OHIO-1 ,3-Lactamase Is Part of the SHV-1 Family

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1990, p. 1570-15760066-4804/90/081570-07$02.00/0Copyright © 1990, American Society for Microbiology

OHIO-1 ,3-Lactamase Is Part of the SHV-1 FamilyDAVID M. SHLAES,l 2.3* CHARLOTTE CURRIE-McCUMBER,2 ANNE HULL,3

I. BEHLAU,3 AND M. KRON3'4

Infectious Diseases Section' and Research Service,2 Veterans Administration Medical Center,10701 East Boulevard, and Department of Medicine3 and Division of Geographic Medicine,4

Case Western Reserve University, Cleveland, Ohio 44106

Received 15 November 1989/Accepted 20 April 1990

The 011IO-1 ,B-lactamase gene was subcloned in a 1.16-kilobase TaqI fragment in the 2.4-kilobase chimericplasmid pSK04. After directional subcloning into M13, the DNA sequence of this fragment was determined.The results showed an open reading frame of 858 base pairs (bp) encoding a protein of 286 amino acids. Thestructural gene showed 95, 87, and 60% DNA sequence identity with SHV-1, LEN-1, and TEM-1, respectively,and 93, 85, and 62% predicted amino acid sequence identity, respectively. The 87 bp upstream of the OHIO-istructural gene had 96% identity with the upstream flanking sequence of SHV-1, including the -35 and -10consensus sequences and the putative ribosomal binding site. A 223-bp DNA probe derived from a Psfi-HaeHlfragment in the C-terminal sequence of OHIO-1 had predicted 96, 88, and 61% sequence identity with SHV-1,LEN-1, and TEM-1, respectively. This probe hybridized to SHV-1 and poorly to LEN-1, but not to TEM-1 or

a variety of other plasmid-mediated I-lactamase genes, under stringent conditions. Screening of plasmid DNAderived from 40 ampicillin-resistant clinical isolates by Southern hybridization with the 223-bp probeuncovered no strains encoding OHIO-1. Isoelectric focusing of the same collection did identify two strainsproducing enzymes resembling SHV-1, however. We have also performed a kinetic comparison of OHIO-1,SHV-1, and TEM-1. OHIO-1 and SHV-1 were indistinguishable from each other but could be distinguishedfrom TEM-i. These data clearly place OHIO-1 within the SHV-1 family of 13-lactamases.

The OHIO-1 P-lactamase is a group 2b enzyme with asubstrate and inhibitor profile resembling those of TEM-1and SHV-1 (8, 28). Recent immunochemical data (34) andinhibitor profiling data (A. A. Medeiros, personal communi-cation) suggest that OHIO-1 belongs within the SHV-1family of P-lactamases. LEN-1 is a ,B-lactamase encoded onthe chromosomes of some strains of Klebsiella pneumoniaewith a pI of 7.0 and a nucleotide sequence resembling that ofSHV-1 (2, 22). OHIO-1 has a pI similar to that ofLEN-1 butis plasmid mediated. OHIO-1 appears to have been geo-graphically restricted within the state of Ohio (17, 28, 29). Tobetter understand the evolution of this restricted resistancemechanism, we have sequenced the structural gene and itsflanking sequences. To arrive at a better understanding ofstructure and function among P-lactamases, we havesearched for differences in substrate specificities amongthese enzymes and compared these with differences in theirsequences. Finally, we have developed an intragenic DNAprobe from the OHIO-1 gene sequence.

MATERIALS AND METHODS

Bacterial strain and plasmids. Standard plasmids in Esch-erichia coli or Pseudomonas aeruginosa hosts were used forthe following plasmid-mediated t3-lactamases: TEM-1,TEM-2, HMS-1, SHV-1, OXA-1, OXA-2, OXA-3, PSE-1,PSE-2, PSE-3, and TLE-1 (7, 9-11, 14, 18, 20, 21, 27, 32).ROB-1 was carried in Haemophilus influenzae (24), andPSE-4 (14; G. A. Jacoby, personal communication) was achromosomal marker in PU21 pMG19. LEN-1 was a chro-mosomal gene in K. pneumoniae LEN-113 (2; kindly pro-

* Corresponding author.

vided by A. Medeiros). The OHIO-i-bearing plasmidspDS075 and pDS076 have been previously described andwere utilized in E. coli C600 or HB101 hosts (19). pSK02, achimeric plasmid derived from cloning OHIO-1 in a 2-kilobase AvaI fragment into pMK20, has also been previ-ously described (29).

