JOURNAL OF BACTERIOLOGY, May 1992, p. 2891-28970021-9193/92/092891-07$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 174, No. 9

The Chloramphenicol Acetyltransferase Gene of Tn2424:a New Breed of cat

ROGER PARENT AND PAUL H. ROY*

Departement de Biochimie, Faculte des Sciences et de Ge'nie, Universite6 Laval, Sainte-Foy,Que'bec, Canada, GIK 7P4, and Laboratoire et Service d'Infectiologie, Centre de

Recherche du CHUL, Sainte-Foy, Quebec, Canada, GlV 4G2

Received 10 September 1991/Accepted 8 February 1992

We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79.This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of thechromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosaand of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent tothe ermG macrolide-lincosamide-streptogramin resistance gene of BaciUlus sphaericus and the vgb virginiamy-cin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase)and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobiumleguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltrans-ferase genes, such as that ofTn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacAlaaminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn2l except for the presence of this region.Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived"integrons" by a site-specific recombinational mechanism.

Tn2424 is a multiresistance transposon, 25 kb in length,from IncFII plasmid NR79. This transposon is closely re-lated to Tn21, and both code for resistance to mercuricchloride, streptomycin and spectinomycin (aadA4), and sul-fonamide (sull). Tn2424 additionally contains two segmentsof DNA. One is 4.5 kb in length and codes for resistance toamikacin and other aminoglycosides through a 6'-N-acetyl-transferase (AacA) and for resistance to chloramphenicol;the other, 1.8 kb in length, is an insertion sequence, IS161(Fig. 1) (28, 29, 47).Evidence from electron microscopy, restriction mapping,

and, more recently, sequencing indicates that various resis-tance structural genes are precisely inserted at specific sitesin an antibiotic resistance operon (31, 42, 49) and that theintegration is mediated by the product of a gene adjacent tothe antibiotic resistance operon which is a member of thephage integrase family of recombinases (26, 27, 32, 38). Theantibiotic resistance genes and the integrase are together in aregion called an "integron" (38), which is also found inplasmids such as R46 and R388. In these plasmids, it is notpart of a transposable element.

Resistance to chloramphenicol is usually mediated by achloramphenicol acetyltransferase (CAT) (37) but can alsobe due to a nonenzymatic mechanism (7, 13). Several CATgenes (cat; from Escherichia coli, Proteus mirabilis, Staph-ylococcus aureus, Bacillus pumilus, Clostridium difficile,Clostridium perfringens, Campylobacter coli, and Strepto-myces acrimycini) have been sequenced, and all show a gooddegree of similarity, especially around the active site (2).They differ, however, in regulation; some gram-positiveenzymes are inducible, while the gram-negative enzymes areconstitutive. Additionally, the cat gene present on Tn9 issubject to cyclic AMP-mediated catabolite repression (20).The cat gene of Tn2424 has been localized to the part of

the 4.5-kb insert closest to the sulfonamide resistance gene

* Corresponding author.

(29). We have sequenced the cat gene and adjacent regionsof Tn2424 and show here that the cat gene differs substan-tially from all but one of the previously sequenced cat genesand belongs to another family of acetyltransferases.

(These results were presented in part at the 30th AnnualInterscience Conference on Antimicrobial Agents and Che-motherapy, Atlanta, Ga., 1990 [33].)

MATERUILS AND METHODS

Bacterial strains and plasmids. Plasmid pUB307::Tn2424 instrain E. coli W677 (thr leu thi lac mal gal) (29) was a kindgift of B. Wiedemann. The plasmid was isolated by themethod of Beaulieu et al. (3) and purified by isopycnicultracentrifugation on a cesium chloride-ethidium bromidegradient. Molecular cloning of restriction digests of Tn2424was done using phagemids pTZ18R and pTZ19R (Pharmacia-LKB). Plasmid pLQ892, used as a control, is a multicopyvector in which the Tn9 cat gene is substituted for theampicillin resistance gene. E. coli NM522 [hsdA5 A(lac-pro)(F' pro' lacIPZAM15)] and DH1 (supE hsdR recA endAgyrA thi-1 relA) served as host strains for cloning.

