THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 hy The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 7, Issue of March 5, pp. 4126-4130,1991 Printed in U. S. A.

Cloning, Nucleotide Sequence, and Expression of the Nitroreductase Gene from Enterobucter cZoucue*

(Received for publication, July 16, 1990)

Christopher Bryant$, Lauren Hubbard, and William D. McElroy From the Department of Chemistry, “001, University of California, San Diego, La Jolla, California 92093

The “classical” nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously de- scribed. Analysis of the 5“untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.

The “classical” nitroreductases of enteric bacteria have been implicated in the activation of nitroaromatic compounds to mutagenic and carcinogenic metabolites (1-3). The mech- anism of this activation involves a reduction of the nitro functional group to the hydroxylamino intermediate (4-6). This hydroxylamino species has been proposed to undergo an acid-catalyzed deprotonation to an electrophilic nitrenium ion which can readily form an N-(deoxyguanosin-8-y1)-1-amino adduct of the parent compound with the guanine bases of DNA (7). Several bacterial nitroreductases have been identi- fied, including those from Escherichia coli (8-lo), Salmonella typhimurium ( l l ) , and Bacteriodes fragilis (12). However, detailed studies concerning the structural and functional properties of these enzymes have not been reported. We have recently reported the purification and preliminary character- ization of a nitroreductase from Enterobacter cloacae (13). This enzyme is active as a monomer with a molecular mass of approximately 27 kDa. Cofactor analysis has shown that 1 mol of FMN is bound/mol of active enzyme. The enzyme can utilize either NADH or NADPH as a source of reducing equivalents and has a rather broad substrate specificity with

*This work was supported by United States Army Contract DAAK70-86-K-0031. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Marlene A. DeLuca who passed away November 18,1987. Her presence in our lives as teacher, scientist, mentor, and friend will be deeply missed.

The nucleotide sequencefs) reported in thispaper has been submitted

M3 7085. to the GenBankTM/EMBL Data Bank with accession number($

$ To whom correspondence should be addressed.

regards to the electron accepting molecule. Preliminary in- vestigations into the mechanistic details of the electron trans- fer as catalyzed by this enzyme indicate that the reduction of nitroaromatic compounds and quinones proceeds in an oblig- atory two-electron process. Thus, in many respects this en- zyme is similar to the quinone reductases found in most mammalian tissues. Detailed studies of this enzyme have been hampered by the relatively minute quantities of purified pro- tein available. In an attempt to circumvent this problem and gain information concerning the primary structure of the protein, we report here the cloning, expression, and purifica- tion of the cloned E. cloacae nitroreductase in E. coli.

MATERIALS AND METHODS

Reagents-DNA-modifying enzymes and their respective buffers were purchased from standard suppliers including Bethesda Research Laboratories, Stratagene (San Diego, CAI, Pharmacia LKB Biotech- nology Inc., and New England BioLabs (Beverly, MA). XZAP phage vector, E. coli strains XL1 and BB4, pBluescript plasmids, and the X phage packaging kit Gigapack Gold were purchased from Stratagene. Sequenase DNA sequencing kit was purchased from Unites States Biochemical (Cleveland, OH). Accugel40 sequencing grade polyacryl- amide solution was purchased from National Diagnostics (Manville, NJ). Nitrocellulose and an Elutrap electroelution apparatus were purchased from (Schliecher & Schuell). Goat anti-rabbit IgG alkaline phosphatase conjugate was acquired from Bio-Rad. Immobilon-P electroblotting membrane was provided by Millipore (Bedford, MA). 5-Bromo-4-chloro-3-indoyl-phosphate, nitroblue tetrazolium, and isopropyl-P-o-thiogalactoside were purchased from Bethesda Re- search Laboratories. All other reagents were supplied from various sources and were of the highest purity available unless otherwise specified. The nitroreductase-deficient E. coli strain NFR 502 was provided by D. W. Bryant (McMaster University, Hamilton, Ontario, Canada). E. coli strain TB1 was obtained from T. Baldwin (Texas A&M University, College Station, TX).

