JOURNAL OF BACTERIOLOGY, Mar. 1997, p. 1497–1504 Vol. 179, No. 5 0021-9193/97/$04.00ϩ0 Copyright ᭧ 1997, American Society for Microbiology

Identification and Characterization of acoK, a Regulatory Gene of the Klebsiella pneumoniae acoABCD Operon

1 2 1 2 HWEI-LING PENG, YIN-HSIU YANG, WEN-LING DENG, AND HWAN-YOU CHANG * Departments of Microbiology and Immunology1 and of Molecular and Cellular Biology,2 Chang Gung College of Medicine and Technology, Kwei San, Tao Yuan, Taiwan

Received 8 August 1996/Accepted 26 November 1996

By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies

of NtrC recognition sequence [CAC-(N11 to N18)-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.

Many bacteria are able to utilize acetoin as a carbon source. homologous to acoR is found in the corresponding region in In some of the bacteria, dissimilation of acetoin proceeds via the C. magnum acoABXCL operon (19). However, the func- an oxidative cleavage process which is catalyzed by an acetoin tion of the gene product has not been determined. We have dehydrogenase enzyme system with acetaldehyde and acetyl also identified an open reading frame, designated acoK, lo- coenzyme A as the end products (13, 22). The acetoin dehy- cated upstream but transcribed from a divergent promoter to drogenase enzyme system is composed of E1␣ and E1␤,an the K. pneumoniae acoABCD operon. In this article, we report acetoin-dependent dichlorophenolindophenol oxidoreductase the complete nucleotide sequence of the acoK gene and dem- (Ao:DCPIP OR); E2, a dihydrolipoamide acetyltransferase; onstrate the regulatory role of AcoK on the expression of the and E3, a dihydrolipoamide dehydrogenase. Structural genes K. pneumoniae aco operon. encoding E1␣,E1␤, E2, and E3 of the acetoin dehydrogenase enzyme system have recently been isolated from several bac- MATERIALS AND METHODS terial species (11, 19, 25, 28). In Alcaligenes eutrophus, the ␣ ␤ Bacterial strains, plasmids, media, enzymes, and chemicals. Bacterial strains structural genes identified for E1 (acoA), E1 (acoB), and E2 and plasmids used in the study are listed in Table 1. The bacteria were grown at (acoC) are clustered with acoX, which encodes a protein of 37ЊC in Luria broth (LB) unless otherwise indicated. Restriction endonucleases unknown function in the order of acoXABC (28). The struc- and T4 DNA ligase were obtained commercially and used as directed by the suppliers. All chemicals were reagent grade and were purchased from either tural gene for E3 which is not found in A. eutrophus aco Sigma Chemical Co. (St. Louis, Mo.) or Merck (Darmstadt, Germany). The operon, however, is present as acoL in Pelobacter carbinolicus Sequenase kit and radioactive nucleotides were obtained from Amersham Corp. acoABCLS (25) and Clostridium magnum acoABXCL (19) (Buckinghamshire, England). operons and as acoD in the Klebsiella pneumoniae acoABCD Determination of Ao:DCPIP OR activity. For Ao:DCPIP OR activity assay, the acetoin-induced bacterial cells were harvested by centrifugation, resus- operon (27). Amino acid sequences deduced from these genes pended in 100 mM potassium phosphate buffer (pH 7.5), and permeabilized by exhibit significant homology to those of the respective compo- adding 20 ␮l of toluene. The assay mix contained 100 mM potassium phosphate nent of various 2-oxo acid dehydrogenase complexes, suggest- (pH 7.5), 0.08 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, 0.10 ing an evolutionary relationship among these catabolic sys- mM DCPIP, 0.40 mM acetoin, and 100 ␮l of the toluene-permeabilized bacteria in a final volume of 1.0 ml. The rate of DCPIP reduction was recorded at 578 nm tems. by a Jasco EHC-363 spectrophotometer at 37ЊC. The protein concentration was Despite the wealth of information on acetoin dehydroge- determined by assuming that 109 cells yield approximately 150 ␮g of protein, as nases, how these aco operons are regulated is less understood. estimated previously (23). The acoR gene located upstream and transcribed in the same Tn1000 mutagenesis. Mutagenesis of acoK was performed in Escherichia coli A. eutrophus acoXABC by using the recombinant clone as follows. Plasmid pHP657, which contains acoK direction as the operon is found to be and the acoABC genes, was initially introduced into E. coli CSH41 and then required for expression of the operon (18). On the basis of mobilized by F-mediated conjugation (21) to E. coli HB101. The transconjugants sequence analysis, the AcoR is likely a regulatory protein re- were selected on LB plates supplemented with streptomycin (20 ␮g/ml) and quired for ␴54-dependent transcription of acoXABC. A gene ampicillin (100 ␮g/ml), and the activities of Ao:DCPIP OR of the acoK::Tn1000 mutants were measured. The location of each Tn1000 insertion in acoK was determined by restriction endonuclease digestion followed by sequencing anal- ysis. * Corresponding author. Mailing address: Department of Molecular Recombinant DNA techniques and DNA sequencing. DNA manipulation and and Cellular Biology, Chang Gung College of Medicine and Technol- plasmid DNA isolation were performed as described previously (33). DNA ogy, Kwei San, Tao Yuan, Taiwan. Phone: 886-3-3283016, ext. 5160. sequence determination was carried out by the dideoxy chain termination Fax: 886-3-3283031. E-mail: [email protected]. method (34) with the Sequenase kit. Both universal M13 primer and synthetic