Microbiological media and antibiotics. All microbiologicalmedia (Difco) were obtained from BDH. Ampicillin wasobtained from Ayerst. Chloramphenicol was obtained fromICN Biochemicals. X-Gal (5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside) was purchased from Boehringer Mann-heim Canada Ltee.Enzymes. Restriction endonucleases were purchased from

Boehringer Mannheim Canada Ltee, Bethesda ResearchLaboratories, or New England BioLabs and used accordingto the manufacturers' conditions. T4 DNA ligase was pur-chased from Bethesda Research Laboratories.

Transformation and screening of recombinant plasmids.Transformation of CaCl2-treated E. coli strains was per-formed by the method of Maniatis et al. (25). Selection ofdesired clones was done on solid media containing ampicillin(50 ,ug/ml) or chloramphenicol (25 ,ug/ml) or both and X-Gal.

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aUd1 adAl lnt 9' tnDRmor opronTn21A

4~ I ...*r-"E *-fa aga a-

_I~~~~~~~~~~I

Bad A2IS161

Tn 2424

= m-1kb

FIG. 1. Maps of Tn2J and Tn2424 showing the two additional regions carried by Tn2424: IS161 (1.8 kb) and a 4.5-kb region carrying anaacAla amikacin resistance gene plus an ORF (34) and a cat gene. Solid bars represent the 38-bp terminal inverted repeats of the transposons,and open bars (on stalks) represent the 25-bp repeats at the ends of a 10.9-kb insertion (8) corresponding to the integron (see text) insertedinto Tn2613 (44) to form Tn2l. M, tnpM gene (17); 4 and 5, two ORFs flanking the sulfonamide gene (38).

Screening of recombinant plasmids was performed by theminiscale method of Birnboim and Doly (4).

Preparation of plasmid DNA and DNA sequencing. Large-scale preparation of plasmid DNA from recombinant clones,preparation of single-stranded pTZ template, and DNAsequencing reactions were performed as previously de-scribed (5). Nucleotide sequence data and derived proteinsequences were analyzed by the Genetics Computer Groupsoftware (11) and by the programs RDF (23), FASTA, andTFASTA (35).Assay of acetylation of chloramphenicol. CAT activity was

assayed on crude cell extracts prepared by sonication in 1mM Tris HCI, pH 7.6. The CAT activity was measured bythe method of Kingston and Sheen (19). Acetyl coenzyme Aresuspended in 20 mM sodium-phosphate, pH 7.1, was usedas cofactor. Thin-layer chromatography was performed us-ing silica gel H (Mandel) preheated at 110°C for 30 min andcooled to room temperature before use. Samples werespotted 1.8 cm above the edge of the plastic-backed thin-layer chromatography sheets.

Nucleotide sequence accession number. The sequence de-scribed in this paper has been assigned GenBank accessionnumber M80188.

RESULTSMolecular cloning of the cat gene from plasmid pUB307::

Tn2424. Meyer et al. (29) have shown from restrictionmapping data that the amikacin and chloramphenicol resis-tance genes of Tn2424 are part of a 4.5-kb segment of DNAwhich is inserted between the streptomycin and sulfonamideresistance genes. The amikacin resistance gene is locatedclose to the streptomycin resistance gene, while the chlor-amphenicol resistance gene is close to the sulfonamide

cat

|' J~~~~~~~~~~~~~~~I

Xbal Hindlill PvIlPst I Smal

resistance gene. An 8.3-kb BamHI fragment (29) containingall four resistance genes was cloned into pTZ19, as was a

7.4-kb EcoRI fragment coding for amikacin, chlorampheni-col, and sulfonamide resistance. The region common to thetwo fragments was mapped, and several subclones were

constructed and used in the sequencing strategy shown inFig. 2.

