Identification of the gene encoding lipoate-protein ligase A of ...

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carya, the Bacteria, and the Archaea (Herbert and Guest,. 1975;. Dupre et al., 1980; No11 and Barber, 1988). In the bacterium. Escherichia coli, the two primary ...
THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269,No. 23, Issue of June 10,pp. 16091-16100, 1994 Printed in U.S.A.

Identification of the Gene Encoding Lipoate-Protein Ligase A of Escherichia coli MOLECULAR CLONING AND CHARACTERIZATION OF THE 1plA GENE AND GENE PRODUCT* (Received forpublication, January 21, 1994, and in revised form, April 1, 1994)

Timothy W. Morris*, Kelynne E. Reed*§, andJohn E.Cronan, Jr.aI1 From the Departments of $Microbiology and IBiochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 R(+)-Lipoic acid is a cofactor required for function of only when covalently attached to the respective transacylase the a-keto acid dehydrogenase and glycine cleavage (E21 en- subunits of lipoate-dependent enzymes, thus servingas a classical “swinging arm” for shuttling reaction intermediates zyme complexes. The naturally occurring form of lipoate is attached by amide linkage to the r-amino group of a between different active sites within large multisubunit enspecific lysine residue within conserved lipoate-acceptzyme complexes (Perham, 1990). The free carboxyl group of ing protein domains. Lipoate-protein ligase(s) catalyze lipoic acid is joined by amide linkage to the €-amino group of a the formation of this amide bond between lipoyl groups specific lysine residue within each lipoate-accepting protein and specific apoproteins.We report the isolation of the domain. This specific attachment of a lipoyl group to only a lplA gene which encodes an Escherichia colilipoate-pro- single lysine of E 2 apoprotein domains is catalyzed by the litein ligase. Strains with lplA null mutations transport poate-protein ligase(s). Reed et al. (1958) first described lilipoic acid normally but have severe defects in the in- poate-protein ligases inStreptococcus faecalis as well as E. coli corporation and utilizationof exogenously supplied li- and postulated that lipoate is joined to protein in a two-step poic acid and lipoic acid analogs. These strains are also ATP-dependent reaction with lipoyl-AMP as an activated interhighly resistant to selenolipoate (a growth-inhibiting limediate. This reaction sequenceanalogous is to thatof the well poate analog) and contain no detectable lipoate-protein ligase activity in cell extracts. The lplA gene has been characterized biotin-protein ligase of E. coli (Cronan, 1989; cloned, sequenced, and physically mapped to min99.6 Abbott and Beckett, 1993). Further work in mung bean seed(4657 kilobases) of the E. coli chromosome. Upon over- lings (Mitra and Burma,1965) and bovine liver (Tsunoda and 1967) indicated that a similar pathway operates in expression, the 38-kDa lplA gene product was purifiedYasunobu, to homogeneity and shown to have a mass, N-terminal se- eukaryotes, although thecomposition and detailedenzymology fully described in any system. quence and amino acid composition consistent with theof the ligaseenzyme has not been of E. coli deduced 337 residue primary sequence. Enzyme assays However, recent progress in the characterization genes involved in lipoate synthesis (Reed and Cronan, 1993; show that purified LplA catalyzes the ATP-dependent attachment of [36Sllipoic acid to apoprotein, thus con- Hayden et al., 1993; Vanden Boom et al., 1991) as well as the A. Anal- ready availability of plasmid clones for the overproduction of firming that lplA encodes lipoate-protein ligase ysis of lplAnull mutants also indicates the existence of a lipoate-accepting apoproteinshavestimulated renewed bioE. chemical studies of protein lipoylation in E. coli (Brookfield et second(lplA-independent)lipoyl-ligaseenzymein coli. This is the first identification of a lipoate ligase al., 1991) and bovine mitochondria (Fujiwara et al., 1992). Degene and the first analysis of a purified lipoate ligase spite this renewed interest, no gene encoding a known lipoyl enzyme. ligase has been identified from any organism. Here, we have isolated an E. coli lipoate-protein ligase gene (IplA) by transposon mutagenesis and enrichmentfor mutants unable to utiLipoic acid (1,2-dithiolane-3-pentanoic acid) is an essential lize exogenous lipoate. Molecular cloning and overexpression of disulfide cofactor in the central metabolism of an extremely lplA resulted in the ready purification of its gene product, liwide range of organisms, including representativesof the Eu- poate-protein ligase A. Unexpectedly, characterization of lplA carya, the Bacteria, and the Archaea (Herbert and Guest, 1975; null mutants also indicates that E. coli contains a separate Dupre et al., 1980; No11 and Barber, 1988). In the bacterium lipoyl-ligase enzyme other thanthat encoded by the lplA gene. Escherichia coli, the two primary lipoic acid-dependent enEXPERIMENTAL PROCEDURES zymes are the pyruvate and &-ketoglutarate dehydrogenase Bacterial Strains and Media-The E. coli K-12 strains used in this protein complexes (Vanden Boom et al., 19911, both of which are work were derived from strain JKl(rpsL), a streptomycin-resistantprocritical for proper functionof the citricacid cycle during aerobic totrophic strain from the laboratory of J. Konisky. Strain constructions growth. It haslong been appreciatedthat lipoate is functional utilizing bacteriophagePluir transductional crosses werecarried out by conventional methods (Miller, 1972). Strains KER176 (rpsL lipA::dKn), (rpsL lipA::dKnfadE), and KER310 (rpsL1ipA::dKn lipB::dTc) * This work was supported in part by Grant AI 15650 from the Na- KER296 tional Institutes of Health. The costs of publication of this article were were described previously (Reed and Cronan, 1993). Strains 143, 144, 148, and 329 (rpsL 1ipA::dKn IplA::dTc) were isolated as tetracyclinedefrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordancewith 18 resistant derivatives of KER176 by the negative enrichment procedure outlined below. The ZplA::dTc alleles of strains 143, 144, 148, and 329 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted were then transduced into KER296 to give the rpsL 1zpA::dKn 1plA::dTc to the GenBankmIEMBL Data Bank with accession number(s) L27665. fadE strains TM129 (lplA143), TM130 (lplA144),TM131 (lplA148), B Present address: Dept. of Microbiology, University of Texas, Austin, TM132 (lplA329),respectively. The tetracycline resistance markers of TX 78712. strains 148 and 329 were also transduced into JK1 to give the rpsL 11 To whom correspondence should be addressed. Tel.: 217-333-0425; 1plA::dTc strains TM134 (ZplA148) and TM135 (lplA329), respectively. Fax: 217-244-6697. Strains KER176andKERB10wereeachtransformed with plasmids