1497 1498 PENG ET AL. J. BACTERIOL.

TABLE 1. Bacterial strains and plasmids used in the study

Strain or plasmid Relevant characteristics Source or reference E. coli Ϫ HB101 F ⌬(gpt-proA)62 leuB6 supE44 ara-14 galK2 lacY1 ⌬(mcrC-mrr) rpsL20 xyl-5 mtl-1 recA13 33 ϩ ϩ CSH41 FЈ lacI proA B ⌬(lac proAB)galE thi 23 Ϫ ϩ Ј q ⌬ NovaBlue(DE3) endA1 hsdR17(rk12 mk12 ) supE44 thi-1 recA1 gyrA96 relA1 lac [F proAB lacI Z M15::Tn10 Novagen (Tetr)](DE3) Plasmids pACYC184 Cmr Tcr 5 pET30c Kmr Novagen pUC18 Ap r 33 pHP654 pHC79 derivative containing acoK and acoABCD 11 pHP657 Deletion derivative of pHP654 containing acoK and acoABC This study pHP669 pUC18 derivative carrying 4.6-kb PstI fragment from pHP657 This study pHP679 Deletion derivative of pHP669 This study pHP738 pHP657; acoK::Tn1000 This study pHP748 pHP657; acoK::Tn1000 This study pHP754 pHP657; acoK::Tn1000 This study pHP756 pHP657; acoK::Tn1000 This study pHP761 pHP657; acoK::Tn1000 This study pHP788 Tcr; 3.6-kb NcoI-EcoRV fragment with acoK in pACYC184 This study pHPA31 Kmr; 2.7-kb PCR product containing acoK in pET30c This study pHPA90 Apr; 0.73-kb PCR product containing aco promoter region in pUC18 This study

