Vol. 37, No. 10ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, OCt. 1993, p. 2119-21250066-4804/93/102119-07$02.00/0Copyright X 1993, American Society for Microbiology

Identification of the satA Gene Encoding a Streptogramin AAcetyltransferase in Enterococcus faecium BM4145ROSANNA RENDE-FOURNIER,1 ROLAND LECLERCQ,2* MALRC GALIMAND,1

JEAN DUVAL,2 AND PATRICE COURVALIN'Unite des Agents Antibacteriens, Centre National de la Recherche Scientifique, EP J0058,Institut Pasteur, 75724 Paris Cedex 15,1 and Service de Bacteriologie-Virologie-Hygiene,

H6pital Henri Mondor, Universite Paris XII, 94010 Creteil Cedex;2 France

Received 22 March 1993/Returned for modification 11 June 1993/Accepted 29 July 1993

Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group Aantibiotics by inactivation. The strain harbored a plasmid containing a gene, sat4, responsible for this resistance;this gene was cloned and sequenced. It encoded SatA, a protein deduced to be 23,634 Da in mass and homologouswith a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichiacoli, Pseudomonas aeruginosa, and Staphylococcus aureus. The similarity of SatA to other acetyltransferases,LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E. coli, and to two putativeacetyltransferases, NodL from Rhizobium kguminosarum and Urfl from E. coli, was also observed in a regionconsidered to be the enzyme's active site. Acetylation experiments indicated that acetyl coenzymeA was necessaryfor SatA activity and that a single acetylated derivative of pristinamycin IIA was produced. Other members ofthe streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme. Weconclude that resistance to the streptogramin A group of antibiotics in E. faecium BM4145 is due to acetylationby an enzyme related to the novel chloramphenicol acetyltransferase family.

The streptogramin antibiotics, also designated syner-gistins, are a family of natural compounds. They are pro-duced by Streptomyces spp. as a mixture of componentsclassified into two groups: streptogramin A and strepto-gramin B. Members of the streptogramin A group includepristinamycin IIA, RP54476, virginiamycin M, and strepto-gramin A and are polyunsatured cyclic macrolactones; the Bcomponents, comprising pristinamycin IA, RP57669, virgin-iamycin S, and streptogramin B, are cyclic depsipeptides. Inclinical practice, the A and B components are administeredin combination because they act synergistically (4). TIwocomplexes, pristinamycin and virginiamycin, are commer-cially available in certain European countries, and a newsemisynthetic injectable formulation, RP59500 (a combina-tion of RP54476 and RP57669), is under development. Thestreptogramins have a narrow spectrum of activity thatincludes gram-positive organisms (in particular staphylo-cocci and streptococci), gram-negative cocci, Mycoplasmaspp., Ureaplasma spp., Chlamydia spp., and Rickettsia spp.(4, 11, 18). Nearly all strains of the species Enterococcusfaecalis display intrinsic resistance to streptogramin A com-ponents (12). The biochemical mechanism for this resistanceis unknown. It is unlikely to be drug modification, sincewhole cells or crude extracts of E. faecalis strains do notinactivate streptogramin A components when tested by amicrobiological technique (unpublished data). By contrast,most Enterococcusfaecium isolates are susceptible to strep-togramin A components and to the members of the strepto-gramin complex (12).Acquired resistance to streptogramin A and B components

can be due to various mechanisms, but only resistance tostreptogramin A or to both components A and B is respon-sible for a marked loss of antibacterial activity of thecombination. Three mechanisms are known to be responsi-

* Corresponding author.

ble for resistance to streptogramin B components: enzymaticmodification of the drug, active antibiotic efflux, and modi-fication of the drug target. Resistance by enzymatic hydro-lysis of the depsipeptidic ring is conferred by the vgb gene(virginiamycin B) (2, 19). This rare plasmid-mediated resis-tance has only been found in staphylococci (12). Activeefflux due to the inducible staphylococcal msrA gene (mac-rolide streptogramin resistance) leads to resistance to 14-and 15-carbon-ring macrolides including erythromycin;these compounds are also inducers (26). Streptogramin Bcomponents are not inducers, but induction with erythromy-cin yields cross-resistance to these drugs. In most cases,resistance to streptogramin B components is due to methyl-ation of the 23S rRNA, which confers cross-resistance tomacrolide, lincosamide, and streptogramin B antibiotics, theso-called MLSB phenotype (36). Until now, acquired resis-tance to streptogramin A components appeared to be con-fined to staphylococci; this is due to two mechanisms,putative efflux or inactivation of the drug. The vga gene(virginiamycin A), which confers resistance to streptograminA components, has recently been sequenced (1). The homol-ogy of the deduced amino acid sequence with that of proteinsacting as ATP-binding efflux pumps suggested that activeefflux was responsible for resistance. Inactivation of strep-togramin A components occurs by acetylation of the hy-droxyl group (7, 20), but the corresponding gene has not yetbeen characterized.Here we report plasmid-mediated resistance to the strep-