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16092

E. coli Gene Encoding Lipoate-Protein Ligase

A

pKR56 (with selection for ampicillin resistance) and pMS421 (with88- washed four times with 2 ml of wash buffer (minimal E salts plus 50 lection for spectinomycin resistance) to form strains TM140 and TM178. pg/ml chloramphenicol and 1% Brij-58) toremove unincorporated lipoic Strain TM169 was constructed by transduction of JK1with the linked acid. Dried membranes were countedin 4 ml of Biosafe Counting Scintetracycline resistance and lip&? markers from KER56 (zbd:: TnlO tillant (Amersham Corp.). lipA.2). A tetracycline-sensitive derivative of TM169 was isolated by Radiolabeling of CulturesinPSILipoate, [l-*4C]Octanoate, or fusaric acid selection (Maloy and Nunn, 1981) and savedTM170. as The ~5SelSelenolip,oate-Strains were cultured overnight in minimal E glutetracyclineresistanceelementfrom TM134 wastransducedinto cose medium with acetate plus succinate and diluted 1:lOO into 1 ml of TM170 to give TM171 (rpsL lipA.2ZplA::dTc). This strain was then fresh media containing0.03-0.7 pCi of [35Sllipoate( 5 5 4 4 0 mCi/mmol), transduced to kanamycin resistance by a P1 lysate of GP150 (T. Sil- 3-5 pCi of [l-'4Cloctanoate (55 mCi/mmol), or 4.5 x lo4 counts/min of havy) to yield the recombination defective derivative TM172 (rpsL lip& [75Selselenolipoate(specific activityundetermined).Aftergrowthto 1plA::dR recA::Kan). Plasmids pTM61-i and pserB59-1 were transsaturation (6-18 h), cells were precipitated with 10% trichloroacetic formedinto strain TM134 to form theampicillin-resistantstrains acid, washed with1%trichloroacetic acid or acetone, and the resulting TM181 andTM183, whereas plasmids pET16b andpTM70 were trans- samples were boiled in denaturing SDS buffer and separated on 10% formed into the E. coli B strain BL21 A DE3 (ompTrB-mB-att::A DE3; acrylamide SDS gels. Specifically labeled [35S]lipoyl-proteins, Studier and Moffat, 1986) to form the ampicillin-resistant derivatives ['4Cloctanoyl-proteins, and [75Selselenolipoyl-proteins were visualized TM201 and TM202. by fluoroautoradiography. [l-'4C10ctanoate was purchased from AmeriCulture media were rich broth (Davis etal., 1982) or minimal salts can Radiolabeled Chemicals. [WILipoate and [75Selselenolipoate were medium E (Davis etal., 1980) supplemented with0.4% glucose and, as synthesized and purified as described previously (Reed et al., 1994). needed, 0.5-50,000 ng/ml DL-a-lipoic acid (Sigma),5 rn sodium acetate, Lipoate Protein Ligase Assay-LPL activity was assayed by using 5 mM sodium succinate,0.1% vitamin-free casein hydrolysate(Difco),30 [35Sllipoicacid as the substrate for incorporation into a n artificially pg/ml streptomycin, 25 pg/ml kanamycin, 3-10 pg/ml tetracycline, 100 enriched preparation of lipoate-accepting ACP-E2p apoprotein. Compg/ml ampicillin, 30 pg/ml spectinomycin, and 30pg/ml chloramphen- plete LPL reactions included 0.018-500 pg of extracts or fractions to be icol. Selenolipoic acid (1,2-diselenolane-3-pentanoicacid) was syntheassayed for LPL activity, heat-treated ACP-E2p apoprotein from 6.0 t o sized as previously described (Reed et al., 1994) and added to minimal 500 pg of extract, 0.75-6.0 pv [35Sllipoate(4-210 mCi/mmol), 1.5 rn E glucose media a t 1 0 4 0 0 0ng/ml. Solid media contained 1.5% agar. All Na, ATP, 1.5 mM MgCI,, 0.3 mM dithiothreitol, and25-100 mM sodium cultures were grown aerobically at 37 "C. Growth of liquid cultures was phosphate, pH7.0, in a volume of 0.1 or0.2 ml. Assays were initiatedby monitored with a Klett-Summerson colorimeter equipped witha green the additionof [35S]lipoateto prewarmed reaction mixtures and allowed filter or by measuring the optical densityat 600 nm. to proceed for up to 60 min at 37 "C. Assays of crude material generally Dansposon Mutagenesis and AmpieillinICycloserine Enrichmentsrequired 6-60 min incubations while assays of purified fractions proStrain KER176(1ipA)was mutagenizedby transposition of a mini TnlO ceeded for 2-6 min. During the initial development of the assay and element (TnlOdTc) into the bacterialchromosome from the bacterioph- characterization of the activitypresent incrudeextracts,reaction age A suicide vector ANK1098 (Wayet al., 1984). Cultures ofKER176 (10samples were pipetted onto 25-mm Whatman 3MM filter discs presatuml) in rich broth plus 5 mM acetate and 5 mM succinate were infected rated with 10% trichloroacetic acid and 1 mM unlabeled lipoate. After with ANK1098 particles during mid-logarithmic growth phase a t a ratio drying, the filters were washed four times with a 3:6:1 (by volume) of 0.3 A particledcell and transposition of the TnlOdTc element was mixture of chlorofondmethanoYacetic acid (5 mvfiltedwash) and two induced upon infection by the addition of 1 mM IPTG.' Cultures were times with 95% ethanol to remove free unbound [35Sllipoate.The dried then shaken for 90 min, residual A particles were removed by washing filters were then counted twice in 4 ml of counting scintillant. For cells twice in minimal E salts, and thecells were spread on minimal E subsequent assays, the procedure was modified by pipetting reaction glucose plates supplemented with acetate, succinate, and tetracycline samples directly into 