oligonucleotides were used, and the sites of banding compressions were resolved cloned into the HincII site of pUC18, and its sequence was confirmed. The by using dITP. Transposon insertion sites in acoK were determined by using a inserted DNA was excised from the resulting plasmid pHPA90 with EcoRI and synthetic oligomer, 5Ј-AACAACGAATTATCTCCTTA-3Ј, corresponding to HindIII and then end labeled with [␥-32P]ATP using T4 polynucleotide kinase. the inverted repeat sequence of Tn1000 (16). The nucleotide and amino acid Linearized pUC18 DNA was used as a nonspecific competitor in the binding sequences were analyzed by using the DNASTAR program (DNASTAR Inc., assay. Serial dilutions of the purified AcoK were incubated with the radioactively Madison, Wis.) on a Macintosh LC computer. labeled DNA in 20 ␮l of a buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM RNA preparation and primer extension. Total RNA was isolated from an MgCl2, 100 mM KCl, 2 mM CaCl2, 10% glycerol, and 2 mM dithiothreitol at early-log-phase culture of either K. pneumoniae CG43 (11) or E. coli 25ЊC for 30 min. Electrophoresis of the reaction samples was performed essen- HB101(pHP657) grown in LB medium supplemented with 0.2% acetoin. Primers tially as described previously (4), that is, on a 4% polyacrylamide gel in 0.5ϫ TBE AR10 (5Ј-AGACCAGTGAGGGTGATTG-3Ј) and AR11 (5Ј-ACTGAGAAGC (33) for 2 to4hat160V. TGAAGCAG-3Ј) are the reverse complements of the regions from bp 29 to 46 ATPase activity assay. ATPase activity of AcoK was determined according to and 139 to 156, respectively (see Fig. 2). The assay mixture for primer extension the method of Jordan and McMacken (17) with some modifications. The ATPase contained 10 pmol of the synthetic oligonucleotide; 20 ␮g of cellular RNA; 0.2 activity was measured in a reaction mixture (30 ␮l) containing 33 mM Tris-HCl ␮ ␣ 32 ϳ ␮ ␮ mM (each) dATP, dTTP, and dGTP; 10 Ci of [ - P]dCTP ( 3,000 Ci/mmol); (pH 7.9), 10 mM MgCl2, 66 mM KCl, 0.5 mM dithiothreitol, 25 M ATP, 1 Ci 5 U of RNasin; and5UofMoloney murine leukemia virus reverse transcriptase of [␥-32P]ATP (5,000 Ci/mmol), and serial dilutions of the purified AcoK. After (Life Technologies, Gaithersburg, Md.). The reaction was performed at 37ЊC for incubation at 37ЊC for 30 min, aliquots (1.5 ␮l) of each reaction mixture were 20 min. An excess of cold dCTP was added, and the reaction was continued for spotted onto a polyethyleneimine (PEI)-cellulose plate and developed in 1 M another 10 min. The primer extension product was analyzed on a sequencing gel formic acid–0.5 M LiCl, and the signals were detected by autoradiography. The by using the sequence ladder generated by the same primer. radioactive areas were excised from the PEI-cellulose plate and placed in a In vivo synthesis of AcoK in E. coli and purification of the recombinant scintillation vial containing 1 ml of Ultima-Flo scintillation fluid (Packard, Me- protein. The coding region of the acoK gene was amplified by PCR with the riden, Conn.), and the radioactivity was determined in a liquid scintillation high-fidelity Pfu DNA polymerase (Stratagene, La Jolla, Calif.) with primers counter. A His-tagged LysR-type regulatory protein, MdcR (unpublished result), 5Ј-ATGAAGCCATTGGATTTAGAA-3Ј, beginning at the first in-frame initia- which was purified under identical conditions, was used as a negative control. tion codon of the gene, and 5Ј-TTAATCCAGTAATTTCATCTC-3Ј, a reverse Calf intestine alkaline phosphatase (Boehringer GmbH, Mannheim, Germany) complement of the sequence containing the termination codon of acoK. The was the positive control. One unit of ATPase activity was defined as the amount PCR product was purified from an agarose gel and cloned into a T7 promoter- of protein that hydrolyzed 1 nmol of ATP under the conditions described above. based expression vector pET-30c (Novagen). The construct would produce an Nucleotide sequence accession number. The GenBank accession number of AcoK with in-frame fusion of a polyhistidine tag to its N terminus that allowed acoK is U10553. the fusion protein to be purified through affinity binding to the nickel-charged resin. E. coli NovaBlue(DE3) carrying the recombinant plasmid was grown in LB supplemented with 25 ␮g of kanamycin per ml at 37ЊC until the optical density RESULTS at 600 nm reached 0.4. Isopropyl-thio-␤-D-galactopyranoside (IPTG) was added Њ to a final concentration of 1.0 mM, and the incubation was continued at 37 C for A DNA fragment upstream of acoABCD is required for ace- 90 to 120 min. Bacterial cells were then harvested by centrifugation, resuspended in 4 volumes of ice-cold binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM toin-inducible expression of the operon. The K. pneumoniae of Tris-HCl [pH 7.9]), and disrupted with a sonicator (Ultrasonic Processor XL; acetoin dehydrogenase E1 component is encoded by the first Heat Systems, Farmingdale, N.Y.). The centrifuge-clarified extracts were applied two genes of the acoABCD operon. The Ao:DCPIP OR activ- to a previously charged 2.5-ml column of His-Bind metal chelation resin (Nova- gen) and washed with 6 volumes of washing buffer (60 mM imidazole, 500 mM ity associated with the E1 enzyme was used in this study as a NaCl, 20 mM Tris-HCl [pH 7.9]) to remove the unbound proteins. The proteins reporter system for measuring the expression level of the bound to the resin were then eluted with 6 volumes of eluting buffer (1 M operon. In a previous study, an approximately fourfold in- imidazole, 500 mM NaCl, 20 mM Tris-HCl [pH 7.9]). The purity of AcoK in each crease in Ao:DCPIP OR activity was found in E. coli carrying fraction eluted from the column was verified on a sodium dodecyl sulfate (SDS)- polyacrylamide gel (20). The protein profiles on the gel were visualized with a cosmid clone of the K. pneumoniae acoABCD operon Coomassie brilliant blue R-250. The protein concentration was determined by (pHP654) upon induction with 0.01% acetoin for 1 h (11). the method of Bradford (3). Initially, restriction endonuclease-mediated deletion of DNA mobility shift assay. The DNA fragment containing the putative control pHP654 was performed to localize the region required for the region of the K. pneumoniae aco operon was amplified by PCR with the primers 5Ј-TCTGATCTCCCGCATCTT-3Ј (AR13; see Fig. 2) and 5Ј-ATGAGCCCGG acetoin-inducible expression. The smallest clone, pHP657, CCAGCAGTT-3Ј (AR38; see Fig. 2), located within the coding sequences of which remains capable of conferring the acetoin-inducible Ao: acoA and acoK, respectively. The amplified 0.73-kb DNA fragment was sub- DCPIP OR activity in E. coli was isolated and analyzed further VOL. 179, 1997 A REGULATORY GENE OF THE K. PNEUMONIAE acoABCD OPERON 1499