Amino-terminal Protein Sequence Analysis-A 50-pg sample of partially purified nitroreductase was subjected to sodium dodecyl- polyacrylamide gel electrophoresis as described by Laemmli (14). Gels were soaked in electroblotting transfer buffer (25 mM Tris base, 192 mM glycine, 15% (v/v) methanol, pH 8.2) for 30 min. A sheet of Immobilon-P membrane was cut to fit the gel, prewet in 100% methanol for a few seconds, and washed in deionized water for 5 min. Transfer of the SDS’ polyacrylamide gel electrophoresis separated proteins onto the Immobilon-P membrane was performed at 70 V for 2 h. Electroblotted proteins were visualized by staining with 0.2% Coomassie Blue R-250, 45% methanol, 10% acetic acid. The band corresponding to the nitroreductase was excised, destained with 40% methanol, 15% acetic acid, and washed in water. The excised band was transferred to the loading cartridge of an Applied Biosystem Instruments model 470A gas-phase sequenator equipped with a model 120A on-line phenylthiohydantoin amino acid analyzer.

Preparation of Anti-nitroreductme Antiserum-Anti-nitroreduc- tase antibodies were collected from young male New Zealand White rabbits subsequent to exposure to purified nitroreductase. An immune response to the nitroreductase was elicited through a combination of lymph node, intramuscular, and subcutaneous injections of the nitro-

’ The abbreviations used are: SDS, sodium dodecyl sulfate; MES, 2-(N-morpho1ino)ethanesulfonic acid kb, kilobase(s).

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Cloning the E. cloacae

reductase. The nitroreductase was initially administered by an intra- lymphnodal injection of 20 pg of purified nitroreductase emulsified in 0.2 ml of Freund's complete adjuvant into each hind leg. Six weeks later, the animal was boosted by a single intramuscular and multiple intrascapular injections of 70 pg of nitroreductase emulsified in 0.2 ml of Freund's incomplete adjuvant. Booster injections were repeated at 3-week intervals thereafter. Anti-nitroreductase IgG was isolated from 50 ml of whole blood collected 10 days after the third boost. The serum was fractionated by precipitation between 25-37% saturated ammonium sulfate. The resulting pellet was dissolved in a minimum volume of water and dialyzed against several liters of water. The dialysate was microcentrifuged to remove a slight precipitate (largely IgM), and the supernatant was redialyzed against 10 mM sodium phosphate buffer, 0.15 M NaCl, 0.02% sodium azide, pH 7.8. Antibod- ies reacting with E. coli strain NFR 502; and phage proteins were removed from the purified IgG fraction by incubation with E. coli and phage lysates immobilized on nitrocellulose (15).

Construction of Genomic DNA Library-E. cloacae genomic DNA was isolated essentially as described by Kuliopulus et al. (16). Sub- sequently, 500 pg of the purified genomic DNA was precipitated by the addition of 0.5 volumes of 7.5 M sodium acetate and 2 volumes of anhydrous ethanol and incubated on dry-ice for 2 h. The DNA was collected by microcentrifugation, resuspended in 1 X EcoRI digestion buffer and partially digested with EcoRI at 0.25 units/pg of DNA for 1 h at 37 "C. The EcoRI-digested DNA was size fractionated by electrophoresis on a 0.8% agarose gel. The portion of the gel contain- ing EcoRI DNA fragments of approximately 4-7 kb in size was excised and the DNA extracted by electroelution using an Elutrap apparatus. The DNA was precipitated, resuspended in water, and 0.2 pg of DNA was ligated with 1 pg of AZAP phage DNA using the manufacturer's suggested conditions. The ligation mixture was packaged into A phage coat proteins using a Gigapack Gold packaging kit. The initial library was found to contain approximately lo6 recombinant clones and was subsequently amplified from plate lysates grown on E. coli BB4.

Immunoscreening of Genomic DNA Library-The XZAP DNA expression library was initially screened by the method of Young and Davis (17) using an anti-nitroreductase IgG and a chromogenic im- munodetection assay (18). Approximately 2 X lo4 clones were screened by plating on 85-mm lawns of E. coli BB4 at a density of 5 X io3 plaque-forming units/plate. Immunopositive clones were iso- lated and purified by multiple rounds infections and screenings until a homogeneous population of immunopositive plaques was achieved.