4 ml of ethanovacetic acidlBrij-58 (9 vo1umes:l (10 pg/ml) to selectfor recipients of the TnlOdTc element. After3 days volume:l% by weight)standing above 25-mm GN-6 membranes. of incubation the 2000-4000 tetracycline-resistant coloniedplate were Samples were then immediately filtered through the membranes by pooled, diluted to 2 x lo7 celldm1 into fresh minimal E glucose media vacuum pressure and washed four times under vacuum with 2-ml porsupplemented with acetate, succinate, and tetracycline and shaken at tions of the ethanol-acetic acid-Brij-58 mixture. Dried membranes were 37 "C until exponential growth was observed. Cells from these cultures then counted as above. The reported values for LPL activities were were then washedtwice, resuspended at 1 x lo8 celldml in prewarmed corrected for background noncovalent binding of lipoate to denatured minimal E glucose media containingno supplements, and shaken until proteins trappedby the filtersas well as for the 33% countingefficiency growth ceased due to starvation for acetate and succinate. After 2 of h of filter-bound [35Sllipoyl-proteinrelative to soluble %-labeled samples. starvation, cultures were diluted to1 x 10' cells/ml in 100 ml of fresh Strains t o be assayedfor LPL activity were cultured to mid-logarithminimal E glucose media containing 5 ng/ml lipoic acid. This supple- mic growth phase in minimal E glucose media supplemented with viment restored the activity of the lipoate-dependent a-ketoacid dehydro- tamin-free casein hydrolysate and5 ng/ml lipoate, washed twice in 0.1 genases and thus eliminated the need for supplementation with acetate culture volume of 50 mM sodium phosphate, pH 7.0, and resuspended in plus succinate.Following two generations of lipoate-stimulated growth, 0.01 culture volume of the same buffer plus 10% glycerol and 1 mM the rapidly dividing cells in these pooled cultures wereselectively lysed phenylmethylsulfonyl fluoride. After two passages through a French by the addition of 1 mg/ml ampicillin plus 1 m~ D-cycloserine (Miller, press at 18,000 pounds/square inch, lysates were centrifuged at 100,000 1972).When the opticaldensity of the ampicillinplus cycloserinex g for 1h at 4 "C. These clearedcell extracts were either held on ice for treated cultures haddeclined by approximately 50-fold, the remaining immediate assay of LPL activity or flash frozen a t -70 "C for subseintact cells were collected by filtration, washed extensively to remove quent assay. Control reactions demonstrated that extracts frozen imampicillin and cycloserine, and plated on minimal E glucose tetracy- mediately after preparation retained full LPL activity. Lipoate-acceptcline media supplemented with acetate plus succinate. Enrichments ing apoprotein substrate was provided by identically prepared extracts from four independent cultures yielded a totalof approximately 5000 from strains that overexpressed the ACP-E2p recombinant fusion protetracycline-resistant colonies. tein from plasmid pKR56. For assays of LPL activityin crude extracts, Assay of Lipoic Acid Dansport by Whole Cells-Strains were grown apoproteinwasobtained from lysates of the overproducing strain t o mid-logarithmic growth phase in minimal E glucose supplemented TM140 which had been heat treated (70"C for 5-15 min) to inactivate withvitamin-freecaseinhydrolysateandacetateplussuccinate, endogenous LPL activity, centrifuged a t 10,000 x g for 10 min toremove washed twice in ice-cold starvation buffer (minimal Eglucose plus 50 aggregated material, and then used immediately or stored a t -70 "C. pg/ml chloramphenicol), resuspendedt o a constant optical density of 3.0 For assays of purified LpL4 protein, ACP-E2p apoprotein was from (OD,,, ",) and held on ice. Cells were then prewarmed to 37 "C for 1-5 strain TM178 (IipA ZipB). Briefly, the 100,000 x g supernatant from a min, and uptake reactions were initiated by the addition of l/lOth strain TM178 extract was fractionated by ammonium sulfate precipivolume of [35Sllipoate (80 mCilmmo1) in prewarmed starvation buffer. tation and the 40-80% ammonium sulfate pellet was resuspended in 20 The final concentrationof lipoate in the assay was 750 nM. At indicated mM Tris-HC1, pH 7.5, dialyzed against the same buffer, heat treated time points0.2-ml samples were removed, filtered immediately through (70 "C for 15 min), centrifuged at 10,000 x g for 10 min to remove 25-mm GN-6 cellulose estermembranes(GelmanSciences),and denatured protein, and the heat-soluble 10,000 x g supernatant was concentrated by ultrafiltration to a final protein concentration of 9.4 a t -20 "C and thawed just prior to use. Theabbreviationsusedare:IPTG, isopropyl p -thiogalactoside; mg/ml. This material was stored Proteinconcentrationsweredetermined by the microbiuretassay PAGE, polyacrylamide gel electrophoresis; ACP, acyl carrier protein; (Itzhaki and Gill, 1964) againsta standard of bovine serum albumin. E2p, lipoamide transacetylase subunit; E20, lipoamide transsuccinyRecombinant DNA Methods-Plasmid DNA was isolated and malase subunit; Brij-58,polyoxyethylene 20 cetyl ether; LPL, lipoate-protein ligase; ORF, open reading frame; kb, kilobase(s); bp, base pair(s). nipulated by standard procedures (Sambrook etul., 1989). Preparation