FIG. 1. Molecular organization of acoK and acoABCD. Structures of the deletion and Tn1000 insertion plasmids are also included. Ao:DCPIP OR activities of these acoK mutants were determined and are shown on the right. ND, not detectable. N, NcoI; RI, EcoRI; P, PstI; RV, EcoRV. by using restriction enzyme site mapping. In addition to the the region contained in pHP669. The results indicated that the acoABC genes, pHP657 was found to contain an approximately region required for regulating the expression of acoABCD 5-kb upstream region of the aco operon (Fig. 1). extends at least 3 kb upstream of the acoA gene. Two subclones with a deletion in the upstream region were Nucleotide sequence of the regulatory gene. Nucleotide se- constructed to identify the location of the regulatory region for quences of the 3-kb upstream DNA and the five Tn1000- the acetoin-inducible expression of the aco operon. The plas- insertion sites were determined (Fig. 2). The sequence re- mid pHP669, constructed by subcloning a PstI fragment from vealed an open reading frame transcribed from a divergent pHP657 into pUC18, contains complete acoABC genes and an promoter of the acoABCD operon. The open reading frame, approximately 850-bp sequence upstream of the first in-frame designated acoK, is capable of encoding a polypeptide of 921 ATG codon of acoA. Plasmid pHP679, derived from pHP669 amino acid residues with a calculated molecular mass of 104 by exonuclease BAL 31 digestion from the EcoRI site of the kDa. There was no obvious ribosomal binding sequence in the linker, contains the acoABC genes and approximately 350 bp region upstream of the acoK, whereas a potential transcription of the upstream DNA. As shown in Fig. 1, the Ao:DCPIP OR terminator was found in the region downstream of the stop activity detected in the cell extract of either E. coli codon (Fig. 2). In the 253-bp intergenic region between acoA HB101(pHP669) or E. coli HB101(pHP679) was not affected and acoK, a putative NtrC activator recognition sequence by the exogenously added acetoin. The activity in E. coli [CAC-(N11 to N18)-GTG] (15) located immediately upstream HB101(pHP669) was maintained at a basal level as in E. coli of the Ϫ35 sequence of the potential promoter of the aco HB101(pHP657) without acetoin induction. In contrast, the operon (11) was found. Ao:DCPIP OR activity detected in the E. coli carrying pHP679 Mapping of the transcription initiation site of acoK. Primer was comparable to that of the acetoin-induced E. coli extension was used to determine the precise initiation site of HB101(pHP657). The results indicated that the upstream the acoK transcript. RNA samples from two different sources, 850-bp region contained in pHP669 is not sufficient for con- K. pneumoniae CG43 and E. coli HB101(pHP657), and two trolling the acetoin-inducible expression of the aco operon. independent primers (AR10 and AR11) were used in the re- In order to localize the upstream regulatory region further, action. In any combination of the primers and RNA samples, pHP657 was subject to Tn1000 mutagenesis. Five pHP657 de- two transcription start sites were identified (Fig. 3). The major rivatives with a Tn1000 insertion in the upstream region were product (S1) was mapped to a G located in the acoA coding identified. The Ao:DCPIP OR activities in E. coli strains car- region which is 319 bp upstream of the acoK start codon. rying these mutated plasmids were measured and found to be Approximately 50 bp downstream to the S1, the minor product much lower than that of the parental plasmid, suggesting a (S2), which was mapped to a thymidyl residue, was found. positive regulatory role for AcoK. Mapping of the Tn1000 Examination of the upstream sequence of both transcription insertion sites in the upstream region was then done. As shown starts revealed two possible ␴70-type promoters (32), GGAA in Fig. 1, the Tn1000 insertion sites all were mapped beyond TA-N16-CCTGAT and ATCTCC-N18-TGCAAT. Interest- 1500 PENG ET AL. J. BACTERIOL.