Nucleotide sequencing of the cat gene. The nucleotidesequence of the 2.7-kb region between a PstI site upstreamof the cat gene and a BamHI site in the open reading frame(ORF) ORF5 downstream of the sull gene was determined.The complete nucleotide sequence is shown in Fig. 3. Thefirst 1,035 nucleotides (nt) are from the inserted DNAspecific to Tn2424, while nt 1036 to 2685 are identical to thecorresponding "3' conserved segments" from R388 (42),R46 (38), and pVS1 (6, 7). A putative promoter (TGGTCT. ...N17... .TATGGT) is located at nt 185 to 213 and isfollowed by a 59-base element (9) (a potential stem-loopstructure typical of the downstream end of gene cassettesinserted into integrons) at nt 237 to 301 and a consensuscassette junction, GTTAGGC (38), at nt 297 to 303. The catORF is from nt 344 to 976; this is immediately followed byanother 59-base element at nt 973 to 1040 and anothercassette junction, GTTAGAT, at the beginning of the 3'conserved segment. There is no evidence for any leaderpeptide involved in translational regulation, as is seen insome cat genes (12, 24) and in the cmlA nonenzymaticchloramphenicol resistance gene of Tn1696 (39).

Acetylation of chloramphenicol. Tests of CAT were per-formed by the method of Kingston and Sheen (19) on crudecell extracts prepared by sonication from the clone (pLQ161)containing the 7.4-kb EcoRI fragment of Tn2424, the clone(pLQ162) containing the 8.3-kb BamHI fragment, and a

orf4 sul 1 orf5

Hindill Hirndlll EcoRV Ps11 BgWlI Nrul Nhe1 Nru1 BamH1

cfnolBCp1 Asp 7001

= -1OObpFIG. 2. Restriction map of sequenced regions or recombinant plasmids containing parts of Tn2424. Lengths and directions of thin arrows

represent the extent of sequencing reactions. Arrows starting with an asterisk indicate reactions primed by custom-made oligonucleotides.Arrows starting with a dot indicate reactions from sites found from sequencing data and for which not all sites are mapped.

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CTGCAGTGGCCGGCCGAGCTX;GAACAGATAGTAAAGTTCGGGTTTGCCGGATCAGCCTTGCCGGCCTGTTGCTCGCTAATCTGGTTACGTGGCAACAAC

CTGACGCTTTTACTGGCCAGTGCCAGATAATGTTTCCTGTGACGCAAGGCGGGTCAAGCGAAAATAATCTACCAAGAAGTATGGTCTTGTAACGGGT

TGATCCCTATGGTCTAATCGGGCAA rAACATGTTA

faGCGACGCGTGGAGTCGCTCTAATTCGTACAAATTTTATGACGAATTA rGGAGTCCCTCAGGGAAGCTrCTGACTGAGCAGGTGAAGCat-> M T N Y F E S P F K G K L L T E Q V K

AATCCGAACATCAAGGTAGGGCGGTATAGCTACTATTCCGGCTATTACCQTGGGCACTCGTTTGATGATTGTGCTCGCTACCTTCTACCQGACCGTGATGN P N I K V G R Y S Y Y S G Y Y H G H S F D D C A R Y L L P D R D D

ACGTTGATCAGCTGATTATCGGCAGCTTCTGCTCCATCGGATCAGGCGCACGTTATTATGGCTGGGAATCAAGGCCACCGATATGATTGGGTCTCTTCV D Q L I I G S F C S I G S G A R F I M A G N Q G H R Y D W V S S

TTCCTTTTc VCTATGAACGAGGAGCCCGCGTTTCAAAATCAGTCGATGCATTCCAGCGGGCTt GCGACACGTTATAGGAAGTGATGTGTG;GATCF P F F Y M N E E P A F A K S V D A F Q R A G D T V I G S D V W I

GGTTCGGAGGCCATGATCATGCCCGGGATCAAGATCGGGCATGGAGCGGTGATAGGTAGCCGCGCTTIGGTTGCCAAAGACGTGGAACCCTACACCATAGG S E A M I M P G I K I G H G A V I G S R A L V A K D V E P Y T I V