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E. coli Gene Encoding Lipoate-Protein Ligase A of bacterial chromosomal DNAwas as described by Wilson (1987).DNA sequence was generated by the dideoxy chain terminating method of Tabor and Richardson (1987; 1989)using Sequenase 2.0 (U.S. Biochemical Corp.) and double-stranded plasmid templates. The plasmid subclones used for DNAsequencing are described in Fig. 5 A . Chromosomal DNAfrom strain TM134 (lplAl48::TnlOdTc)was digested to completion with a variety of restriction enzymes, separated on 1%agarose gels, and transferred by capillary action to Nytran membranes (Schleicher and Schuell). Membranes were hybridized at 68 "C to a digoxigenin labeled NdeI-BglI restriction fragment of pCTV613 (Vanden Boom et al., 1991) which carries a 1.45-kbportion of the TnlOdTc transposon. Membranes were then washed and positive bands detected with the GeniusnumiPhos 530 system (Boehringer Mannheim). For direct mapping of the lplA gene to an ordered set of phage A clones of the E . coli chromosome (Kohara et al., 19871, the 5.1-kb BamHI-KpnI fragment of plasmid pTM56 (containing chromosomalDNA adjacent to the TnlOdTc element 148) was labeled with digoxigenin (Boehringer Mannheim), hybridized at 68 "C to an ordered array of the Kohara A miniset bound to nitrocellulose (Takara Biochemical), and positive clones were detected with Lumi-Phos 530. Plasmids and Plasmid Constructions-ChromosomalDNAfrom strain TM134 was digested with BamHI and gel-purified fragments of 8-10 kb were ligated into the BamHI site of pSU19 (a derivative of plasmid pSU2719; Martinez et al., 1988) and transformed into strain XL1-Blue (Bullock et al., 1987) with selection for both 50 pg/ml chloramphenicol resistance (encoded by pSU19) and 3 pg/ml tetracycline resistance (encoded by the TnlOdTc element within the desired chromosomal BamHI fragment). The resultant plasmid contained an 8.3-kb BamHI insert and was designated pTM55. Likewise, a 5.1-kb BamHIPvuII chromosomal fragment also carrying the TnlOdTc 148 element from strain TM134 was gel purified and inserted between the BamHI and SmaI sites of pSU19 to form pTM56. The 2.3-kb EcoRI fragment containing the right half of the TnlOdTc element was deleted from pTM56 t o yield the chloramphenicol-resistant and tetracycline-sensitive plasmid pTM58. This plasmid was digested with PstI and BamHI and then treated with exonuclease I11 (Promega) to form a series of nested deletion plasmids for sequencing of the lplA gene. For further DNA sequencing, the 1.7-kb NruI-EcoRI and 1.1-kb SalI-NruI fragments ofpTM58 were deleted to give plasmids pTM21 and pTM24, respectively. Plasmid pserB59-1(Roeder and Somerville, 1979) was provided byR. Somerville and carries the wild type lplA gene on a 5.2-kb BamHI fragment of chromosomal DNA inserted into the BamHI site of pBR322. This 5.2-kb BamHI fragment from pserB59-1 wasthen inserted into the BamHI site of pSU19 to form pTM59.A 1.25-kbHpaI fragment from pTM59 which carried the entire lplA gene was ligated into the SmaIsite of pKK223-3 (Brosius and Holy, 1984)to form plasmids pTM61-1 and pTM614, which placed the ZplA coding sequence under control of the tac promoter (pTM61-4) or in the opposite orientation (pTM61-1) to allow constitutive low level expressionof 1plA. The 1.25-kb EcoRI-Sal1 fragment from pTM61-4was then inserted between the EcoRI and XhoI sites of pMTL22 (Chambers et al., 1988) to yield pTM69, thus allowing lac promoter-driven expression of the lplA gene. The 1.25-kb XbaI-BamHI fragment ofpTM69 was then ligated into pET16b (Novagen)to place the ZplA gene under control of the powerful bacteriophage T7 promoter and ribosome binding sequences (Studier and Moffat, 1986) in the resulting plasmid pTM70. Plasmids pTM60, pTM65, and pTM68 were constructed in vivo by recombination of the TnlOdTc elements from the chromosomes of strains TM129, TM130, and TM132,respectively,ontoplasmidpTM59 using the method of Vanden Boom et aZ. (1991).Plasmid pKR56 was used to overexpress a recombinant lipoate-accepting fusion protein composed of the E . coli acyl carrier protein (ACP) and the lipoamide transacetylase (E2p) subunit of pyruvate dehydrogenase. The 217-bp EcoRI-ClaI fragment of a synthetic acpP gene frompMR16 (Rawlings and Cronan, 1988) was fused to the 684-bpXhoII-Hind111 fragment of the aceF genefrom pGSlOl (Guest et al., 1985)and placed downstream of the IPTG-inducible tac promoter in the expression vector pKK223-3. The expressed ACP-E2p fusion protein consisted of the first 69 residues of ACP fused to the first two lipoate-accepting domains (residues 19-249) of the E2p subunit. Plasmid pMS421 (Grana et al., 1988) carried the lacP repressor gene and was used to regulate transcription of the acp-aceF fusion gene. Overproduction, Purification, and Analysis of LplA Protein-Strains bearing plasmid subclones of the lplA gene were cultured to mid-logarithmic growth phase and (as needed) LplA overproduction was induced by the addition of 0.5 m~ IPTG for2.0-3.5 h and cleared extracts were prepared as described above.Extracts from strain TM202 (which overexpressed the ZplA gene via the T7 expression system) were utilized as