Activity Screening of Immunopositiue Clones-Immunopositive clone were subcloned into pBluescript plasmids using the automatic excision process outlined by the manufacturer (Stratagene). The resultingplasmids were screened for their ability to generate increased levels of nitroreductase activity. 50-ml cultures of E. coli strain BB4 harboring a pBluescript plasmid containing a cloned insert were grown at 32 "C while shaking. After 4 h each culture was adjusted to 1 mM isopropyl-P-D-thiogalactopyranoside so as to induce the lactose promoter present on the pBluescript vector. The cultures were al- lowed to incubate an additional 4 h after which they were harvested by centrifugation at 2,000 X g for 10 min. The cell pellets were resuspended in 1 ml of cold Tris-buffered saline, and cellular extracts were prepared by sonication followed by centrifugation to remove cellular debris. The resulting extracts were assayed for nitroreductase activity using standard assay conditions as previously described (13).

Immunoblot Analysis of Positive Clones-Clones which tested pos- itive for nitroreductase activity were further analyzed in an attempt to characterize the protein(s) reactive with the antibody elicited toward E. cloacae nitroreductase. The proteins were separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto ni- trocellulose paper (19). Rabbit anti-nitroreductase IgG was used in sufficient quantity to detect at least 100 pg of nitroreductase when visualized by incubating the nitrocellulose blot with goat anti-rabbit IgG conjugated with alkaline phosphatase followed by incubation with 5-bromo-4-chloro-3-indoyl-phosphate and nitroblue tetrazolium (18).

Restriction Analysis and Subcloning-Restriction mapping of the cloned DNA insert was accomplished through the analysis of DNA size patterns on 0.8% agarose gels subsequent to single or double digestion with various restriction endonucleases. Comparison of pat-

' E. coli strain NFR 502 is a mutant strain which is deficient in both type I nitroreductases, i.e. nfr A-B- (8). This strain was utilized in an attempt to reduce loss of antibodies specific to the E. cloacae nitroreductase due to interactions with E. coli nitroreductases present in extracts used during immunoaffinity purification of the antisera.

Nitroreductme Gene 4127

terns with known reference points within the vector DNA allowed for the construction of a linear map of the restriction sites within the cloned insert. The relative position of the nitroreductase gene within the cloned DNA fragment was ascertained by the measurement of nitroreductase activity present in cell-free extracts of E. coli harboring the subcloned fragments.

DNA Sequence Analysis-Restriction fragments of the cloned DNA insert were subcloned into pBluescript and sequenced using the dideoxy chain termination method of Sanger et al. (20). Single- stranded DNA templates for the sequencing reactions were prepared from the phagemid form of the pBluescript vectors. The reactions were primed with 17-mer oligonucleotides complementary to specific regions within the multiple cloning site of the vector. In addition, some reactions were primed with 19-mer oligonucleotides comple- mentary to regions within the cloned insert determined during prior rounds of sequencing. Sequence information was entered into a com- puter with the aid of a sonic digitizer and was stored and analyzed using GENEPRO software (Riverside Scientific) implemented on an IBM compatible personal computer.

Overexpression of E. cloacae Nitroreductuse in E. coli-Plasmid pCB18 was constructed by the deletion of a BgZIII-XhoI fragment such that the 5'-end of nitroreductase gene, including the natural ribosome-binding site, was proximal to the T7 promoter of the pBluescript vector. This plasmid was transformed into E. coli strain TB1 and selected on plates supplemented with 250 pg/ml penicillin. Plasmid pGP1-2, which contains the T7 RNA polymerase gene situated behind the PL promoter of X phage (21), was then trans- formed into E. coli strain TB1 harboring a copy of pCB18. Those cells containing both plasmids were selected on plates supplemented with penicillin (250 pg/ml) and kanamycin (50 pg/ml). E. coli TB1/ pCB18/pGP1-2 was grown in LB broth supplemented with 0.5% glucose, penicillin (250 pg/ml), and kanamycin (50 pg/ml). Flasks containing 1 liter of the above media were inoculated with 20 ml of an overnight culture grown at 30 "C. The bacteria were grown at 30 "C with constant shaking to a density such that the absorbance at 600 nm was approximately 2. At this point the X promoter was heat induced by the addition 1 liter of media which was heated to 60 "C and was followed by incubation at 42 "C for 4 h. Cells were harvested by centrifugation and washed with 3 volumes of Tris-buffered saline, pH 7.5. The final pellet was resuspended in approximately 100 ml of extraction buffer (0.1 M Tris-HC1, 1 mM EDTA, 1 mM 2-mercapto- ethanol, pH 7.5).