TGGGGGGAAACCClXAAGTCGATTIAGGAAGCGC CTTTTAATTTAGwqlAGATATGGCTTGGTGGGATTGGCCGCTGGAACAAATG G N P A K S I R K R F S E E E I S M L L D M A W W D W P L E Q I

CAAGGAAGCAATGCtrl7T7sClTTTGTTCTGGCATTGCCAGCCTGTATCGTCGCTGGCAAGGCACAAGCGCACAAGTCCCGCATK E A M P F L C S S G I A S L Y R R W Q G T S A

__CGTGQG CG CTA-TCCTAAGCACTAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTATT

TTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATATATCAT AAAGGCTGGC7TTTCTTGTTATCGCAATAGTTGGCGAAGTAATCGCAACATCORF4-> M K G W L F L V I A I V G E V I A T S

CGCATrAAAATCTAGCGAGGGCTTTACTAAGCTTGCCCCTTCCGCCGTTGTCATAATCGGTTATGGCATCGCAQTTATTTTCTTTCTCTGGTTCTGAAAA L K S S E G F T K L A P S A V V I I G Y G I A F Y F L S L V L K

TCCATCCCTGTCGGTGTTGCTTATGCAGTC'TGGTCGGGACTCGGCGTCGTCATAATTACAGCCQTTGCCTGGTTGCTTCATGGGCAAAAGCTTGATGCGTS I P V G V A Y A V W S G L G V V I I T A I A W L L H G Q K L D A W

GGGGCTAGGTATGGGGCTCATAATTGCTGCCTTTTTTGCTCGCCCGATCCCCATCGTGGAAGTCGCTGCGGAGGCCGACGCCATGGTGACGGTGTTCG F V G M G L I I A A F L L A R S P S W K S L R R P T P W *

Sul-> M V T V F

GGCATTCT AATCTCACCGAGGACTCTTTTCGATGAGAGCCGGCGGCTAGACCCCOGCCGGCGCTGTCACCGCGGCGATCGAAATGCTGCGAGT,CGGATG I L N L T E D S F F D E S R R L D P A G A V T A A I E M L R V G S

CAGACGTCGTGGATGTCGGACCGGCCGCCAGCCATCCGGACGCGAGGCCTGTATCGCCGGCCGATGAGATCAGACGTATTGCGCCGCTCTTAGACGCCCTD V V D V G P A A S H P D A R P V S P A D E I R R I A P L L D A L

GTCCGATCAGATGCACCG7TGT AATCGACAGCTTCCAACCGGAAACCCAGCGCTATGCGCTCAAGCGCGGCGTGGGCTACCTGAACGATATCCAAGGAS D QM H R V S I D S F QP E T QR Y A L K R G V G Y L N D I Q G

TTTCCTGACCCTGCGCTCTATCCCGATATTGCTGAGGCGGACTGCAGGCTGGTGGTTATGCACTCAGCGCAGCGGGATGGCATC:GCCACCCGCACCGGTCF P D P A L Y P D I A E A D C R L V V M H S A Q R D G I A T R T G H

ACCTTCGACCCGAAGACGCGCTsCGACGAGATTGTGCGGTTCTTCGAGGCGCGGGTTTC'CGCCTTGCGACGGAGCGGGGTCGCTGCCGACCGGCTCATCCTL R P E D A L D E I V R F F E A R V S A L R R S G V A A D R L I L

CGATCCGGGGATGGGAT.li-.L-4.GAGCCCCGCACCGGAAACATCGCTGCACGTGCTGTCGAACCTls:AAAAGCT,GAAGTCGGCGTTGGGGCTTCCGCTAD P G M G F F L S P A P E T S L H V L S N L QK L K S A L G L P L

TTGGTCTCGGTGTCGCGGAAATCCTTCTTGGGCGCCACCGTTGGCCTTCCTGTAAAGGATCTGGGTCCAGCGAGCCTTGCGGCGGAACTTCACGCGATCGL V S V S R K S F L G A T V G L P V K D L G P A S L A A E L H A I G