an enriched source of LplA for the two purification schemes described below. For determination of the amino acid composition,LplA was prepared by precipitation in 2040%ammonium sulfate and chromatography through a 1-ml Mono Q column (Pharmacia Biotech Inc.) in 10% glycerol 50 m~ sodium phosphate, pH 8.0, with the LPL activity emerging in the flow-throughvolume. This material waselectrophoresed through a 10% polyacrylamide SDS gel and the predominant 38 kDa protein band was electroblotted to polyvinylidene fluoride membrane foraminoacidcomposition analysis by the AminoQuant system (Hewlett Packard). In subsequent preparations, LplA was purified by a modified procedure. Briefly, a cleared extract in 1mM phenylmethylsulfonyl fluoride, 100 mM ammonium chloride, 10% glycerol, 20 mM TrisHC1, pH 7.5, was loaded directly onto a 25-ml heparin-agarose (type 11, Sigma) column and eluted with a linear gradient of 100-400 mM ammonium chloride. Fractions containing LplA (180-200 mM ammonium chloride) were desalted by ultrafiltration and applied to a Mono Q column run in 10% glycerol, 20 mM Tris-HC1, pH7.5, and developed with a linear gradient of M O O mM ammonium chloride.The LplAprotein (as well as theLPL activity) eluted from this column at 150 mM ammonium chloride. This purified material was concentrated and desalted by ultrafiltration prior to amino acid microsequencing, mass spectroscopy, and analysis of the LPL reaction requirements. N-terminal sequencing was performed by automated Edman degradation in anApplied Biosystems 470ASequenator.Molecular weight determinations were byelectrospray ionization and matrix assisted laser desorption TOF mass spectroscopy using VG Quattro and VG TofSpec mass spectrometers. RESULTS