FIG. 2. Nucleotide and deduced amino acid sequences of K. pneumoniae acoK. The predicted amino acid sequence is shown above the nucleotide se- quence. The positions and directions of acoK and acoA and the primers including AR10, AR11, AR13, and AR38 are also indicated. The possible transcription initiation sites as determined by primer extension are shown in boldface letters with designations of S1 and S2. The putative promoters of acoK and the potential Ϫ35 region of acoABCD (11) are boxed. Putative NtrC-like upstream activator sequences are underlined. The potential transcription terminator is indicated by inverted arrows. The insertion sites of Tn1000 and the junctions of pHP669 and pHP679 are shown (ö).

identity of 58.4% to the maltose binding protein (MalT), a positive transcriptional regulator of E. coli maltose regulon (9), was found. As shown in Fig. 4, the conserved sequences include

an ATP binding domain (Gly-X2-Gly-X-Gly-Lys-Thr-Thr) at the N terminus and a helix-turn-helix motif of LuxR-type reg- ulatory proteins (14) at the C terminus. Besides the ATP and DNA binding domains, it is interesting to note that a high leucine content (59 Leu out of 228 conserved residues) was found in the two proteins. No homology, however, was found either between AcoK and the AcoR of A. eutrophus (18) or between AcoK and the NtrC (12). AcoK is capable of regulating the expression of acoABCD operon in trans. To demonstrate a regulatory role for AcoK, the NcoI-EcoRV fragment (Fig. 1) containing the entire acoK gene was subcloned into the NcoI-ScaI sites of pACYC184 (5), a plasmid carrying the origin of replication from plasmid p15A that allows coexistence of the vector with another plasmid carrying the ColE1 origin. The resulting plasmid, pHP788, was cotransformed with each of the acoK::Tn1000 plasmids into E. coli HB101. The cotransformants were induced with 0.01% acetoin for 90 min, and their Ao:DCPIP OR activity was mea- sured. Approximately two- to sixfold increases in the Ao: DCPIP OR activity upon induction were observed in the co- transformants, suggesting that the acoK is capable of regulat- ing the expression of acoABCD operon in trans. In addition, we also examined whether the cotransformants carrying pHP788 and one of the deletion mutants, pHP669 and pHP679, re- spond to acetoin induction. As shown in Table 2, upon acetoin ingly, there are also several copies of putative NtrC recognition induction, the Ao:DCPIP OR activity of the E. coli strain sequence located in front of the acoK promoters (Fig. 2). carrying both pHP788 and pHP699 increased to a level com- Amino acid sequence comparison. The deduced amino acid parable to that in E. coli HB101(pHP657). The result is con- sequence of acoK was used to search for homologous data files sistent with the notion that acoK is capable of activating aco- in GenBank. Significant homology with an overall amino acid ABCD expression in trans. A somewhat conflicting result was VOL. 179, 1997 A REGULATORY GENE OF THE K. PNEUMONIAE acoABCD OPERON 1501

to exert its function. To verify the possibility, the ATPase activity of the purified AcoK was determined. As shown in Fig. 7, the purified AcoK clearly possesses ATPase activity. No such activity was detected by using a different His-tagged protein, MdcR, which was purified through an identical procedure, indicating that the ATPase activity is intrinsic to AcoK. Fur- thermore, addition of acetoin (1 mM) or its derivatives such as acetate (1 mM), acetalaldehyde (10 ␮M), 2,3-butanediol (1 mM), and diacetyl (1 mM) to the assay mixture did not signif- icantly affect the ATPase activity of AcoK (data not shown), suggesting that the ATPase activity is independent of any of the molecules.