GCAAIsGCGCTGACTACGTCCGCACCCACGCGCCTGGAGATCISCGAAGCGCAATCACCTrCTCGGAAACCCTCGCGAAATTTCGCAGTCGCGACGCCAGN G A D Y V R T H A P G D L R S A I T F S E T L A K F R S R D A R

AGACCGAGGGTTAGATCATGCCTAGCATTCACCTTCCGGCCGCCCGCTAGCGGACCCTGGTCAGGTTCCGCGAAGGTGGGCGCAGACATGCTGGGCTCGTD R G L D H A*

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2401 CAGGATCAAAC25CACTATGAGGCGGCGGTTCATACCGCGCCAGGGGAGCGAAGACAGCGAGGAGCCTCCGAACGTTCGGG00GCCCTCGGGTGAT2500ORF5-> M D S E E P P N V R V A C S G D

2501 ATCGACGAGGTTGTGCGGCTGATGCACGACGCTGCGGCGTGGATGTCCGCCAAGGGAACGCCCGCC%GGACGTCGCGCGGATCGACCGGACATTCGCGG 2600I D E V V R L M H D A A A W M S A K G T P A W D V A R I D R T F A E

2601 AGACCTTCGTCCTGAGATCCGAGCTCCTAGTCGCGAGTTGCAGCGACGGCATCGTCGGCTGTTGCACCTTGTCGGCCGAGGATCC 2685T F V L R S E L L V A S C S D G I V G C C T L S A E D

FIG. 3. Nucleotide sequence of the 2.7-kb PstI-BamHI fragment of Tn2424 containing the cat cassette, parts of the preceding cassettecontaining the putative promoter, and the 3' conserved segment coding for ORF4, sull, and ORF5. The putative promoter and the GTTsequences at the cassette boundaries are overlined; the potential stem-loop structures (59-base elements) are underlined. nt 1036 to 2685 areidentical to the corresponding sequences of the 3' conserved segments of the integrons from R388 (42), R46 (38), and pVS1 (7).

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- 1.3-diacetoxy-Cm

- 3-acetoxy-Cm

- 1-acetoxy-Cm

Tn2424catPacatAtcatBsorfSaorf

Tn2424catPacatAtcatBsorfSaorf

- Cm

1 2 3 4 5FIG. 4. Thin-layer chromatography of the ['4C]chloramphenicol

(Cm) CAT assay products. The CAT assay was performed by us-ing crude sonicates as described in Materials and Methods. Lanes:1, [14C]chloramphenicol; 2, DH1(pLQ162); 3, DH1(pLQ161); 4,NM522(pLQ892); 5, DH1(pTZ19R).

clone (pLQ892) containing the pBR325 (Tn9-type) cat gene.Figure 4 shows the separation by thin-layer chromatographyof chloramphenicol from its acetylated derivatives and dem-onstrates the acetylation of chloramphenicol by the clonedTn2424-encoded cat gene product.

Similarity of the sequence to those of the Agrobacteriumtumefaciens cat gene and other ORFs. The derived proteinsequence of the cat gene was compared with those of otherknown cat genes. No significant similarity was observedexcept with a recently described chromosomally encodedcat gene fromA. tumefaciens (45). A search of the GenBankand EMBL DNA data bases by the program TFASTArevealed three other strong similarities, all with ORFs (Fig.5). These three ORFs are adjacent to the genes that are theobjects of the respective publications and are not reported asORFs by the authors.The strongest similarity is with an ORF from Pseudomo-

nas aeruginosa chromosomal DNA. This ORF is down-stream of the regA gene, which regulates exotoxin produc-tion (16), and begins in the middle of another ORF, whichhas been identified as regB (46). The Pacat translation in Fig.5 is from an extended sequence from strain PAO1, kindlyfurnished by Susan Kaye (18), which contains the entireORF. In strain PAO1, in which regB is not expressed,chloramphenicol resistance is expressed (40). The Tn2424cat shows 72.9% amino acid identity with the P. aeruginosacat and 67.4% identity with theA. tumefaciens cat; the lattertwo show 70.3% identity with each other.The other two ORFs showing strong similarity to the

Tn2424 cat are partial; the first, Bsorf, is located immedi-ately upstream of the Bacillus sphaenicus chromosomalermG gene, encoding macrolide-lincosamide-streptograminB resistance (30); the second, Saurf, is immediately down-stream of an S. aureus plasmid gene, vgb, encoding resis-tance to virginiamycin (1). In addition to their strong simi-larity to the three gram-negative cat genes, these partialORFs show an especially strong similarity to each other(Fig. 5).