Zsolation of Null Mutations in the lplA Gene-In order to identify the gene(s) required for protein lipoylation in E. coli, we reasoned that strains which lacked a functional lipoateprotein ligase would be unablet o utilize lipoic acid provided in the culture medium. By mutagenesis of a strain that was unable t o synthesize lipoate due t o a null mutation in the 1 i A gene, we sought to generate a doubly mutant strain which would grow only under conditions that bypassed any requirement for the lipoate-dependent enzymes. (Although the 1 i A gene is required for the de novo synthesis of lipoate, lipA null mutants are proficient in the uptake and utilization of lipoic acid provided in the medium, Vanden Boom et al., 1991) Accordingly, the lipA strain KER176 was subjected to transposon mutagenesis, and the resulting pools of tetracycline-resistant mutants were passed through a n enrichment scheme designed to remove cells able t o utilize exogenously supplied lipoate. Survivors of this ampicillidcycloserine enrichment were initially propagated on minimal E glucose media supplemented with acetate plus succinate (to provide metabolic bypasses of thepyruvateanda-ketoglutarate dehydrogenases, respectively) and then screened for growth on minimal E glucose tetracycline plates supplemented with all possible combinations of lipoic acid (5 ng/ml), acetate, andsuccinate. This screen identified four candidates that grew only when supplemented with both acetate and succinate (regardless of the presence or absence of lipoic acid). These four isolates were designated strains 143,144,148, and329 and retainedfor further study as putative lipoate-protein ligase deficient (1plA) mutants. Phage P1 lysates were prepared from the four candidates as well as the parent strain KER176. When all possible transductional crosses between these strains were performed, we found that only the lysate from the KER176 parental strain contained transducing particles that restored lipoate-dependent growth to the putative lplA isolates, suggesting that allfour mutants fell into the same linkage group. Furthermore, P1-mediated transduction of the tetracycline resistancemarkers from strains 143,144,148, and 329 into a second lipA strain (KER296) yielded new strains (TM129 t o TM132, respectively) with the same growth defects as the original isolates, indicating that the lipoate utilization defect was conferred by the TnlOdTc insertions. Growth studies further showed that in contrast to strains KER176 and KER296, strains TM129 to

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E. coli Gene Encoding Lipoate-Protein LigaseA TABLEI

Lipoate related growth defects in lipA and lipA lplAnull mutants Strains were cultured in minimal E glucose media supplemented with acetate plus succinate and then dilute inocula were transferred to fresh media containing supplementsas indicated. Growth phenotypes were scored a s (++) for the rapid growth (-70 min doubling time) andhigh culture 3.3) observed for strains with functional lipoylated enzymes; (+)for the much slower growth (-200 min doubling time) and lower densities (OD, culture densities (OD,,, 2.2) observed when lipoate-deficient strains were supplemented with acetate and succinate; and (-1 for no detectable growth. Supplements were: 5 6 0 , 0 0 0 ng/ml lipoate, 5 mM sodium acetate, 5 mm of sodium succinate, 3-8 pM 8-thiooctanoate, and 48-480 p~ 6-thiooctanoate.

-

-

Supplements added to minimal E glucose media Defective functions

JK1 (wild type) KER176 KER296 (1ipA) TM131 TM132 (lipA 1plA )