Restriction endonuclease mapping, cloning, subcloning, andDNA sequencing. Restriction endonucleases were used todigest CsCl-ethidium bromide density equilibrium gradient-purified plasmid DNA or minipreparation DNA according tothe instructions of the manufacturer. Minipreparations wereprepared according to the Ish-Horowicz modification of theprocedure of Doly and Birnboim (19). M13mpl8 andM13mpl9 subcloning was performed according to proce-dures outlined by the supplier of M13 DNA (Life Technol-ogies, Gaithersburg, Md.). DNA sequencing was performedaccording to the method of Sanger et al. (26) and was carriedout with the Sequenase kit (United States BiochemicalCorp., Cleveland, Ohio). Purified custom synthetic oligonu-cleotides which were used as primers in the sequencingreaction were purchased from Case Western Reserve Uni-versity School of Medicine. They were prepared by using anautomated system based on the P-cyanomethyl-phosphora-midite method (Applied Biosystems, Inc., Foster City,Calif.).DNA probing. Plasmid DNA was isolated as described

above and was blotted from agarose gels to nitrocellulosefilters (Millipore Corp., Bedford, Mass.). DNA fragmentswere labeled by the random primer method with an oligonu-cleotide labeling kit (U.S. Biochemical). Southern hybridiza-tions (30) were performed under stringent conditions (3) with50% formamide at 42°C for 18 to 24 h, and washing was

performed under stringent conditions with 0.2x SSC (lx

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SSC is 0.15 M NaCI plus 0.015 M sodium citrate) at 65°C.Filters were developed by autoradiography with KodakXAR X-ray film (Eastman Kodak Co., Rochester, N.Y.).

P-Lactamase extraction and isoelectric focusing. ,-Lacta-mase extraction and isoelectric focusing were performed asdescribed by Vecoli et al. (33) with gels with a pl range of 3to 10. For those extracts requiring concentration beforeanalysis, a miniconcentrator from Amicon Corp. (Danvers,Md.) was used for up to a 25-fold concentration.

Antibiotics. Aztreonam and potassium penicillin G weresupplied by E. R. Squibb & Sons (Princeton, N.J.), cefazolinwas supplied by Eli Lilly & Co. (Indianapolis, Ind.), andcefoperazone and sulbactam were supplied by J. B. Roerig(New York, N.Y.). Cefotaxime, cephaloridine, and cloxacil-lin were purchased from Sigma Chemical Co. (St. Louis,Mo.). Cefoxitin and imipenem were provided by MerckSharp & Dohme (Rahway, N.J.), ceftazidime was providedby Glaxo, Inc. (Research Triangle Park, N.C.), ceftriaxonewas provided by Hoffmann-La Roche Inc. (Nutley, N.J.),clavulanic acid was provided by Beecham Laboratories(Bristol, Tenn.), mezlocillin was provided by Miles Inc.,Pharmaceutical Div. (West Haven, Conn.), and piperacillinwas provided by Lederle Laboratories (Pearl River, N.Y.).Nitrocefin was obtained from BBL Microbiology Systems(Cockeysville, Md.).

i-Lactamase kinetics. Cells were disrupted by sonicationor by agitation with glass beads and, if necessary, wereconcentrated as described above. Hydrolysis of,B-lactamswas performed at 30°C in a temperature-controlled DUspectrophotometer (Beckman Instruments, Inc., Fullerton,Calif.) with the following wavelengths: for penicillin, me-zlocillin, and piperacillin, 233 nm; for cefoperazone, 265 nm;for cephaloridine, 255 nm; for nitrocefin, 432 nm. We deter-mined extinction coefficients by hydrolysis to completionwith NaOH or TEM-1. AE233 was 1 mM-1 cm-1 for penicil-lin, 6 mM-1 cm-' for mezlocillin, and 7.6 mM-1 cm-' forpiperacillin, AE255 was 8.6 mM-1 cm-' for cephaloridine,and AE265 was 6.8 mM-1 cm-' for cefoperazone. To deter-mine the kinetics of hydrolysis, substrates were used inconcentrations ranging from 10 to 100,uM. Lineweaver-Burkplots (6) were prepared, and Km and Vmax were calculatedfrom the linear regression. For inhibition kinetics, the en-zyme and inhibitor (10 to 600,uM) were preincubated for 10min at 30°C before the addition of nitrocefin to a finalconcentration of 100,uM. Standard 1/v versus [1] (concen-tration of inhibitor) plots (Dixon plots) (6) were prepared byusing linear regression, and the Ki was calculated.

RESULTS

DNA and predicted amino acid sequence of the OHIO-1gene. The restriction endonuclease map of pSK04 is shownin Fig. 1. The 1,164-base-pair (bp) TaqI fragment containingthe OHIO-1 structural gene was subcloned by using PstI andTaqI digestions and ligating it into M13mpi8 and M13mpi9vectors. Sequencing was performed in both directions forthe 5' end of the fragment and in only one direction for thedistal end. Synthetic, custom oligonucleotides derived fromthe sequence were used to "walk" along the fragment ofinterest to confirm the derived sequence. The original TaqIfragment was probably the result of partial digestion, sincewe found two additional TaqI sites in our sequence of the1,164-bp fragment and have confirmed their existence bysubsequent restriction endonuclease digestion. The DNAand predicted amino acid sequences of the OHIO-1 genecompared with the published sequences of the SHV-1 gene

