyme recognition sites, which divide the protein into three sections of 22%, 39%, and 39%. Using site-directed mutagenesis, we created SalI and PstI sites in the identical locations of the gene for the B. megaterium beta subunit. We were then able to create genes coding for chimeric beta subunits containing all combinations of these three regions from the two different beta subunits, as shown in Fig. 1. The vectors for these constructs consisted of either pUC19 (6) (ampicillin-resistant; for pMAS112, pSRM103, pSRM104, and pRAS009) or pACYC184 (7) (chloramphenicol-resistant; for pWSB39, pSRM107, pRAS007, or pRAS008).


Figure 1: Chimeric beta subunits. The location of the SalI and PstI sites in the E. coli beta gene, and engineered into the B. megaterium beta gene, are indicated at the top of the figure. These two restriction sites divide beta into three fragments containing 22%, 39%, and 39% as shown. The designations for the plasmids containing each chimera are shown on the left, and the identity of each section is designated by either a BM (B. megaterium) or EC (E. coli). Plasmid pWSB39 contains the intact E. coli beta gene, and plasmid pMAS112 contains the intact B. megaterium beta gene.



Each of these plasmids was transformed into two E. coli beta mutants, strain AN818, an uncD mutant that fails to assemble an F(1) on the membrane(8) , and JP17, a chromosomal deletion of uncD(9) . The transformants were tested both genetically and biochemically for ATPase function. Genetically, cells were analyzed for their ability to grow either aerobically on minimal succinate medium or anaerobically on LB medium containing glucose. Aerobic growth on succinate is a test for the ability of the ATPase to synthesize ATP from a proton gradient. Similarly, without an ATPase, cells will not grow anaerobically, presumably because they are unable to couple ATP hydrolysis to proton pumping. For biochemical assays, cells were grown in rich medium to an A of 1, and membranes were isolated and assayed for ATPase activity and respiration-driven ATP synthesis activity. The results are shown in Table 1. The intact E. coli beta gene in pWSB39 and the intact B. megaterium beta gene in pMAS112 complemented both mutants well for growth on succinate. Two of the chimeric beta subunit constructions, pRAS007 and pRAS008, also complemented both mutants. As was the case for pWSB39, both chimeric constructions allowed both mutants to grow aerobically on succinate and anaerobically on rich medium.



Biochemical assays of ATPase and ATP synthase activities also showed that the four plasmids that complemented the mutants (pWSB39, pMAS112, pRAS007, and pRAS008) also restored measurable ATPase activities to membranes isolated from the plasmid-bearing mutants. The activities measured in the uncD deletion strain JP17 were, for most of these plasmids, lower than those assayed in the point mutant AN818 carrying the same plasmid.

The hybrid beta subunit coded for by pRAS007, consisting of the center section from B. megaterium between the N- and C-terminal sections from E. coli, acted the most like the E. coli beta subunit alone, indicating that assembly of a beta subunit into an F(1) complex is affected very little by the differences in amino acid sequence between the center sections of the two beta subunits.

The opposite construction in pSRM104, consisting of the N- and C-terminal sections from B. megaterium combined with the center section from E. coli, did not complement the AN818 mutant as well as pMAS112. Growth on succinate was very poor, as indicated by the +/- in Table 1. This growth was only marginally better than that produced by the negative control culture carrying pUC18. ATPase and ATP synthase activities produced by cells carrying this construction, however, were about half of those produced by cells carrying pMAS112, which carries the gene for the intact B. megaterium beta subunit. The deletion strain JP17 carrying pSRM104, however, produced no measurable ATPase or ATP synthase activity and did not grow at all aerobically on succinate medium or anaerobically on rich medium.

The other construction that was capable of genetically or biochemically complementing both of the beta mutants was the chimera produced by pRAS008, consisting of the first two sections from B. megaterium and the C-terminal section from E. coli. Cells carrying pRAS008 grew both aerobically on succinate and anaerobically on rich medium, but not as well as cells carrying pRAS007. The assays of ATPase and ATP synthase activities produced by membranes isolated from cells carrying pRAS008 contained about half the activity of membranes isolated from cells carrying pRAS007 or pMAS112 and 15-20% of the activity in membranes from cells carrying pWSB39, the intact E. coli beta plasmid. Membranes isolated from the deletion strain JP17 carrying pRAS008 had the same ATP synthesis activity as JP17 cells carrying pRAS007 but had virtually no measurable ATPase activity. All other plasmids carrying chimeric beta genes produced no genetic complementation and no measurable ATPase or ATP synthase activities in either of the beta mutants.


DISCUSSION

The beta subunit of the ATPase has been the subject of much investigation, as it contains nucleotide binding sites and the catalytic site for ATP synthesis or hydrolysis(10, 11) . It is the most evolutionarily conserved of the ATPase subunits(2) . The effects of many naturally occurring and site-directed mutations in the beta subunit gene have been analyzed. Chemical modification studies on the ATPase have been focused largely on reactive residues in the beta subunit(12) . The recent structural determination of the bovine mitochondrial ATPase has provided the most detail about the structure and function of the beta subunit(13) . Since we demonstrated that the B. megaterium beta subunit could substitute for the E. coli beta subunit(3) , we have attempted to develop a system for constructing chimeric beta subunits to analyze the effects of the many amino acid differences between the two beta subunits on the assembly and function of beta. Of particular note is the energy-coupling defect seen in the E. coli beta mutant complemented with the B. megaterium beta subunit. The resultant E. coli are capable of aerobic growth on succinate, but cannot grow anaerobically on rich medium, and have therefore become obligate aerobes as a result of carrying hybrid ATPases containing B. megaterium beta subunits(4) . A goal of our chimeric beta studies is to localize this coupling defect to a region and possibly to a small number of amino acid differences between the subunits.