Purification of Clorwd Nitroreductuse-The nitroreductase ex- pressed from the cloned gene was purified by a modification of the protocol previously reported (13). Briefly, the cells were homogenized with a Branson model 350 Sonicator. The particulate matter was removed by centrifugation at 12,000 X g for 30 min. The clarified cell lysate was then fractionated between 40 and 70% (v/v) with acetone at -30 "C and brought up in 50 mM Tris-HC1, 1 mM 2-mercaptoeth- anol, 1 mM EDTA, 10 p~ FMN, pH 7.5. This sample was loaded onto a Q300 anion-exchange column (2.5 X 24.5 cm) and developed with a 0-1 M linear gradient of NaCl. Those fractions containing nitrore- ductase activity were pooled and loaded onto a 1 X 110-cm column of preparative grade Superose 12 (Pharmacia LKB Biotechnology Inc.) preequilibrated in 50 mM acetate, 50 mM MES, 100 mM Tris, pH 7.0. Those fractions containing nitroreductase activity were pooled and stored at -70 "C.

RESULTS

Genomic Cloning of DNA Coding for E. cloacae Nitroreduc- tase-Immunoscreening of 2 x lo4 plaques of the genomic DNA library resulted in the isolation of 13 positive clones (0.07% of the plaques screened). These clones were converted into the plasmid form of the pBluescript vector and screened directly for the expression of nitroreductase activity. Four of the 13 immunopositive clones were found to direct the expres- sion of nitroreductase activity at levels approximately 100- fold greater than that of control cells transformed with the vector only. Restriction analysis with EcoRI indicated that the cloned inserts for all four clones were of identical size (-7 kb). Thus, subsequent analysis was performed on a single clone which was both immunopositive and activity positive. The plasmid containing the entire 7-kb cloned insert was designated pCB1. Interestingly, the expression of nitrore-

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4128 Cloning the E. cloacae Nitroreductase Gene

ductase activity was independent of induction or the orien- tation of the cloned insert relative to the lac promoter of the pBluescript vector. Western blot analysis of the crude extracts of E. coli harboring PCB1 indicated that the increased level of nitroreductase activity associated with the presence of this plasmid was also accompanied by the appearance of a single major immunoreactive protein which, as seen in Fig. 1, was found to comigrate with the nitroreductase from E. cloacae.

Restriction Analysis and Subcloning-Restriction analysis of the cloned insert resulted in the mapping of several restric- tion sites within the cloned DNA insert. The subcloning of several restriction fragments and the correlation of nitrore- ductase activity associated with their presence in E. coli allowed for the localization of the nitroreductase gene to a 1.6-kb BgZII-NsiI segment of the cloned insert subsequently referred to as pCB9. The deduced restriction map of PCB1 and the analysis of activity associated with various subcloned fragments is presented in Fig. 2.

DNA Sequence Analysis of the E. cloacae Nitroreductase Gene-A number of restriction fragments from PCB9 were subcloned into pBluescript plasmids to facilitate the sequenc- ing of the entire coding region of the nitroreductase gene on both strands. The strategy employed in determining the DNA sequence of the subcloned fragment present in plasmid PCB9 is presented in Fig. 3. The resulting nucleotide sequence and the deduced amino acid sequence of the major open reading frame presumed to code for the nitroreductase is presented in Fig. 4.