HPH H B

FIG. 1. Restriction endonuclease map of pSK04. B, BglI; H,HaeII; Hf, Hinfl; P, PstI; Pv, PvuI; T, TaqI. Kb, Kilobases.

are shown in Fig. 2. The upstream flanking sequencesincluding the -35 and -10 consensus sequences and theputative ribosomal binding sites are also shown. These twogenes share 95% DNA sequence identity and 96% sequencehomology across the 858-bp coding sequence and 90%identity across the 87-bp upstream sequence. The promotorsequences are identical for the two genes. Figure 3 shows thepredicted amino acid sequence of OHIO-1 as compared withthe directly determined amino acid sequence of SHV-1(PIT-2) (5) and the predicted amino acid sequences of SHV-1(22) and LEN-1 (2). SHV-1 has a triplet insert encoding anadditional glycine at position 189 that is not present inOHIO-1, LEN-1, or the amino acid sequence of SHV-1(PIT-2). OHIO-1 and LEN-1 share 85% amino acid sequenceidentity, while OHIO-1 shares 93% amino acid sequenceidentity and 97% sequence homology with SHV-1. OHIO-1shares 60 and 62% DNA and amino acid sequence identitywith TEM-1 (27) (data not shown).

Kinetic comparison of OHIO-1, SHV-1, and TEM-1. Theresults of kinetic comparison of OHIO-1, SHV-1, and TEM-1 are summarized in Tables 1 and 2. These three enzymesseem quite similar, with SHV-1 and OHIO-1 resemblingeach other more closely than either resembles TEM-1. Noneof the enzymes hydrolyzed any of the newer 3-lactams(aztreonam, cefotaxime, ceftazidime, ceftriaxone, and imi-penem) or the cephamycin (cefoxitin) tested at detectablerates (>0.1,umol min-'). These 3-lactams were used asinhibitors against the hydrolysis of nitrocefin in concentra-tions ranging from 10 to 600,uM. None of these compoundshad detectable affinity for the enzyme as determined by Ki,explaining the lack of hydrolysis when cefoxitin and thenewer cephalosporins were tested as substrates. Clavulanatewas a more efficient inhibitor of SHV-1 and OHIO-1 thanwas sulbactam.Development and use of an intragenic DNA probe. Our

previous work had shown that a 1.2-kilobase probe bearingthe 858-bp OHIO-1 gene did not hybridize under stringentconditions to R1010 bearing SHV-1. However, given the factthat the genes share 95% homology, the overall homology of

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ANTIMICROB. AGENTS CHEMOTHER.

Upstream flanking sequence-35

OHIO-1 CGAGTAATACTCACATGGCACTGACGCAAAATCATCGATTTATTCAACAAAGCCATTGTGAASHV-1 AATAAAAGATGAAAAATGATGAAGGAAAAAAGAGOAATTGTGAA

-10

OHIO-i TCAGCAAAACGCCGGGTTATTCTTATTTGTCGCTTCTTTACTCGCCTTTATCGGCCSHV-1 TCAGCAAAACGCCGGGTTATTCTTATTTGTCGCTTCTTTACTCGCCTTTATCGGCC

RBSOHIO-1 CTCACTCAAGGATCTACCGTGGTTSHV-1 CTCACTCAAGGATGTATTGTGGTT

Coding sequence

OHIO-1 ATGCGTTATTTTCGCCTGTGTATTATCTCCCTGTTAGCCACCCTGCGGCTGCGGGTACACSHV-1 ATGCGTTATaTTCGCCTGTGTATTATCTCCCTGTTAGCCACCCTGgcGCTGGCGGTACACOHIO-1 F RSHV-1 I A

OHIO-1SHV-1OHIO-1SHV-1

OHIO-1SHV-iOHIO-1SHV-1




GCCGGACCGCAGCCGCTTGAGCAAATTAAACTAAGCGAAAGCCAGCTGTCGGGCAGCGTAGGGCCaGcCCGCAGCCGCTTGAGCAAATTAAACTAAGCGAAAGCCAGCTGTCGGGCcGCGTAGG

G RS S

CATGATAGAAATGGATCTGGCCAGGCCCGGCACGCTGACCGCCTGGCGCGCCGATGAACGCTTCATGATAGAAATGGATCTGGCCAGcggCcGCACGCTGACCGCCTGGCGCGCCGATGAACGCTT

R P GS G R

TCCCATGATGAGCACCTTTAAAGTAGTGCTCTGCGGCGCAGGTCTGGCGCGGGTGGATGCCTCCCATGATGAGCACCTTTAAAGTAGTGCTCTGCGGCGCAGtgCTGGCGCGGGTGGATGCC

GV

GGTGACGAACAGCTGGAGCGAAAGATCCACTATCGCCGACAGGATCTGGTGGACTACTCGCCGGGGTGACGAACAGCTGGAGCGAAAGATCCACTATCGCCagCAGGATCTGGTGGACTACTCGCCGG