Similarity to the LacA-CysE-NodL family of acetyltrans-ferases. The carboxyl half of the Tn2424 cat shows signifi-cant similarity to three proteins: the thiogalactoside acetyl-transferase LacA of E. coli (15), the serine acetyltransferaseCysE of E. coli (10), and a putative acetyltransferase NodLof Rhizobium leguminosarum (43). Downie (14) has shownthe latter three proteins to be related to each other. The

Tn2424catPacatAtcatBsorfSaorf

1 50MTNYFESPF KGKLLTEQVK NP ..... NIK VGRYSYYSGYMGNYFESPF RGKLLSEQVS NP ..... NIR VGRYSYYSGYMENYFESPF RGITLDKQVK SP ..... NLV VGKYSYYSGY

+++DHKMSLLNLNNDH GPDPENILPI KGN.RNLQFI KPTITNENIL VGEYSYYDSK

5* *t ** 1 #* t# nt#

51 100YHGHSFDDCA RYLLPDRDDV DQLIIGSFCS IGSGARFIMA GNQGHRYDWVYHGHSFDDCA RYLMPDRDDV DKLVIGSFCS IGSGAAFIMA GNQGHRAEWAYHGHSFEDCA RYLLPD.EGA DRLVIGSFCS IGSGAAFIMA GNQGHRNEWINSPEKFYDNI EHHY..EFIG DKLIIGKFCA IAEGVKFIMN GAN.HRMDGIRG.ESFEDQV LYHY..EVIG DKLIIGRFCS IGPGTTFIMN GAN.HRMDG.

101 150SSFPFFYMNE EPAFAKSVDA FQR.AGDTVI GSDVWIGSEA MIMPGIKIGHSTFPFHFMHE EPVFAGAVNG YQP .AGDTLI GHDVWIGTEA MFMPGVRVGHSTFPFFFMPE VPEFENAANG YLP.AGDTVI GNDVWIGSEA IIMPGITVGDTTYPFNIFGC GWEKVTPTIE QLPFKGDTVI GNDVWIGQNV TIMPGVIIGDSTYPFHLFRM GWEKYMPSLK D+++** * 555*5 5 55555 *555* *15

151 200Tn2424cat GAVIGSRALV AKDVEPYTIV GGNPAKSIRK RFSEEEISML LDMAWWDWPL

Pacat GAIIGSRALV TGDVEPYAIV GGNPARTIRK RFSDGDIQNL LEMAWWDWPLAtcat GAVIGTRALV TKDVEPYAIV GGNPAKTIRK RFDDDSIALL LEMKWWGWPABsorf GAIIAANSTV VKSVEPYSIY SGNPAKFIKK RFSDEKIEFL LKLEWWNWSG

55*5** * 5 5555*5 *5555* *5 55 * 5 5 5 * 55 5

Tn2424catPacatAtcatBsorf

201 231EQIKEAMPFL CS. .SGIASL YRRWQGTSAADIEAAMPLL CT. .GDIPAL YRHWKQRQAT AERLKAAMPLM TS. . GNVAAL YRFWRSDSLEEIFDNLEIL TSEAGLEELM NKYSKRDAIN

FIG. 5. Amino acid similarities between the plasmid-specifiedCAT protein of Tn2424 and chromosomally specified CAT proteinsfrom P. aeruginosa (18, 40) and A. tumefaciens (45) and partialsequences of presumptive CAT proteins from B. sphaericus (30) andS. aureus (1). The last two sequences are unidentified readingframes adjacent to other antibiotic resistance genes which were theobjects of the publications cited. Symbols: ++ +, ends of partialsequences; #, conserved residues; *, conservative substitutionsamong the CAT proteins. Z values are as follows: Tn2424cat-Pacat,111.70; Tn2424cat-Atcat, 102.84; Tn2424cat-Bsorf, 46.15; Tn2424cat-Saorf, 16.30; Pacat-Atcat, 112.98; Pacat-Bsorf, 45.20; Pacat-Saorf, 20.62; Atcat-Bsorf, 41.30; Atcat-Saorf, 16.67; Bsorf-Saorf,30.71.