Purification of Expressed E. cloacae Nitroreductase in E. coli-The nitroreductase expressed in E. coli under the T7 promoter is produced at levels 20 and 200 times higher than in E. cloacae under trinitrotoluene-induced and -uninduced conditions, respectively. Under the conditions used here, the nitroreductase constitutes approximately 5% of the total sol- uble cellular protein as judged by specific activity. A summary of the purification protocol developed for the cloned nitrored- uctase is presented in Table 11. This four-step procedure yields 21 mg of purified protein from 6 liters of cells.

DISCUSSION

Genomic Cloning of the Nitroreductase Gene-Several lines of evidence suggest that the fragment of E. cloacae DNA which has been cloned and expressed in E. coli contains the

1 2 3 4 MWt. rs I(

97.4 - 66.2 -

42.7 -

31.0 - ~

4

21.5 -

14.4 - FIG. 1. Western blot analysis of the cloned nitroreductase

gene product. Lane I, partially purified preparation of E. cloacae nitroreductase. Lane 2, crude extract of E. coli BB4 harboringplasmid pCB1. Lane 3, crude extract of E. coli BB4 harboring the parent plasmid pBluescript. Lane 4, crude extract on E. coli BB4. All samples contained 0.1 mg of protein and were separated on a 12.5% SDS- polyacrylamide gel electrophoresis gel, electroblotted onto a nitrocel- lulose membrane, and immunostained as described under “Methods and Materials.” MWt, molecular weight.

Plasmid Activity -

PCB’ I I I I I ; : I I II : I I (+I E S m K B g S K Ns Nc Bg E

Bm Bm S Em

E Ne I : I : I I I I I I (4

Nc E PCB6 H-1 (-1

S S PCB7 - (-1

E S PCB5 - (-1

Bg w - Nc (+I

Bg Ns PCB9 - (+I

FIG. 2. Restriction mapping and subcloning of E. cloacae genomic DNA. The restriction map of the original clone PCB1 was generated through the analysis of size patterns resulting from single and double digests of the DNA with various restriction endonucleases. Localization of the gene coding for the nitroreductase involved sub- cloning of the above fragments and the correlation of the nitrore- ductase activity in the crude extracts of E. coli harboring plasmids containing them. Restriction enzymes: E, EcoRI; Sm, SnaI; Bm, BamHI; K, KpnI; Bg, BglII; S, StyI; Ns, NsiI; Nc, NcoI.

””“ - - ” K x s K K SC

FIG. 3. DNA sequencing strategy. The DNA from plasmid PCB9 was sequenced by subcloning various fragments using the restriction sites indicated. The shaded region spans the coding region of the nitroreductase gene and was sequenced on both strands. Se- quencing reactions were primed using oligonucleotides complemen- tary to regions within the multicloning region of the Bluescript vector and sequences internal to the coding region of the nitroreductase gene. Restriction sites used in the construction of subclones are denoted as follows: K , KpnI; X , XhoI; S , StyI; Sc, ScaII.

gene coding for the nitroreductase previously purified from that microorganism. (i) A comparison of the N-terminal amino acid sequence of the nitroreductase purified from E. cloacae, shown in Table I, with that deduced from the nucleo- tide sequence of the major open reading frame presumed to code for the nitroreductase, reveals an exact match for the 13 amino acids determined. (ii) Western blot analysis (Fig. 1) indicates that the cloned gene codes for a protein which is similar to the E. cloacae nitroreductase both immunogenically and on the basis of its migration rate on SDS-polyacrylamide gels. (iii) The specific activity of the overexpressed nitrore- ductase is equivalent to that of the E. cloacae protein when purified to homogeneity (see Table 11). (iv) The absorption spectrum of the purified cloned protein in both the ultraviolet and visible regions is identical to that of the enzyme isolated from E. cloacae (data not shown). Cumulatively, this evidence clearly indicates that the cloned gene from E. cloacae codes for the nitroreductase previously purified from this microor- ganism.