RQ

TCAGCGAAAAACACCTTGCCGACGGCATGACGGTCGGCGAACTCTGTGCCGCCGCCATTACCATCAGCGAAAAACACCTTGCCGACGcaATGACGGTCGGCGAACTCTGcGCCGCCGCCATTACCA

GA

FIG. 2. DNA and predicted amino acid sequences for the structural genes and flanking sequences for the OHIO-1 and SHV-1 genes.Nucleotide differences are noted by lowercase letters in the SHV-1 sequence. Where such differences result in a change in amino acid, thedifferences are indicated below the nucleotide sequences. The PstI and HaeII sites surrounding the 223-bp fragnent used as a probe areshown. The -35 and -10 consensus sequences and the putative ribosomal binding site (RBS) are indicated. Apparent deletions or insertionsin the downstream flanking sequences are indicated.

the probe might have been only about 66% and might nothave formed a stable duplex under our conditions (37°C, 50%formamide). Given the similarity of the DNA sequences ofOHIO-1, SHV-1, and LEN-1, it was not possible to developan intragenic DNA fragment probe that would recognizeonly OHIO-1. A 223-bp DNA probe derived from a PstI-HaeII fragment in the C-terminal sequence of OHIO-1 (Fig.1) had predicted 96, 88, and 61% sequence identity withSHV-1, LEN-1, and TEM-1, respectively. This probe hy-bridized to SHV-1 and poorly to LEN-1, but not to TEM-1or a variety of other plasmid-mediated 1-lactamase genes(listed in Materials and Methods), under stringent conditions(data not shown).We also used this probe to screen plasmid DNA derived

from 40 ampicillin-resistant gram-negative bacilli collectedfrom the clinical microbiology laboratory. We were carefulto eliminate duplicate isolates from the same patient. South-ern blots were prepared with the parent plasmid pSK04 (andsometimes pDS075 as well) as a positive control and pBR322as a negative control on each gel. These blots were thenprobed with the 223-bp intragenic fragment. Only the pSK04and pDS075 controls were positive (data not shown). Toconfirm the finding that none of these strains encoded

OHIO-1, we surveyed culture sonic extracts for p-lactamaseby using nitrocefin hydrolysis and, for those producingdetectable P-lactamase, with isoelectric focusing. Twenty-eight strains produced ,B-lactamase, according to the nitro-cefin hydrolysis test. None had an enzyme with a pl of 7.0(OHIO-1 or LEN-1), while most (19 of 28 or 68%) producedenzymes resembling TEM-1 (pl, 5.4) and two producedenzymes resembling SHV-1 (pI, 7.6). Two had pls of 5.2 andappeared to be similar to TEM-101 (12). The remaining twodid not focus. These data were consistent with our probedata, although a K. pneumoniae isolate and an E. coli isolateproducing SHV-1 were negative with our probe, suggestingthat these genes might be located on chromosomal DNArather than on the plasmid DNA we screened. The data alsosuggested that while OHIO-1 may be common on certainwards of our hospital (28), it has not disseminated through-out the hospital.

DISCUSSION

The first plasmids encoding the OHIO-1 1-lactamase wereisolated in the Cleveland Veterans Administration MedicalCenter in 1981. The enzyme was first reported in 1986 (29).

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TGAGCGATAACAGCGCCGCCAATCTGCTGCTGCCAGCCGTCGGCGGCCCCGCAGGATTGACTGCTGAGCGATAACAGCGCCGCCAATCTGCTaCTGgCcaCCGTCGGCGGCCCCGCAGGATTGACTGC

P AA T

OHIO-1 CTTTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCGCTGGGAAACGGAACTGASHV-1 CTTTTTGCGCCAGATCGGCGACAACGTCACCCGCCTTGACCGCTGGGAAACGGAACTGA


ATGAGGCGCTTCCCGGCGACGCCCGCGACACCACTACCGCCCGCAGCATGGCCGCGACCCTATGAGGCGCTTCCCGGCGACGCCCGCGACACCACTACCcCggcCAGCATGGCCGCGACCCT

A RP A

PstIV

OHIO-1 GCGCAAGCTG CTGACCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTGGATSHV-i GCGCAAcgTt CTGACCAGCCAGCGTCTGAGCGCCCGTTCGCAACGGCAGCTGCTGCAGTGGAT

GGCOHIO-1 K LSHV-1 N V G


OHIO-1SHV-1

GGTGGACGATCGGGTCGCCGGACcgTTGATCCGCTCCGTGCTGCCGGCGGGCTGGTTTATCGCCRp

GATAAGACCGGAGCTGGCGAgCGGGGTGCGCGCGGGATTGTCGCCCTGCTTGGCCCGAATAACA

OHIO-1 AAGCAGAGCGGATTGTGGTGATTTATCTGCGGGATACGCCGGCGAGCATGGCCGAGCGAAATCSHV-1 AAGCAGAGCGcATTGTGGTGATTTATCTGCGGGATACcCCGGCGAGCATGGCCGAGCGAAATC