togramin A group of antibiotics in E. faecium BM4145. Wesequenced the resistance gene and show that drug acetyla-tion is the biochemical mechanism involved.

MATERIALS AND METHODS

Bacterial strains and plasmids. The strains and plasmidsused in the present study are listed in Table 1. E. faeciumBM4145 was isolated from a urine sample in 1984 at the

2119

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.

2120 RENDE-FOURNIER ET AL.

TABLE 1. Strains and plasmids used in the present study

Strain or plasmid Relevant characteristicsa Source or reference

E. faecium BM4145 Pc Km MLSB Tc Str; pIP815, Tra- SA Wild typeE. faecalis BM4110 Str SA 5S. aureus BM3002 pIP524, Cd Km SA SB Tm 13E. coliDBlO thiA leu rna gyrA rpsL MLSAS Fas 6JM83 araA(lacproAB) rpsL [480A(1acZ)MJ5] 38

PlasmidspAT15 pBR322QOHindIII 3.9 kb from pIP815 This workpAT421 pUC18QlSau3AI-HindIII 1,618 bp from pAT15 This workpAT422 pUC18QSau3AI-HindIII 953 bp from pAT421 This workpAT423 pUC18fQSau3AI 193 bp from pAT421 This workpAT424 pUC19QEcoRI-HincII 966 bp from pAT421 This workpUC18/19 Tra- Mob- Ap 38pBR322 Tra- Mob- Ap Tc 3

a Plasmid designations and characters are aligned. Abbreviations: Ap, ampicillin resistance; Cd, cadmium resistance; Fa, fusidic acid resistance; Km,kanamycin resistance; MLSA, macrolide, lincosamide, and streptogramin A resistance; MLSB, macrolide, lincosamide, and streptogramin B resistance; Pc,penicillin resistance; superscript s, susceptible; SA, streptogramin A group resistance; SB, streptogramin B group resistance; Str, streptomycin resistance; Tc,tetracycline resistance; Tm, tobramycin resistance; Tra-, non-self-transferable; Mob-, nonmobilizable.

Henri Mondor Hospital, Creteil, France. The strain wasidentified as an Enterococcus isolate by Gram staining, theabsence of catalase, inability to produce gas, presence ofLancefield antigen group D, and growth in 40% bile, in 6.5%sodium chloride, in 0.1% methylene blue, and at pH 9.6.Species identification (14) was based on the absence ofpotassium tellurite reduction, pigmentation, and motility at30°C and on the results of biochemical tests in the API 50 CHgallery (bioMerieux, Marcy l'Etoile, France).Media and culture conditions. Brain heart infusion broth

and agar (Difco Laboratories, Detroit, Mich.) were used.Susceptibility tests were done on Mueller-Hinton agar(Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France).All incubations were performed at 37°C. Transformation ofEscherichia coli DB10 and JM83 was performed as describedpreviously (27).

Antibiotic susceptibility testing. Antibiotic susceptibilitywas determined by the disk diffusion technique. The Steersmethod with 104 CFU per spot was used to determine theMICs of antibiotics on solid medium as described previously(17). The kinetics of inactivation of pristinamycin IIA bywhole cells were determined in liquid medium as describedpreviously (17). Cells suspended in 0.01 M phosphate buffer(pH 7.0) containing 100 p,g of pristinamycin IIA per ml wereincubated at 37°C for various periods of time. The pH of thissuspension was monitored and remained constant. Inactiva-tion of the antibiotic was monitored by a microbiologicaltechnique by using Bacto-Medium I agar (Difco Laborato-ries) and Micrococcus luteus ATCC 9341.