alignment is shown in Fig. 6. The program RDF (23) withktuple = 2 and 100 shuffles of the shorter sequence againstthe longer yields Z values of 13.11 for CAT-LacA, 11.28 forCAT-NodL, 8.87 for CAT-CysE, 8.74 for NodL-CysE, and6.81 for LacA-CysE. NodL and LacA are closely related(14) and give a Z value of 42.56. These four proteins, alongwith the homologs of Tn2424 cat shown in Fig. 5, form a newfamily of acetyltransferases.

DISCUSSION

Tn2424 is another example of a multiresistance integronwhich has evolved by site-specific integration of discreteunits (cassettes), principally containing antibiotic resistancegenes, between two conserved segments, i.e., a 5' conservedsegment containing a common antibiotic resistance promoter(and, in the opposite direction, a gene encoding a site-specific recombinase of the phage integrase family) and a 3'conserved segment containing ORF4, sull, and ORF5. Anancestral integron consisting of only the 5' and 3' conservedsegments has been described for P. aeruginosa plasmidpVS1 (6, 7). While some integrons contain single insertionsof resistance gene cassettes, most contain multiple tandeminsertions which include either the Tn21-type (aad41) or thepSa-type (aadA2) streptomycin-spectinomycin resistancegene. The integration of an aadA4 gene was presumably anearly event in integron evolution, as was the insertion of anintegron into Tn2613, an ancestral mercury transposon, tocreate Tn2J (44) (the order of these two events is undeter-

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CHLORAMPHENICOL ACETYLTRANSFERASE GENE OF Tn2424 2895

Tn2A2ACat 104 AFQRAGDTVIGSDMWIGiEAMIMPGlKIGHLAVIGSRALVAKDyEDYZIVGGNPAKSIEKR: 1: 11 111: 1:1:11: 11 IIII: ::I::I1:: 1111:

NodL 125 GLQLGRPVSIGRHAWIGGGAIILPGVTIGDHAVIGAGSVVTRDVPAGSTAMGNPARVKAGG: I::II III: :1 1111111 11

LacA 125 GEMYSFPITIGNNYWIGSHVVINPGVTIGDNSVIGAGSIVTKDIP2NVVAAGVPCRVIBEI

CysE

Consensus

: : : : : I : I I : : I I : :186 KSGGDRHPKIREGYMIGAGAKILGNIEVGRfaKIGAGSVVLQPYP-HITAAGVPARIVGEP

IG--VWIG--A-I-PG--IG--AVIGAGS-V--DVPP---A-G-PAR

FIG. 6. Amino acid similarities between the CAT protein of Tn2424, the probable acetyltransferase NodL of R. leguminosarum (43), andthe LacA thiogalactoside acetyltransferase and CysE serine acetyltransferase of E. coli. The similarity of the last three proteins was notedby Downie (14). Amino acid identities are indicated by vertical lines, and similarities are indicated by colons. Consensus amino acids,occurring in at least three of the four proteins, are shown.

mined). Several integrons with multiple insertions containtheir other resistance gene(s) upstream of aadA; Tn2424 isone of two integrons with an insert between aadA and sull,the other being TnJ696, whose cmLA gene is similarly located(5, 39). Unlike Tn1696, the 4.5-kb insert of Tn2424 consistsof multiple cassettes, including a 2.6-kb region containing noevident ORFs between the amikacin resistance gene and thecat gene (33, 34). It is thus unlikely that the cat and sullgenes of Tn2424 would be expressed from the commonpromoter in the 5' conserved segment; however, a putativepromoter has been located in the cassette immediatelyupstream of the cat cassette. Although most gene cassettesin integrons contain no promoter, one has been shown toexist in the cmLA cassette of Tn1696 (5).The cassette immediately upstream of the cat cassette and