Western Blot Analysis-At least three oxygen-insensitive nitroreductases have been identified in the crude extracts of

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Cloning the E. cloacae Nitroreductase Gene 4129

5' CTATCGCGGGATAGAGCCGGCTACCATCTGAGTAAAAAGCACTGGATCTCC

CTTTACGGCACCGACGACGTGACGCCGGAACTGGTTACCGACCTCATTAATGATTCG~G

GAATCTGGTGGTGGATAAGCTGCCGAAAAAAGACCAGAAGTGGATCCGTCCGGTCTGAT

TGTTCGTCTGA-AGATTAAGCGCTCGAATGCACTTGATCTTTTTCCGCGTCCC -35 -10

GCTGTAATCTGCTTTCCTTTCGCGCCTCGAGCGGGCGAATTTCATCACC GGAGTT TT PF ATG GAT ATC ATT TCT GTC GCC CTG AAA CGC CAC TCT ACC AAG GCG Met Asp Ile Ile Ser Val Ala Leu L 3 Arg H i 3 Ser Thr Lys Ala

V81 Gfn Tyr

TTC GAC GCA AGC AAA AAA CTG ACC GCG GAA GAA GCG GAA AAA ATC Phe Asp Ala Ser Lys Ly3 Leu Thr Ala Glu Glu Ala Glu Ly3 Ile

Pro **P

AAA ACC CTG CTG CAG TAC AGC CCG TCC AGC ACC AAC TCC CAG CCG Ly3 Thr Leu Leu Gln Tyr Ser Pro Ser Ser Thr Asn Ser Gln Pro

TGG CAC TTC ATT GTA GCC AGC ACC GAG GAA GGA AAA GCG CGC GTG Trp H i 3 Phe Ile Val Ala Ser Thr Glu Glu Gly Lys Ala Arg Val

GCG AAG TCC GCT GCG GGC ACC TAT GTG TTC AAC GAA CGC AAA ATG Ala Lys Ser Ala Ala Gly Thr Tyr Val Phe Asn Glu Arg Lys Met

Aan Thr

CTG GAT GCT TCC CAC GTG GTG GTG TTC TGC GCG AAA ACC GCG ATG Leu Asp Ala Ser H i 3 Val Val Val Phe Cys Ala Lys Thr Ala Met

GAT GAC GCC TGG CTG GAG CGC GTC GTG GAT CAG GAA GAG GCC GAT Asp A3p Ala Trp Leu Glu Arg Val Val Asp Gln Glu Glu Ala Asp

AaP

GGC CGT TTC AAC ACG CCG GAA GCC AAA GCC GCA AAC CAT AAG GGC Gly Arg Phe Asn Thr Pro Glu Ala Lys Ala Ala A3n His Lys Gly

Ala ASP

CGC ACC TAC TTC GCC GAC ATG CAC CGC GTG GAT CTG AAA GAT GAC Arg Thr T r Phe Ala Asp Met H i 3 Arg Val Asp Leu Ly3 A3p Asp

AEg PK. Ser

GAC CAG TGG ATG GCG AAG CAG GTT TAC CTG AAC GTC GGC AAC TTC Asp Gln Trp Met Ala Lys Gln Val Tyr Leu A3n Val Gly ASn Phe Bin

CTG CTG GGC GTG GGC GCG ATG GGT CTG GAC GCG GTA CCA ATT GAA Leu Leu Gly Val Gly Ala Met Gly Leu Asp Ala Val Pro Ile Glu Ala

GGT TTC GAC GCC GCT ATT CTC GAC GAA GAG TTT GGC CTG AAA GAG Gly Phe Asp Ala Ala Ile Leu Asp Glu Glu Phe Gly Leu Lys Glu Glu V81 Ala

Ly3 Gly Phe Thr Ser Leu Val Val Val Pro Val Gly His H i 3 Ser AAA GGC TTC ACC AGC CTG GTG GTG GTA CCG GTT GGG CAC CAC AGC

T W

GTG GAA GAT TTC AAC GCC ACG CTG CCG AAA TCT CGC CTG CCG CTG Val Glu Asp Phe A3n Ala Thr Leu Pro Lys Ser Arg Leu Pro Leu