HaeIIv

AGCAAATCGCCGGGATCGCCGGGGCGCTGATCGAGCACTGGCAACGCTAAAGCAAATCGCCGGGATCGgCaaGGCGCTGtaCGAGCACTGGCAACGCTAA

Iy

Downstream flanking sequence

OHIO-1 CCCGGCGGTGGGCGCGCGCGTTATCCGGCTCGTAGCACCTCGCAGGCGTGCCGGCGATATGACSHV-1 CCCG CGTGGC CGCGCGTTATCCGGCcCGcAGCACCTCGCAGcgtgcCgGGCGATATGAC

OHIO-1 TGGCGGCGGCATCGGAGAGATGGCTTCTGGCTAATGATGGTCGGTGCAACCTGGGTGAAAGGTSHV-1 TGGCGGCGGCATCGGAaAGATG CcggTcGGTAATGATGGT GGTG AACC GGGTc

OHIO-1 AACGCCATAAACGGTGGCCACCTGATTGTGTTTCTFIG. 2-Continued.

At that time, we had received from Columbus, Ohio, plas-mids also encoding this enzyme. Within three years of itsdiscovery, conjugative plasmids encoding OHIO-1 werefound disseminated in virtually all species of the familyEnterobacteriaceae on at least one ward in our medicalcenter (28). We later showed that all of these plasmids,regardless of origin, were derived from a single progenitor(17). These observations were consistent with the lack ofobservable transposition of OHIO-1 (D. M. Shlaes and G.Jacoby, unpublished observations).We cloned and sequenced the OHIO-1 gene as a first step

in trying to understand its evolution, to identify differencesamong the group 2b (8) enzymes that might correlate withdifferences in substrate specificity, and to identify an intra-genic probe. The DNA sequence of OHIO-1 confirms itsclose relationship with LEN-1 and SHV-1 and its somewhatmore distant, but important, homology with TEM-1. TheDNA and amino acid sequence identity is greater betweenSHV-1 and OHIO-1 (95 and 93%) than between LEN-1 andOHIO-1 (87 and 85%). Thus, OHIO-1 is more closely relatedto SHV-1 than to LEN-1 even though its pI is closer to thatof LEN-1. The numerous amino acid substitutions in thesequence of OHIO-1 when compared with the sequence ofSHV-1 show that OHIO-1 is not a simple single-pointmutation of SHV-1 but has probably evolved from SHV-1

over many years. These homologies also suggest that OHIO-1 is not some intermediate between SHV-1 and LEN-1.Recent immunochemical data suggest that OHIO-1 be-

longs to a family of group 2b enzymes containing SHV-1 andits derivatives (34). The sequence of a K. pneumoniaechromosomal P-lactamase with a pI of 7.0 to 7.1, LEN-1,was also recently published (2). It appears closely related toPIT-2 (SHV-1), the amino acid sequence of which wasreported by Barthelemy and co-workers (5), and to the DNAsequence of SHV-1 reported in the companion to this paper(22). Vedel et al. (34) studied P-lactamases encoded by thechromosome of K. pneumoniae which resemble LEN-1.They concluded, on the basis of a two-dimensional, charge-based electrophoresis, that these enzymes are more closelyrelated to OHIO-1 than OHIO-1 is to SHV-1 (34). However,this could be related more to overall charge similarities thanto specific primary structures.An examination ofnumerous potential P-lactam substrates

and inhibitors shows little difference among the specificitiesof the three enzymes examined. It seems, however, thatOHIO-1 and SHV-1 are more similar to each other than toTEM-1 against the substrates tested. None of these enzymesbound or hydrolyzed (data not shown) the newer cephalo-sporins at detectable rates.DNA or amino acid sequence data are now available for a

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1574 SHLAES ET AL. ANTIMICROB. AGENTS CHEMOTHER.

H R Y F R LMERXYIERL

10C I I S L LC I I S L L

M R Y V R L C V I S L L

20A T L P L R V H A GA T L P L A V H A S

sA T L P L V V Y A G

I40 50

E S Q L S G S V G M I E M D L A R PE S Q L S G R V G N IjE M D L A S GE S Q L S G R V G M I E M D L A S GE S Q L S G R V G M E MLD L A N G

II70

N T F K V V L C G A GMSNT F K V V L C G A V

S.T F K V V L C G A V* SIT F K V L L C G A V

III IV100

D L V D S P V S E K HD L V D Y S P V S E K HD L V D Y S P V S E K HD L V DWYS P V SE KJH

8L A RL A RL A RL A R

110L A DL A DL A DL V D

.0

G T L TR T L TR T L TR T L A

V D A G D E QV D A G D E QV D A G D E QV D A G L E Q

G N T V G E LA M T V G E LG M T V G E LG M T I G E L

30P Q P L E Q I K L SP Q P L E Q I K L SP Q P L E Q I K L SP QP L E Q I K Q S

60A W R A D E R F P NA W R A D E R F PKA W R A N E R F PMA W R A D E R F P N

90L E R KL E R KL E R KL D R R

120C A A AC A A AL A A AC A A A

I H Y R R QI H Y R Q QI H Y R Q QI H Y R Q Q

I T M S D NI T M S D NI T N S D NI T L S D N

130 140 150 160S A A N L L L P A V G G P A G L T A F L R Q I G D N V T R L D RS A A N L L L A T V G G P A G L T A F L R Q I G D N V T R L D RS A A N L L L T A V G G P A G L T A F L R Q I G D N V T R L D RS A G N L L LA TV G GP AG LT A FL R Q I G D NV TR L DR