DISCUSSION

We have shown in this article that a regulatory gene, acoK, FIG. 3. Primer extension analysis of acoK. The transcription initiation site of is required for the acetoin-induced expression of acoABCD acoK was mapped by extension of the primer AR10 (Fig. 2) with reverse tran- scriptase, and the product was analyzed on a 6% polyacrylamide-urea sequencing genes which encode the acetoin dehydrogenase enzyme com- gel. The dideoxy sequencing ladder was generated by the same primer. Lanes C, plex of K. pneumoniae. On the basis of sequence similarities in T, A, and G show the sequencing reaction products. The primer extension the C-terminal helix-turn-helix-containing region, AcoK could products (loaded in lane 1) and the relevant region of the sequence are shown be classified as a member of the LuxR family of transcriptional ء together with the start sites ( ), S1 and S2. activators (14). However, AcoK is a protein of approximately 100 kDa, which is much larger than any other LuxR-type reg- ulators except the MalT protein (30). MalT is so far unique in found in the cotransformant carrying pH679 and pHP788. In the family in the large size of its N-terminal arm (9) that is the E. coli strain, the Ao:DCPIP OR activity remained high comparable to that of AcoK. In addition to the similar molec- and independent of the acetoin induction (Table 2). ular mass, a considerable sequence similarity (54.8%) was Expression and purification of the recombinant AcoK. To found between AcoK and MalT. The putative ATP binding site obtain a sufficient quantity of AcoK for analyzing its biochem- which is located at the N-terminal region of MalT is also ical properties in vitro, the 2,763-bp coding sequence was am- present in the corresponding part of AcoK. Although with no plified by PCR and cloned into the expression vector pET30c known function, a high content of leucine residues is noted in for in-frame fusion with a polyhistidine tag at the N terminus both proteins. Since AcoK is an apparent homolog of MalT, its of AcoK. The constructed plasmid pHPA31 was transformed isolation adds this protein as an additional member to this into E. coli NovaBlue(DE3) and the whole cell proteins were subfamily. The sequence conservation between AcoK and analyzed by SDS-polyacrylamide gel electrophoresis. As dem- MalT also suggests an involvement of a common regulatory onstrated in Fig. 5, a unique protein of approximately 100 kDa mechanism shared by the two proteins. was observed only in E. coli NovaBlue(DE3)(pHPA31) upon Compared to the undetectable Ao:DCPIP OR activity in E. IPTG induction. After purification through a nickel-charged coli strains carrying the acoK::Tn1000 plasmids, the comple- affinity column, a nearly homogeneous AcoK preparation was mentation of the defects by providing AcoK in trans clearly obtained, as shown in Fig. 5. demonstrated a positive regulatory function of the protein. In DNA binding activity of the purified AcoK. We reasoned these cases, the acetoin-dependent activation of the acoABCD that binding to the promoter region of acoABCD by AcoK is an operon was also restored. A similar result was also observed essential step for the protein to regulate the expression of the for the deletion mutant pHP669. In contrast, the E. coli strain operon. A gel mobility shift assay was used to examine the carrying pHP679, in which only 26 codons of acoK were re- possibility. Since the data from the previous experiments (Fig. tained, expressed acoABC constitutively even in the absence of 1 and Table 2) suggest that the region contained in pHP669 but acetoin. Providing an intact acoK gene in trans to the E. coli not in pHP679 is critical for the expression of acoABCD strain did not exhibit any regulatory effect. A likely explanation operon, the region was also included in the study. The 0.73-kb for this phenomenon is that the acoABCD operon is regulated insert DNA from pHPA90, which contains not only the inter- negatively through a cis-acting element which is present in genic region between the acoA and acoK but also extends pHP669 but not in pHP679. The cis-acting element may con- beyond the region contained in pHP679, was therefore se- tain a sequence recognized by AcoK. In the absence of acetoin, lected as the probe for the assay. As the amounts of the puri- binding of AcoK to this region may prevent expression of fied AcoK increased to 80 ng, the extent of the DNA mobility acoABCD operon. This possibility is being investigated. shift became evident (Fig. 6). A shifted complex was still The presence of an enhancer-like element (24) next to the present when an excess of unlabeled pUC18 DNA was used as Ϫ35 regions of the acoABCD (11) and acoK (Fig. 2), as well as a competitor (lane 5 in Fig. 6). The smearing of the bands and in the intergenic region between acoR and acoX of A. eutro- the relatively short shifting distance are possibly due to disso- phus (18), suggests that a similar regulatory mechanism is ciation of the protein-DNA complex during electrophoresis shared by AcoK and AcoR. On the basis of amino acid se- and the relatively large size of the DNA used in the binding quence similarity (18), AcoR belongs to the NifA family of assay. Nevertheless, the results indicated that the AcoK is transcriptional activators which are required for regulation of capable of binding the promoter region of acoABCD operon ␴54-dependent gene expression (24). However, the consensus and the binding is specific. sequence of the NifA and NtrC groups of enhancer binding ATPase activity of the purified AcoK. The finding of an ATP proteins identified in AcoR was not found in AcoK. Also, no binding domain at the N-terminal region of AcoK suggests that apparent sequence similarity was observed between AcoK and the binding and hydrolysis of ATP are required for the protein AcoR. Therefore, although they both are involved in control- 1502 PENG ET AL. J. BACTERIOL.