GlY

AGC ACG ATT GTG ACC GAG TGC TGA TCTCCTCTGGCACGCTGGGTTCCGGCG Glu Thr L.u Ser Thr Ile Val Thr Glu C 3

dl

TGCCTGTTTTCAATACAGCATACTCTAAGGATGCTACACCCTATACGCTCTGCGCTGAG

CATGCGTCAGACCATCATGTTCTCGCCATTACGCCCTCCTCTCGCTGAAGCGTTTCTCT

CAGCGTGGAACATTCAGGTTCTCACCCTAATTTTGTCGTTGTGTATCGATTGCCCTCGT

TACAGTCAAATAATTTGTAACAGCACAGAAATACCCACATCAGAAATTTCGCATACATT

AATCAATAACCCTCAAAATATCCCCATTATTTTTCTCACGTCACGCCAAATATTCCACA

CTGATAGAGCATTAATTATTTCGCTCTTTGCAGGAATTGTTACATGTCTATTTTAGGAT

TCGGGGTTCCGCATGTTTTTATAGGCCGCCACCGCGGTG 3'

51

110

169

228

287

332

317

422

4 67

512

557

602

6 4 7

692

137

782

827

872

917

968

1027

1086

1145

1204

1263

1322

1381

1420

FIG. 4. Nucleotide and deduced amino acid sequence of the cloned nitroreductase gene from E. cloacae. The sequences which are homologous with the -10 and -35 regions of a putative promoter site are shown. The Shine-Delgarno consensus ribosome- binding site is indicated by the boxed region. The arrows span a region which constitutes an inverted repeat which may be involved in the modulation of gene expression. Those amino acid positions at which differences occur between the E. cloacae and S. typhimurium proteins are noted below the E. cloacae sequence in bold letters.

E. coli (10). During the course of this work, a definite back- ground activity present in E. coli strains BB4 and TB1 was observed. An examination of the Western blot presented in

Fig. 1 clearly indicates the presence of a protein in the crude extracts E. coli strain BB4 which is cross-reactive with the antibody raised against the E. cloacae nitroreductase. In ad- dition, the molecular weight of this E. coli protein is similar to that of the E. cloacae nitroreductase (-27 kDa). These data suggest the existence of a nitroreductase in E. coli that is quite similar to that isolated from E. cloacae. However, the smallest nitroreductase thus far identified in E. coli has a molecular mass of 5O-kDa, as determined by size exclusion chromatography (9). Thus, the presumed E. coli nitroreduc- tase detected on Western blots is either a minor component of the total E. coli nitroreductase activity and has therefore not been detected by previous investigators, or this protein migrates at an anomalously high molecular weight under the conditions in which the size exclusion columns were run (i.e., perhaps as a dimer).

Nucleotide Sequence of the Nitroreductase Gene-The se- quencing strategy used allowed for the determination of the nucleotide sequence of the nitroreductase gene and the asso- ciated 5'- and 3'-flanking regions. A total of 1,420 bases were sequenced, approximately 65% of which were established from data derived from both strands of DNA. The nucleotide sequence encompassing the putative nitroreductase coding region was determined from data in which 100% of the DNA was sequenced on both strands. Those portions of the data which were derived from the sequencing of a single strand were confirmed by at least two independent sequencing runs.

As previously noted (13), the expression of nitrocellulose activity in E. cloacae can be enhanced 5-10-fold by the addi- tion of 2,4,6-trinitrotoluene to the growth medium. In addi- tion, an analysis of the activity associated with E. coli strain TB1 transformed with plasmid pCB18 indicated that the deletion of the BglII-XhoI fragment resulted in the complete loss of nitroreductase activity due to the presence of the cloned nitroreductase gene. However, when cotransformed with plas- mid pGP1-2, heat induction at 42 "C resulted in a 100-200- fold increase in the level of nitroreductase activity. Thus, the removal of the nucleotide region upstream of the XhoI site at position 255 was found to reduce the expression of nitrore- ductase activity to background levels. These results strongly suggest the existence of a regulatory site(s) within the 5'- untranslated region of the cloned DNA fragment. The initia- tion codon for the nitroreductase gene is preceded by a se- quence (-AGGAGTT-) which is nearly identical to the Shine- Dalgarno consensus sequence found in E. coli. A putative promoter region consisting of a -35 sequence (TGTTCG) starting at nucleotide 170, with a -10 sequence (TAAGCG) starting at position 192 has been identified based upon ho- mology with consensus promoter sequences from E. coli (22). This same region also contains a possible regulatory site spanning nucleotides 121-140. This region constitutes a per- fect inverted repeat. Palindromic sequences of this type are capable of forming stem-loop structures and are thought to provide a site for interaction between DNA and proteins involved in the modulation of gene expression (23). Which of these possible regulatory sites is actually functional is at this point unknown.