Preparation of DNA and agarose gel electrophoresis. Prep-aration of total DNA (5) and purification of plasmid DNA(27) were as described previously. The DNA restrictionfragments were separated by electrophoresis in gels contain-ing 0.8% agarose type II or type VII (Sigma Chemical Co.,St. Louis, Mo.). Fragments were extracted from the gel byusing a commercially available kit (Geneclean; Bio 101, LaJolla, Calif.).DNA-DNA hybridization. For Southern hybridization (30),

DNA was transferred to Nytran membranes (Schleicher &Schuell, Dassel, Germany). Prehybridization and hybridiza-tion were carried out for 5 and 18 h, respectively, at 65°C in6x SSC (lx SSC is 0.15 M sodium chloride plus 0.015 Msodium citrate) containing 0.5% sodium dodecyl sulfate

(SDS) and 0.05% non-fat dry milk. The probe was radiola-beled by nick translation (27).DNA sequencing and computer analysis of sequence data.

Double-stranded DNA sequencing was performed with uni-versal primers (New England Biolabs, Beverly, Mass.).Synthetic oligodeoxyribonucleotides complementary to thedetermined sequence of the insert were used to complete thesequencing and were synthesized by the methoxy phosphor-amidite method (Unite de Chimie Organique, Institut Pas-teur, Paris, France). Nucleotide sequencing was performedby the dideoxynucleotide chain termination method (28) witha modified T7 DNA polymerase and [a-35S]dATP. Nucle-otide and amino acid sequence data were analyzed with theGenetics Computer Group software (9) and the FASTAprogram (25). The SWISS-PROT data base of the InstitutPasteur (release 23.0, August 1992) was screened, and align-ments were constructed by using the Genetics ComputerGroup software.

Analysis of plasmid-encoded proteins. The proteins speci-fied by the recombinant plasmids were synthesized in acell-free in vitro transcription-translation system (AmershamCorp., Arlington Heights, Ill.) (39). Proteins were labeledwith L-[355]methionine and were processed for electrophore-sis in an SDS-polyacrylamide gel as described previously(16).

Streptogramin A acetylation assays. Strains were grown toan optical density at 600 nm of 0.7 in brain heart infusionbroth. E. faecium BM4145 and Staphylococcus aureusBM3002 were pretreated with 500 ,g of lysozyme per ml and200 ,ug of lysostaphin per ml, respectively. These strains andE. coli DB1O derivatives were lysed by sonication in 1 mMTris HCl (pH 7.6). Cell debris was removed by centrifuga-tion at 100,000 x g for 45 min. The protein concentration inthe supernatants (S100) was determined by using the Brad-ford reagent (Bio-Rad, Munich, Germany).The formation of 5-thio-2-nitrobenzoate, which has a

molar extinction coefficient of 13,600 at 412 nm, by S100 inthe presence of pristinamycin IIA (0.1 mM) and acetylcoenzyme A (0.1 mM) was monitored with a recordingspectrophotometer equipped with a temperature-controlledcuvette chamber set at 37°C. The specific activity of strep-togramin A acetyltransferase was calculated from the rate ofchange in the A412 and by the protein concentration in the

ANTimICROB. AGENTS CHEMOTHER.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.

STREPTOGRAMIN A RESISTANCE IN E. FAECIUM 2121

added S100 fraction (29). The values obtained were themeans of four independent experiments and were expressedin nanomoles of pristinamycin IIA acetylated per minute permilligram of protein. Control reactions did not include theenzyme source, acetyl coenzyme A, or antibiotic. Similarexperiments were performed by using virginiamycin M orRP54476 (each at 0.1 mM) as substrates for the enzyme.For detection of modified pristinamycin IIA, enzymatic

and chemical acetylations of the drug were performed.Enzymatic acetylation of pristinamycin IIA was performedin the presence of [1-14C]acetyl coenzyme A (25 ,Ci/mmol)as a cofactor as described previously (29). Chemical acety-lation of pristinamycin IIA (1 mM) was performed overnightwith a 10-fold excess of acetyl chloride in the presence ofanhydrous pyridine (1 ml) at room temperature. The productwas dried under vacuum and was dissolved in methanol.Modified and unmodified pristinamycin IIA were separatedby thin-layer chromatography on precoated silica gel plates(60F254; E. Merck, Darmstadt, Germany) with chloroform-methanol (83:17) as the solvent. The products of the enzy-matic and chemical acetylations were detected by autora-diography or after spraying with ethanolic H2SO4 andheating at 100°C for 10 min, respectively.Enzymes and chemicals. T4 DNA ligase, restriction endo-