None

++

Lipoate Synthesis

None

++ -

++

Acetate+ succinate

Lipoate

++

++

+

++

++

Lipoate, acetate+ succinate

8-Thiooctanoate

6-Thiooctanoate

++ ++

++

Lipoate Synthesis & Lipoate utilization

TM132 failed to utilize either of the lipoate analogs 6-thiooctanoic acid or 8-thiooctanoic acid as lipoic acid substitutes (Table I). When the lplA null alleles 148 and 329 were transduced into the otherwise wild type strain JK1, the resulting strains TM134 and TM135 were resistant to high concentrations (4000 ngt’ml) of selenolipoic acid (1,3-diselenolane-3-pentanoic acid). In contrast, this selenium-containing lipoate analog was previously shown to inhibit growth of the parental strain JK1 at concentrations as low as 10 ngt‘ml (Reed et al., 1994). Thus, transposon insertions into thelplA gene resulted in defective incorporation of lipoate and lipoate analogs as well as in complete resistance to the growth inhibiting analogselenolipoate. Lipoic Acid Uptake by lplA Null Mutants-The above data 0 10 20 30 indicated that the lplA gene was required for lipoate utilizamlnutes tion, but it was not clear in which metabolic step(s) this gene FIG.1. Lipoate uptake by wild type and ZpIA strains. Uptake of participated. Thelipoate-dependent enzymes are located intra- [35Sllipoic acidby whole cells was determined as described under “Excellularly and thus the growth defects of lplA 1ipA strains could perimental Procedures.” Each point is the average of three separate have been due todefective lipoate transport into the cytoplasm. assays of strains JK1(B, wild type) and TM134 (0,1pZA). Therefore, we examined the kinetics of [35 Sllipoic acid uptake by the wild type strain JK1 and its isogenic 1plA::TnlOdTc SDS gels as described under “Experimental Procedures.” As derivative TM134. As shown in Fig. 1, the rate and extent of evident in Fig. 2, the EplA null mutants all demonstrated a severe defect in the accumulation of [36S]lipoyl-proteinswhen lipoate transport were essentially identical in these strains. compared with otherwise isogenic parent strains. This defect Both the wild type and lplA strains accumulated free (nonprotein bound) lipoic acid to levels approximately 10-fold above was observed in both wild type and 1ipA genetic backgrounds. the extracellular concentration. As assessed by the ability of Since we had previously shown that E. coli K-12 strains effi[35S]lipoate ciently incorporated the lipoate analogs octanoic acid and selwild type strains to subsequently ligate transported (Reed et al.,1994), lplA mutants to intracellular proteins (data notshown), this assay detected enolipoic acid into E2 proteins true uptake rather than nonspecific binding of lipoate to whole were also testedfor the incorporation of these compounds. We cells. Because biotin,a cofactor of similar physical properties, is found that lplA 1 i A double mutants were also severely defecknown to enter theE. coli cytoplasm by simple diffusion when tive for the incorporation of [‘4C]octanoate from the medium present a t high concentrations (Barker and Campbell, 19801, (Fig. 3). Furthermore, the representative strain TM131 (LplA we also tested lplA lipA double mutants for growth on veryhigh lipA) was directly compared with its parent strain KER296 levels of lipoate. We found that all four such strains we tested (lipA) for the ability to incorporate radiolabeled lipoate, octanoate, or selenolipoate. A s shown in in Fig. 4, the lplA strain (TM129 to TM132) failed to grow even when supplemented with 50 pg/ml lipoic acid (a concentration 5 orders of magnitude accumulated almost no [35Sllipoyl-proteins, [‘4C]octanoyl-proabove that required to fully restore growth to the parent strain teins, or [76Se]selenolipoyl-proteins.Quantitation of this gel on KER296). These results strongly suggested that the lplA mu- a Molecular Dynamics PhosphoImager indicated that the lplA mutant incorporated47-fold less lipoate,14-fold less octanoate, tants were not defective in the transport of lipoate. In Vivo Radiolabeling of lplA Mutants-We also examined and at least 65-fold less selenolipoate than the parental wild lpk4 null mutantsfor the ability to ligate transported lipoic acid type strain. LPL Activity in CrudeCell Extracts-Since the invivo radioto the lipoate accepting E 2 proteins of the a-ketoacid dehydrothat the lplA in vivo byculturing cells for five to six labeling defects of lplA strains strongly suggested genases. This was tested gene product participated in the lipoate-protein ligase reacgenerations in minimal E glucose media supplemented with acetate and succinate plus[35S]lipoate.Lysates of radiolabeled tion(s), we sought todirectly measure thelevel of LPL activity cultures were prepared and separatedon 10% polyacrylamide in wild type andlplA cell extracts. Accordingly, we developed an

E. Gene coli

Encoding Lipoate-Protein Ligase A 7sSeLip 35S-Lip 14C-Oct 1 4 8 ..W T1 4 8 . W T1 4 8

35s-Lipoate 1 4134144382W9( T/ p /aAl l e l e )

E2p-Lip

16095 WT ( / p / A a l l e i e )

97 kD-

68 kDE2o-Lip 43 kD-

68 kD-

~E2o-Lip

’-7E20-0ct

29 kD-

43 kD-

FIG.2. In vivo radiolabeling of lpL4 mutants with [“Sllipoic acid. Strains 143, 144, 148, and 329 (with null mutations inlplA and lipA) and the isogenic lipA parent strain KER176 (WT,carrying the wild type lplA gene) were cultured in the presence of labeled lipoate and samples prepared as described under “Experimental Procedures.” All samples were normalized according to optical density of the labeled cultures and analyzedby SDS-PAGE. [3sSlLipoyl-proteins are thelipoamide transacetylase subunit of pyruvate dehydrogenase(E2p)and the lipoamide transsuccinylase subunit of a-ketoglutarate dehydrogenase ( E ~ o indicated ), here as E2p-Lip and E20-Lip, respectively. 14c

35s

Oct Lip 1 4 31 4 41 4 83 2 9W TW T( / p / A

allele)

29 kD-

FIG.4. Defective accumulation of [S6S]-lipoylproteins, [l4C1octanoyl-proteins, and [75Selselenolipoylproteins by an lplA null mutant. Strains KER296 (lipAfudE) and TM131(lipAfudE lplA) were dilutedfrom minimal E glucose plus acetate and succinatemedia into fresh media containing either labeled lipoate, octanoate, selenoor lipoate, and grown for five to six generations prior to analysis by SDSPAGE. Radiolabeled species are asdescribed in Fig. 3.