the cat cassette itself both have, at their 3' ends, a potentialstem-loop structure called a 59-base element (9, 38). Al-though the precise length may vary, every inserted cassettethus far described has such a structure, while the ances-tral pVSl integron has none. While the integrase genelocated in the 5' conserved segment has not yet been shownto mediate resistance gene insertions, it has been shown tomediate excision of the aadA gene (41) and formation ofcointegrates by crossing over at the GTT sequence found atcassette junctions. The presence of a 59-base element in cisin one of the elements involved in the crossing over has beenshown to be necessary for formation of cointegrates to occur(26).Because of the lack of similarity of the Tn2424 ORF

encoding chloramphenicol resistance to the majority of CATproteins, it was desirable to demonstrate CAT activity.Figure 4 shows that chloramphenicol is acetylated by theTn2424-encoded cat gene product. The assay is similar tothat used by Tennigkeit and Matzura (45) for the A. tume-faciens CAT. The Tn2424 CAT shows the same pattern ofderivatives as theA. tumefaciens CAT, that is, the formationof 3-acetoxy-chloramphenicol and 1-acetoxy-chlorampheni-col but practically no 1,3-diacetoxy-chloramphenicol. A testof CAT activity by the phase extraction assay of Pothier etal. (36), in which butyryl coenzyme A is used as the cofactor,showed less than 10% as much activity for the Tn2424 catclones as for the clone of the Tn9-type cat. However, it ispossible that for the Tn2424-like CAT proteins, unlike theTn9-like CAT proteins, butyryl coenzyme A is a veryinefficient substitute for acetyl coenzyme A.The cat gene itself has no homology to other cat genes,

with the exception of the recently described chromosomalcat gene of A. tumefaciens (45). Use of the TFASTAprogram to analyze sequences in the DNA data base indi-cated the presence of another very similar cat gene in P.aeruginosa chromosomal DNA. This gene overlaps the regBtoxin regulatory gene by 139 nucleotides (46). The cat gene

shows a typical P. aeruginosa codon usage pattern with veryhigh G+C content in the third position, while regB does not.Only in strain PAO1, in which regB is not expressed becauseof an ATG--ACG mutation of its start codon, is chloram-phenicol resistance expressed (40). The Tn2424, A. tume-faciens, and P. aenuginosa cat genes are very similar, with67 to 73% amino acid identity among them. The partial ORFsfrom B. sphaenicus chromosomal DNA and from an S.aureus plasmid are slightly less related but are similarenough that they also may be cat genes. All of these proteinslack the conserved HHaVCDG active-site sequence of theTn9 CAT family; acyl transfer in the Tn2424 CAT familymust be mediated by a different base-catalyzed mechanism.The Tn2424, A. tumefaciens, and P. aeruginosa cat pro-

teins are members of the NodL-LacA-CysE acetyltransfer-ase family described by Downie (14). While NodL has notbeen proven to be an acetyltransferase, it is strongly relatedto LacA. One member of this family, CysE, has beencrystallized (48), and its structure is being determined. Sincethe structure of "classic" CAT is known (21, 22), crystalli-zation and structure determination of the Tn2424 CAT mayshed some light on structural similarities among these threeenzymes.

ACKNOWLEDGMENTS

We thank Sylvain Guerin and Rene Roy for help with the CATassay, Solange Brassard for skillful technical assistance, SusanKaye for communicating results prior to publication, and DougStorey for helpful discussions.

This work was supported by grants G-1541 from the NaturalSciences and Engineering Research Council and MT-10652 from theMedical Research Council of Canada to P. H. Roy. R. Parent held agraduate fellowship from the Fonds de la Recherche en Sante duQuebec.

ADDENDUM IN PROOF

The complete open reading frame corresponding to Saorfhas been shown to mediate chloramphenicol resistance whencloned into E. coli (V. Loncle and N. El Solh, personalcommunication).

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