FIG. 4. Amino acid sequence comparison of K. pneumoniae AcoK and E. coli MalT. The exact matches are boxed, and the putative ATP binding motif and the consensus DNA binding domain (H-T-H) are also indicated.

ling an acetoin catabolic system, the regulatory mechanism obvious Shine-Dalgarno sequence in front of the coding se- operated by AcoK is likely different from that of AcoR. quence, the expression of acoK is likely to follow a control In the absence of inducer, both transcription and translation mechanism similar to that of malT. As was found for the mal of malT are limited to a low level because of its poor promoter genes (7, 36), we have found that the expression of aco operon and the unfavorable ribosome binding site (6). It has been is suppressed in the presence of glucose (unpublished obser- demonstrated that CAP stimulates malT expression by pro- vation), whereas the binding domain, TGTGA-N6-TCANA moting the binding of RNA polymerase to the rather poor (7), of the catabolite activator protein was not found in the promoter (8). Since acoK is preceded by two rather poor pro- intergenic region of the two divergently transcribed units. moters compared to the typical ␴70 promoters (32) and has no Thus, how the expression of aco genes is turned off in response VOL. 179, 1997 A REGULATORY GENE OF THE K. PNEUMONIAE acoABCD OPERON 1503

TABLE 2. Complementation of acoK mutant by plasmid-encoded acoK in pHP788

Ao:DCPIP OR (mU/mg of protein)a Plasmid (in E. coli HB101) With Without acetoin acetoin pHP657 150 40 pHP738 ϩ pHP788 39 11 pHP748 ϩ pHP788 66 11 pHP754 ϩ pHP788 35 17 pHp756 ϩ pHP788 42 12 pHp761 ϩ pHP788 63 14 pHP669 ϩ pHP788 120 45 pHP679 ϩ pHP788 190 185