We tested the abilities of these plasmids to complement two different beta mutants. Our initial studies had been performed on AN818, which codes for an assembly-defective beta subunit(8) . One possible interpretation of positive results from those studies is that the B. megaterium beta subunit, or the chimeric beta subunits, were acting as chaperones for the AN818 beta subunit rather than, or in addition to, being assembled into a hybrid B. megaterium-E. coli ATPase complex themselves(14) . We therefore repeated the AN818 studies on JP17, a strain carrying a complete deletion of the uncD gene(9) , thus avoiding this potential problem. The genetic results were identical in both strains. Two chimeras, coded for by pRAS007 and pRAS008, complemented both mutants. The results of the biochemical assays, however, supported the concerns about possible chaperone function. ATPase and ATP synthase activities produced by complementation of JP17 by pRAS007 or pRAS008 produced lower ATPase and ATP synthase activities than in AN818. Additionally, several non-complementing plasmids, specifically pSRM103, pSRM104, and pSRM107, produced measurable ATPase activities in AN818 but not in JP17. These differences in measured activities might have been caused by a secondary effect of the foreign, chimeric subunit on assembly of the AN818 beta subunit, producing functional ATPases containing either the AN818 beta subunit alone or together with the chimeric beta subunit. However, the observed complementation and activities produced by pMAS112, pRAS007, and pRAS008 in JP17 must result from functional ATPase containing exclusively the plasmid-encoded beta subunit.

Fig. 2shows an alignment of the two beta sequences, indicating the locations of the SalI and PstI sites used to construct the chimeras. The center section contains the residues involved in nucleotide binding and catalysis. These residues compose the so-called Walker homology sequences A and B, two sequences found by Walker et al.(15) to be common to nucleotide-binding proteins. Sequence A, GGAGVGKT, also called the P-loop, is believed to actually bind to the phosphoryl groups of ATP, and is identical in E. coli and B. megaterium. The lysine residue at position 155 can be modified by pyridoxyl 5`-diphosphadenosine (16) and 7-chloro-4-nitrobenzofurazan (17) and has been implicated in catalysis and coordination of the -phosphoryl group of bound ATP(18) . The recent structural determination of the bovine mitochondrial F(1) has demonstrated that this residue, together with threonine 156, does indeed coordinate the - and beta-phosphoryl groups of the bound nucleotide in that ATPase(13) . Homology sequence B from E. coli, RDEGRDVLLFVD, is slightly different from the homologous sequence of B. megaterium. The arginine residue found at position 235 is replaced by a glutamine residue in B. megaterium, and leucine 239 is replaced by a phenylalanine. Additionally, there is an insertion of a glutamine between aspartate 233 and glycine 234. As shown by the results with chimeric beta construction pRAS007, this section, between the SalI and PstI sites, can be moved from B. megaterium to E. coli with little apparent change in assembly or function of the resultant ATPase. Although during the course of evolution there have been 51 amino acid changes in this region, none destroys the ability of the chimeric subunit containing the N- and C-terminal regions from E. coli to assemble into a functional ATPase. The chimera in pSRM104 contains the reverse construction, in which the E. coli section was transferred to a B. megaterium beta gene. This chimeric beta subunit does not complement either E. coli mutant as well as the intact B. megaterium beta gene alone, although it does restore a significant amount of ATPase activity to the E. coli beta mutant AN818. As discussed above, the ATPase activity produced in AN818 may be the result of interactions between the AN818 beta subunit and pSRM104 chimeric beta subunit, resulting in the assembly into an ATPase of the AN818 beta subunit in addition to, or instead of, the chimeric beta subunit.


Figure 2: Comparison of the primary structures of the beta subunits from E. coli and B. megaterium. Identical amino acids located in the same position are boxed. The locations of the SalI and PstI restriction sites used in the construction of the chimeras is indicated on the sequence. The amino acids composing Walker homology sequences A and B are shown in boldface type and labeled near the start of each sequence. Sequence A is also referred to as the P-loop.



The other chimera that complemented both beta mutants, coded for by pRAS008, differs from the chimera coded for by pRAS007 by the additional replacement of the first 22% of the E. coli beta with the homologous section from B. megaterium and differs from pMAS112 only in the C-terminal section. However, whereas pMAS112 allowed either mutant to grow aerobically on succinate but not anaerobically on rich medium, cells carrying pRAS008, differing only in this C-terminal section, did grow anaerobically. The amino acid differences that cause the coupling defect may therefore be localized to this last section, containing 124 identical amino acid position out of 181 (E. coli) or 185 (B. megaterium). Systematically converting the B. megaterium section to the E. coli section may therefore define certain beta-specific determinants of energy coupling.

Chimeric proteins consisting of domains assembled from regions of homologous proteins have been used to analyze the function of large sections of proteins in catalysis and assembly (for examples, see (19, 20, 21, 22) ). The construction of chimeras represents an alternative approach to site-directed mutagenesis in the analysis of structure-function relationships in enzymes. The studies described here demonstrate that the evolutionarily conserved beta subunit of F-type ATPases can be divided into interchangeable regions, and chimeras of beta can be constructed for the analysis of the beta structure and function.


FOOTNOTES

*
This research was supported in part by American Heart Association Grant-in-aid 93007630 (to W. S. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Grant 7620010 from the American Heart Association, Maryland Affiliate. Present address: U. S. Environmental Protection Agency, Office of Pesticide Programs, 7507C, 401 M St., S.W., Washington, DC 20460.

To whom correspondence should be addressed: Dept. of Biochemistry, Wayne State University School of Medicine, Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-6659; Fax: 313-577-2765.


ACKNOWLEDGEMENTS

We thank Dr. Alan Senior for providing the beta deletion strain JP17.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.