V1,

OHIO-1 W T E L N E A LSHV-1 W E T E L N E A L

PIT-2 W E T E L N E A LLEN-1 WLEJT A L N E A L

70 180 190P G D A R D T T T A R S M A A T L R K L L T SP G D A R D T T T P A S M A A T L R N V L T S

GP G D A R D T T T P A S M A A T L R K L L T SP G D A R D T T T P A S M A A T L R K L L T A

200Q R L S A R S QQ R L S A R S QQ R L S A R S QQ H L S A R S Q

VI210

R Q L L Q W NV D D R VR Q L L Q W NV D D R VR Q L L Q W NV D D R VQ Q L L Q NV D D R V

VII230

OHIO-1 W F I A DJWjASHV-1 W F I A D K T G APIT-2 W F I A D K T G ALEN-1 W F I A D K 6A

260OHIO-i I Y L R D T P A SSV-1 I Y L R Q T P A SPIT-2I Y L R D T P A SLEN-1 I Y L R D T P A S

240G E R G A R G I V A L L GG E R G A R G I V A L L GG E R G A R G I V A LL GG E R G A R G I V A L L G

270N A E R N Q Q I A G I A GN A E R N Q Q I A G I G KN A E R N Q Q I A G I G AM A E R N Q H I A G I G Q

250P N N KP N N KP N N KP D G K

280A LA LA LR *

A E R I VVA E R I V VA E R I V VP G R I V V

FIG. 3. The predicted OHIO-1 amino acid sequence compared with that of SHV-1, with the directly determined amino acid sequence ofSHV-1 (PIT-2), and with that of LEN-1. The seven boxes of Joris et al. (15) are indicated by boxes.

number of ,B-lactamases and penicillin-binding proteins (1, 2,4, 5, 15, 22, 31). In addition, for a selected few, X-raycrystallography has been performed (13, 15, 16, 25). Fromthis combined information, Joris et al. have shown that allDD-serine peptidases, into which family the penicillin-binding proteins and ,-lactamases fall, share specific regionsof homology (15). Recent information from naturally arisingmutants of the ,B-lactamases suggests that at least some ofthese regions are important in the binding and eventualhydrolysis of the substrate (23). These key positions occur atamino acids 160 and 234 and appear to be important in thebinding of abnormal substrates such as cefotaxime andceftazidime. These positions occur close to boxes V and VII,

respectively, of Joris et al. (15). Comparing our sequence

with that of SHV-1, we note that these two positions as wellas all the boxes of Joris et al. remain identical in both. Noneof the other substitutions are adjacent to any of the criticalseven boxes. As might be expected, none of the substitu-tions resulted in appreciable differences in substrate speci-ficity.The DNA probe we have characterized had predicted 96,

88, and 61% DNA sequence identity with SHV-1, LEN-1,and TEM-1, respectively. Under stringent hybridizationconditions (42°C and 50% formamide for 18 to 24 h), thisprobe hybridized to R1010 encoding SHV-1 and weakly tochromosomal DNA encoding LEN-1. The 223-bp fragment

OHIO-iSHV-1PIT-2LEN-1

OHIO-1SHV-1PIT-2LEN-1

OHIO-1SHV-1PIT-2LEN-1

OHIO-iSHV-1PIT-2LEN-1

OHIO-1SHV-iPIT-2LEN-1

OHIO-1SHV-1PIT-2LEN-I.

A G P L IA G P L IA G P L IA G P L I

220R S V LR S V LR S V LR A V L

P A GP A GP A GP P G

I E H W Q R *Y E H W Q R *I E H W Q R *

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TABLE 1. Hydrolysis of ,B-lactam substrates by OHIO-1,SHV-1, and TEM-1

Substrate and enzyme Vxa Kma

PenicillinOHIO-1 17 25SHV-1 14 25TEM-1 23 74

MezlocillinOHIO-1 2.9 75SHV-1 2.9 97TEM-1 6.8 116

PiperacillinOHIO-1 1.4 44SHV-1 2.5 67TEM-1 4 170

CephaloridineOHIO-1 44 348SHV-1 40 169TEM-1 20 848

CefoperazoneOHIO-1 3.9 76SHV-1 2.1 103TEM-1 3.3 254

a Vma,, is expressed in micromoles of substrate hydrolyzed per minute. Kmis expressed in micromolar concentration. Values are means of at least twoseparate determinations.

was easy to isolate from pSK04 since it was about 10% of thesize of the entire plasmid and was well separated from otherfragments after PstI-HaeII double digestion. Furthermore,the digestion was done in the same buffer, obviating the needfor dealing with two different buffer systems.