a One unit of Ao:DCPIP OR activity is defined as the reduction of 1 ␮mol of DCPIP per min. Specific activity of Ao:DCPIP OR is recorded in milliunits per FIG. 6. Mobility shift assay of the putative aco promoter region with the milligram of protein. purified AcoK. The 730-bp DNA isolated from pHPA90 was end labeled with [␥-32P]ATP and included in each reaction as described above. The purified AcoK was added to the assays in increasing concentrations: 0 (lane 1), 20 (lane 2), and 80 (lanes 3, 4, and 5) ng. Approximately a 50-fold concentration of the unlabeled to glucose remains to be investigated. Nevertheless, the pres- DNA isolated from pHPA90 (lane 4) and linear pUC18 (lane 5) are used as the ence of several copies of the putative NtrC recognition se- competitor. C and F on the right represent the DNA-protein complex form and quence might serve as a binding box for AcoK. The possibility the free form of the labeled DNA, respectively. will be tested later in our laboratory by using a DNase I foot- printing assay. Approximately one-third of the known E. coli ␴70-dependent itively regulated by the acoK gene product in the presence of genes have more than one promoter (10); the presence of two acetoin suggests a likely inducer role of acetoin. Consistent acoK promoters is therefore not surprising. Our preliminary with the finding for MalT, AcoK also exhibited an ability to data using a PacoK-luxAB transcription fusion construct also hydrolyze ATP. However, the purified AcoK alone was found confirmed that that the putative promoter region possessed an to be capable of binding to the putative aco promoter in the AcoK-dependent promoter activity (data not shown). The dis- absence of either acetoin or ATP. It is likely that AcoK binds tance between the transcription initiation sites of acoK and to its recognition sites prior to interacting with the effector(s). acoABCD is 85 or 138 bp, depending on which transcription Hence, we speculate that AcoK binds to the recognition sites start site of acoK is used. The close spacing between the two and then activates open complex formation in transcription RNA polymerase binding sites has been shown in many diver- initiation at the expense of ATP while the inducer is present. gently arranged genes (1) that may facilitate either protein- In spite of a close relationship with E. coli in taxonomy, K. protein interactions or the topology alteration of a neighboring pneumoniae itself has not been subject to extensive study be- DNA sequence for gene regulation. cause of its heavy capsule and multidrug-resistant properties. ATP and maltotriose are two effectors for MalT activity (29, 31). MalT binds to an asymmetric hexanucleotide, the MalT The lack of a rec mutant strain, a convenient vector, and a box (35), only if ATP and maltotriose are present. Since acti- delivery system further precluded the bacterium from many vation of open complex formation by MalT does not require studies. Hence, most of the K. pneumoniae genes identified so ATP hydrolysis (31), the actual role of ATP on MalT activity far were characterized in E. coli. Numerous studies have shown remains unclear. The observation that the aco operon is pos- that many proteins of K. pneumoniae and E. coli are function-

FIG. 5. Expression and the purification of the recombinant AcoK. Whole cell FIG. 7. ATPase activity of AcoK. Approximately 1 ␮Ci of [␥-32P]ATP was protein profiles were analyzed by SDS-polyacrylamide gel electrophoresis. Lanes added in each reaction. Lanes 1 to 3 contained 2.5, 25, and 50 ng of the purified 1 and 3 contain total proteins isolated from IPTG-induced cells carrying pET30c AcoK, respectively. Lane 4 contained 50 ng of the AcoK with 1 mM acetoin in and pHPA31, respectively. The whole cell proteins in lanes 2 and 4 were ob- the reaction mixture. The reaction with 50 ng of the MdcR, a His-tagged protein tained from the cells without IPTG induction. Lane 5 is the AcoK purified which was also purified through His-Bind resin, is in lane 5. No protein was through His-Bind resin. The sizes of the molecular mass markers are shown on added in lane 6. Lane 7 is the positive control with calf intestine alkaline the left. The position of the purified AcoK protein is indicated on the right. phosphatase (0.02 U). 1504 PENG ET AL. J. BACTERIOL. ally interchangeable and the regulatory systems for gene ex- nitrogen fixation genes. Annu. Rev. Genet. 20:567–591. ␥␦ pression are highly homologous (2, 26). In our study, we also 16. Guyer, M. S. 1978. The sequence of F is an insertion sequence. J. Mol. Biol. 126:347–365. found that the transcription initiation start sites of acoK in K. 17. Jordan, R., and R. McMacken. 1995. Modulation of the ATPase activity of pneumoniae are identical to those in E. coli carrying the re- the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat combinant plasmid, which reflects the similarity in transcrip- shock proteins. J. Biol. Chem. 270:4563–4569. tional regulation of the two organisms. Therefore, although the 18. Kru¨ger, N., and A. Steinbu¨chel. 1992. Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcali- study was performed primarily with E. coli, we believe the genes eutrophus H16. J. Bacteriol. 174:4391–4400. results are likely the same for K. pneumoniae. Nevertheless, 19. Kru¨ger, N., F. B. Oppermann, H. Lorenz, and A. Steinbu¨chel. 1994. Bio- analysis of the AcoK in its natural host will be carried out. chemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system. J. Bacteriol. 176:3614–3630. 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of ACKNOWLEDGMENTS the head of bacteriophage T4. Nature (London) 227:680–685. 21. Liu, L., and C. M. Berg. 1990. Mutagenesis of dimeric plasmids by the The study was supported in part by grants from Chang Gung College transposon ␥␦ (Tn1000). J. Bacteriol. 172:2814–2816. of Medicine and Technology (CMRP457 to H.-L.P. and CMRP459 to 22. Lopez, J. M., B. Thoms, and H. 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