source of apoprotein, and extracts of wild type strains as a source of enzymatic activity. The enzymatic activity was sensitive to heat treatment (70 “C for 5 min), and the addition of excess unlabeled lipoate or EDTA severely decreased incorporation of [35S]lipoate(Table 11). Since the E2 subunits of E. coli 97 kD E2p are normally fully lipoylated (Packman et al., 1991), extracts of wild type strains demonstrated littleor no lipoylation activity unless a source of apoprotein substrate was provided by the 68 kO addition of a heat inactivated extractfrom an E2apodomainoverproducing strain. Under theseconditions, the LPL activity 43 kC of wild type extracts was20.5 pmol of lipoate incorporatedlmg of total proteidmin whereas extractsof lplA null strains contained 10.12 pmol/mg/min (the averageof the lower limits of detection in six independent experiments). Thus, lipoate-specific LPL activity was severely (and possibly totally) defective LPL activity of in the lplA null mutant. Furthermore, the 29 kC mixed wild type and lplA extracts was approximatelyhalf that FIG.3. In vivo radiolabeling of lplA mutants with [14C]octanoic of wild type extracts (Table 111, indicating that thelplA extracts acid. Strains TM129 to TM132 (carrying thelplA null alleles 143,144, lacked LPL reaction inhibitors. 148, and 329, respectively) and the parent strain KER296 (WT, carrying Physical Mapping and Cloning of the lplA Locus-The posiwild type lplA gene) were cultured in the presence of labeled octanoate, and radiolabeled samples were analyzed as in Fig. 2. Strain KER296 tion of the lplA gene on the E. coli physical map was located by was also labeled with[35S]lipoateand processed in parallel.All strains a combination of Southern blotting methods. First, a digoxigecarried 1 i A and fudE mutations (fudEmutations limit the degradationnin labeled 1.45-kb NdeI- BglI fragment of the TnlOdTc eleand nonspecific incorporation of [‘4Cloctanoate via the P-oxidation path- ment wasused as a probe for the detection of TnlOdTc-containway (Reed et ul., 1994). Specifically radiolabeled proteins are the E2 ing fragments in digests of chromosomal DNA from strain subunits of the pyruvate(E2p),and a-ketoglutarate(E20) dehydrogenase complexes. The octanoylated and lipoylated forms of the E20 protein TM134 (lplA148::TnlOdTc). By comparing the observed sizes of are indicated a s E2o-Oct and E20-Lip, respectively, whereas both forms TnlOdTc-containing chromosomal DNA fragments with the reof the pyruvate dehydrogenase protein are indicated simply as E2p. striction map of TnlOdTc (Way et al., 1984), a low resolution restriction map wasdeduced for the region of the chromosome in vitro assay for this activity utilizing[35S]1ipoicacid and heat- adjacent to the transposon in strain TM134. Alignment of this treated extracts of TM140 as a source of lipoate accepting ap- map with the chromosomal map of Kohara et al. (1987) sugoprotein(see“ExperimentalProcedures”). Although back- gested that the transposon had inserted into bacterial the chroground noncovalent bindingof [35S]lipoateto filters containing mosome a t 4657 kb (min99.6). This mapposition was confirmed TnlOdTc element denatured protein was never eliminated, prewashing of filters by cloning the bacterialDNA adjacent to the and repeated washing of bound samples lowered this back- as plasmids pTM55 and pTM56 (see“Experimental Proceground sufficiently to allow reproducible detection of covalent dures??) andusingthe 5.1-kb BamHI-KpnI fragment from attachment of [35S]lipoate to apoprotein. When analyzed by pTM56 to directly probe the Kohara miniset of h phage carrying ordered and overlapping regions of the chromosome. HybridSDS-PAGE (not shown), radiolabeled samples from in vitro LPL reactions comigrated with [35Sllipoyl-proteinsradiolabeled izations carried outa t high stringency(68 “C)detected only two in vivo, thus confirming that the in vitro reactions produced phage (A 674 and h 675) which carry overlapping fragments from covalently bound lipoyl-protein. We found that LPL activity the 46544661 kb (min 99.6-99.75) region of the chromosome. required two critical components: an artificiallyenriched A search of the GenBank database revealed that thisregion 200 kD

E. coli Gene Encoding Lipoate-Protein Ligase

16096

A

TABLEI1 LPL activity requirements Assays of LPL activity were as described under “ExperimentalProcedures.”All values are the average of duplicate samples. Complete reactions included 0.1-0.2 mg of extracts containing enzyme to be tested, heat-treated ACP-E2p apoprotein from 0.4 mg of strain TM140 extract, 1.5 PM [35Sllipoate,1.5 mMATP, 1.5 mM MgCl,, and 0.3 mM dithiothreitol. In control reactions enzyme or apoproteinwere omitted, or unlabeled lipoate (0.24 mM) or EDTA (LOO mM) were added, or strain JK1 (IplA‘) extract was heated (5 min at 70 “C)prior to the assay, or 0.1 mg of JK1 extract was mixed with 0.1 mg of TM134 (1plA-l extract. The lower limit of detection in this experiment was 0.3 pmoles of lipoate incorporatiodmg extracumin. Reactions proceeded for 6 min at 37 “C.Assays of purified LplA included 0.3 pg of LplA, 280 pg of ACP-ESP apoprotein preparation from strain TM178,6.0 p~ [35Sllipoate,1.5 mM ATP, 1.5 mM MgCl,, and 0.3 mM dithiothreitol. In control reactions, enzyme or apoprotein or ATP wereomitted, or unlabeled lipoate (2.4mM) or EDTA(10 m)or EDTAplus excess MgCl, (30 mM) were added. The lower limit of detection in this experiment was 31 nmol of lipoate incorporatiodmg enzymelmin. Reactions proceeded for 2 min at 37 “C. Activity in total cell extracts Specific Reaction

IplA’ extract No enzyme No apoprotein