In summary, we have cloned and sequenced the OHIO-1structural gene and flanking DNA. The gene is clearly part ofthe SHV-1 family of P-lactamases and belongs to class A ofAmbler (1) and group 2b of Bush (8). It appears to be mostrelated to SHV-1. The amino acid differences do not occur atpositions within the conserved boxes of Joris et al. (15).They also do not occur at sites known to be important forsubstrate binding, as indicated by the recent data on mutantsof group 2b enzymes able to hydrolyze newer cephalospo-rins (23). Since TEM-1 and SHV-1 seem to be present inhigher proportions than is OHIO-1 among gram-negativebacilli in our hospital, one must continue to wonder whyOHIO-1 evolved and why it persists.

TABLE 2. Inhibition of OHIO-1 and SHV-1 by various ,B-lactams

K, (1±M)Inhibitor

OHIO-1 SHV-1

Clavulanic acid 0.21 1.16Sulbactam 5 8.8Cloxacillin 68 145Aztreonam >500 >500Cefotaxime >400 >400Cefoxitin >500 >500Ceftazidime >400 >400Ceftriaxone >600 >600Imipenem >500 >500

ACKNOWLEDGMENTSWe are grateful to A. Medeiros and Roger Levesque for helpful

discussions and to R. Levesque, G. Vedel, and M. Syvanen forsharing with us their unpublished manuscripts or sequences.

This work was supported by the Department of Veterans Affairs.

LITERATURE CITED

1. Ambler, R. P. 1980. The structure of P-lactamases. Philos.Trans. R. Soc. London B Biol. Sci. 289:321-331.

2. Arakawa, Y., M. Ohta, N. Kido, Y. Fujii, T. Komatsu, and N.Kato. 1986. Close evolutionary relationship between the chro-mosomally encoded ,-lactamase gene of Klebsiella pneumoniaeand the TEM 1-lactamase gene mediated by R plasmids. FEBSLett. 207:69-74.

3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.Seidman, J. A. Smith, and K. Struhl (ed.). 1987. Currentprotocols in molecular biology. p. 2.9.5. John Wiley & Sons,Inc., New York.

4. Barthelemy, M., J. Peduzzi, H. Ben Yaghlane, and R. Labia.1988. Single amino acid substitution between SHV-1 P-lacta-mase and cefotaxime-hydrolyzing SHV-1 enzyme. FEBS Lett.231:217-220.

5. Barthelemy, M., J. Peduzzi, and R. Labia. 1988. Completeamino acid sequence of p453-plasmid-mediated PIT-2 3-lacta-mase (SHV-1). Biochem. J. 251:73-79.

6. Bergmeyer, H. U. (ed.). 1974. Methods of enzymatic analysis,vol. 1. Academic Press, Inc., New York.

7. Bobrowski, M. M., M. Matthew, P. T. Barth, N. Datta, N. J.Grinter, A. E. Jacob, P. Kontomichalou, J. W. Dale, and J. T.Smith. 1976. Plasmid-determined f-lactamase indistinguishablefrom the chromosomal ,B-lactamase of Escherichia coli. J.Bacteriol. 125:149-157.

8. Bush, K. 1989. Characterization of P-lactamases. Antimicrob.Agents Chemother. 33:259-263.

9. Dale, J. W., and J. T. Smith. 1974. R-factor-mediated P-lactamases that hydrolyze oxacillin: evidence for two distinctgroups. J. Bacteriol. 119:351-356.

10. Egawa, R., T. Sawai, and S. Mitsuhashi. 1967. Drug resistanceof enteric bacteria. XII. Unique substrate specificity of penicil-linase produced by R-factor. Jpn. J. Microbiol. 11:173-178.

11. Furth, A. 1975. Purification and properties of a constitutiveP-lactamase from Pseudomonas aeruginosa strain Dagleish.Biochim. Biophys. Acta 377:431-443.

12. Gutmann, L., M. D. Kitzis, D. Billot-Klein, F. Goldstein, G.Tran Van Nhieu, T. Lu, J. Carlet, E. Collatz, and R. Williamson.1988. Plasmid mediated P-lactamase (TEM-7) involved in resis-tance to ceftazidime and aztreonam. Rev. Infect. Dis. 10:860-866.

13. Herzberg, O., and J. Moult. 1987. Bacterial resistance to -lactam antibiotics: crystal structure of 13-lactamase from Staph-ylococcus aureus PC1 at 2.5 A resolution. Science 236:694-701.

14. Jacoby, G. A., and M. Matthew. 1979. The distribution of1-lactamase genes on plasmids found in Pseudomonas. Plasmid2:41-47.