Using the program FASTA (24), a comparison of the nitro- reductase amino acid sequence with those deposited in the NBRF protein database (v.23.0), the translated GenBank database (v.63.0), and the Swiss Protein database (v.13.0) failed to identify any sequences of significant homology. Since the nitroreductase has been characterized as a flavoprotein, a more detailed comparison of this protein with other flavopro- teins of known amino acid sequence was explored. However, only one region of significant similarity could be identified.

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4130 Cloning the E. cloacae Nitroreductase Gene

TABLE I NHAerminal amino acid seouence of E. cloacae nitroreductase

Cycle no. 1 2 3 4 5 6 7 8 9 10 11 12 13

Amino acid Met ASP Ile Ile Ser Val Ala Leu Lvs Arg. His Ser Thr

TABLE I1 Purification of the cloned nitroreductase gene product

Sample Volume Protein” Activityb Specific activity Yield Purification ml w units unitslml % fold

Cell lysate 160 916 21,415 23.4 Acetone fractionation 24 173 16,552 95.8 78 4 Q300 chromatography 74 36 13,245 368.3 62 16 Superose 12 chromatography 25 21 11,204 524.2 52 22

“Protein concentration was measured by the BCA method as recommended by the manufacturer (Pierce

One unit of activity is defined as the oxidation of 1 pmol of NADH/min/ml of enzyme and was determined by Chemical Co.).

measuring the slope of the initial decrease in absorbance at 340 nm (E340 = 6,220 M” cm”).

This region beginning at amino acid 153 is highly homologous with the -G-X-G-X-X-G- consensus sequence which is thought to be involved in the binding of dinucleotides such as NADH or NADPH (25). Given the functional role that these reduced pyridine dinucleotides play in the mechanism of the nitroreductase it seems possible that this sequence may be of structural significance.

Subsequent to the completion of this work the nucleotide sequence of the nitroreductase gene from S. typhimurium was reported (26). A comparison of this sequence to that deter- mined for the E. cloacae gene revealed a remarkable overall homology of 88% with only 24 mismatches evenly distributed throughout the sequence. Furthermore, 9 of the 24 differences shown in Fig. 4 are conservative in nature. The functional significance of the nonconservative changes found between the two proteins cannot be assessed at this time as no infor- mation has been reported concerning the structural and func- tional properties of the S. typhimurium protein.

In conclusion, we have succeeded in cloning the gene which codes for a classical nitroreductase from the enteric bacteria E. cloacae. Sequence analysis of this gene reveals that the gene belongs to an as yet unreported family of flavoproteins. The protein product of this gene was overexpressed in E. coli and purified to homogeneity. The properties of this protein were identical to that of the nitroreductase isolated from E. cloacae. The availability of abundant quantities of the E. cloacae nitroreductase expressed from the cloned gene should allow for more detailed studies regarding the structure and function of this enzyme.

Acknowledgments-We thank Amy Lam and Keith Wood for their advice and encouragement during the course of this work. We also thank Shirlene Jay for her technical assistance in the preliminary stages of the cloning effect. We thank Joe Beuchler for his help in the determination of the N-terminal amino acid sequence. C. B. thanks W. S. Allison for his help and support during the final stages of his graduate work at the University of California at San Diego.

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C Bryant, L Hubbard and W D McElroyEnterobacter cloacae.

Cloning, nucleotide sequence, and expression of the nitroreductase gene from

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