15. Joris, B., J. M. Ghuysen, G. Dive, A. Renard, 0. Dideberg, P.Charlier, J. M. Frere, J. A. Kelly, J. C. Boyington, P. C. Moews,and J. R. Knox. 1988. The active-site serine penicillin-recog-nizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem. J. 250:313-324.

16. Kelly, J. A., 0. Dideberg, P. Charlier, J. P. Wery, M. Libert,P. C. Moews, J. R. Knox, C. Duez, C. Fraipont, B. Joris, J.Dusart, J. M. Frere, and J. M. Ghuysen. 1986. On the origin ofbacterial resistance to penicillin: comparison of a ,-lactamaseand a penicillin target. Science 231:1429-1437.

17. Kron, M. A., D. M. Shlaes, C. Currie-McCumber, and A. A.Medeiros. 1987. Molecular epidemiology of OHIO-1 P-lacta-mase. Antimicrob. Agents Chemother. 31:2007-2009.

18. Labia, R., J. Andrillon, and F. Le Goffic. 1973. Computerizedmicroacidometric determination of P-lactamase Michaelis-Menten constants. FEBS Lett. 33:42-44.

19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual, p. 90 and 504. Cold Spring Harbor

VOL. 34, 1990


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

ANTIMICROB. AGENTS CHEMOTHER.

Laboratory, Cold Spring Harbor, N.Y.20. Matthew, M., and R. B. Sykes. 1977. Properties of the (3-

lactamase specified by the Pseudomonas plasmid RPL11. J.Bacteriol. 132:341-345.

21. Medeiros, A. A., M. Cohenford, and G. A. Jacoby. 1985. Fivenovel plasmid-mediated ,3-lactamases. Antimicrob. Agents Che-mother. 27:715-719.

22. Mercier, J., and R. C. Levesque. 1990. Cloning of SHV-2,OHIO-1, and OXA-6 (-lactamase and cloning and sequencing ofSHV-1 ,-lactamase. Antimicrob. Agents Chemother. 34:1577-1583.

23. Philippon, A., R. Labia, and G. A. Jacoby. 1989. Extended-spectrum ,B-lactamases. Antimicrob. Agents Chemother. 33:1131-1136.

24. Rubin, L. G., A. A. Medeiros, R. H. Yolken, and E. R. Moxon.1981. Ampicillin treatment failure of apparently 13-lactamasenegative Hemophilus influenzae type b meningitis due to a novel,-lactamase. Lancet il:1008-1010.

25. Samrouai, B., B. J. Sutton, R. J. Todd, P. J. Artymiuk, S. G.Waley, and D. C. Phillips. 1986. Tertiary structural similaritybetween a class A 3-lactamase and a penicillin sensitive D-alanyl carboxypeptidase-transpeptidase. Nature (London) 320:378-380.

26. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

27. Sawada, Y., S. Yaginuman, M. Tai, S. lyobe, and S. Mitshu-hashi. 1974. Resistance of 3-lactam antibiotics in Pseudomonasaeruginosa, p. 391-397. In S. Mitsuhashi and H. Hashimoto

(ed.), Microbial drug resistance. University of Tokyo Press,Tokyo, Japan.

28. ShInes, D. M., C. A. Currie-McCumber, and M. H. Lehman.1988. Introduction of a plasmid encoding the OHIO-1 f3-lacta-mase to an intermediate care ward by patient transfer. Infect.Control Hosp. Epidemiol. 9:317-319.

29. Shlaes, D. M., A. A. Medeiros, M. A. Kron, C. Currie-McCum-ber, E. Papa, and C. Vartian. 1986. Novel plasmid-mediated3-lactamase in members of the family Enterobacteriaceae fromOhio. Antimicrob. Agents Chemother. 30:220-224.

30. Southern, E. M. 1975. Detection of specific DNA sequencesamong DNA fragments separated by gel electrophoresis. J. Mol.Biol. 98:503-517.

31. Sutclffe, J. G. 1978. Nucleotide sequence of the ampicillinresistance gene of Escherichia coli plasmid pBR322. Proc. Natl.Acad. Sci. USA 75:3737-3741.

32. Takahashi, I., K. Tsukamoto, M. Harada, and T. Sawai. 1983.Carbenicillin-hydrolyzing penicillinases ofProteus mirabilis andthe PSE-type penicillinases of Pseudomonas aeruginosa. Mi-crobiol. Immunol. 27:995-1004.

33. Vecoli, C., F. E. Prevost, J. J. Ververis, A. A. Medeiros, andG. P. O'Leary, Jr. 1983. Comparison of polyacrylamide andagarose gel thin-layer isoelectric focusing for the characteriza-tion of ,3-lactamases. Antimicrob. Agents Chemother. 24:186-189.

34. Vedel, G., G. Paul, B. Picard, and A. Philippon. 1989. Biochem-ical, immunological and physicochemic4l comparisons betweenOHIO-1 and four SHV-type 3-lactamases. FEMS Microbiol.Lett. 65:5-10.

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OHIO-1 ,3-Lactamase Is Part of the SHV-1 Family