nucleases (Amersham Corp.), and the Sequenase version 2.0DNA sequencing kit (U.S. Biochemical Corp., Cleveland,Ohio) were used according to the recommendations of themanufacturers. Lysostaphin, lysozyme, acetyl chloride, andpyridine were from Sigma Chemical Co. and RNase A(bovine pancreas) was from Calbiochem-Behring (La Jolla,Calif.). Nick-translation kits were from Bethesda ResearchLaboratories, Inc. (Gaithersburg, Md.). Radiolabeled com-pounds were obtained from the Amersham RadiochemicalCentre (Amersham, England). Antibiotic disks and otherbacteriological supplies were from Sanofi Diagnostics Pas-teur. The following antibiotics were provided by the indi-cated manufacturers: penicillin G, pristinamycin IA, pris-tinamycin IIA, RP54476, and streptomycin, Rhone PoulencRorer (Antony, France); virginiamycin M, SmithKlineBeecham (Nanterre, France); ampicillin and kanamycin B,Bristol Laboratories (Syracuse, N.Y.).

Nucleotide sequence accession number. The nucleotidesequence of the insert of pAT421 including the satA genefrom E. faecium BM4145 has been deposited in the GenBankdata library (Los Alamos, N.M.) under accession numberL12033.

RESULTS

Properties ofE.faecium BM4145. E. faecium BM4145 wasresistant to penicillin G (MIC, 16 jig/ml), to high levels ofstreptomycin and kanamycin (MICs, > 1,000 ,ug/ml), totetracycline-minocycline, and constitutively to macrolides,lincosamides, and streptogramin B antibiotics (MIC of pris-tinamycin IA, >128 ,ug/ml). The strain was also resistant tomembers of the streptogramin Agroup of antibiotics (MIC ofpristinamycin IIA, 64 F±g/ml) and to the streptogramin com-plex (MIC of pristinamycin complex, 16 ,ug/ml). When testedby a microbiological assay (17) at an initial concentration of100 ,ug/ml, E. faecium BM4145 inactivated more than99.99% of pristinamycin IIA within 4 h (data not shown). Noinactivation was observed with the control strain E. faecalisBM4110, which is intrinsically resistant to pristinamycin IIA(MIC, 64 ,ug/ml). Analysis of the plasmid content of E.faecium BM4145 showed the presence of three plasmidswith approximate sizes of 27, 7, and 4 kb (data not shown).

EBIS S S

11 1 1@{33H III

_~2 pAT421(pUC18) A

pAT423(pUCI8)

pAT424(pUC19) B

pAT422(pUC18)

01 2 <

3 < *I 0S

-)N m ~200b,

C

FIG. 1. (A) Partial restriction map of the insert in pAT421. Boldline, E. faecium BM4145 DNA; hatched open boxes, pUC18 DNA;closed box, satA gene. Restriction sites: B, BamHI; E, EcoRI; H,HincII; HIII, HindIII; S, Sau3AI. (B) Inserts in pAT422, pAT423,and pAT424. The inserts are represented by the solid thin lines, andthe corresponding vectors are indicated in parentheses. (C) Se-quencing strategy of the 1,618-bp fragment containing satA. Arrowsindicate the direction and extent of the sequencing reactions withuniversal primers; arrows starting with a closed circle indicatesequencing reactions in which synthetic oligonucleotides were usedas primers: 1, 5'-CAGCTACIATTGCACCA-3' (positions 585 to569); 2, 5'-TCAGTAACGAAGAACAT-3' (positions 1122 to 1106);3, 5'-AAGATTGGATAATGATA-3' (positions 331 to 315); 4, 5'-CATCCACCCIT7TTTGAGG-3' (positions 855 to 839); 5, 5'-AGAATCrCAGCTGGTGT-3' (positions 1392 to 1366); 6, 5'-GCTTGCGGAACATTCCG-3' (positions 1261 to 1277); 7, 5'-CTCATATTATGATTCTA-3' (positions 266 to 282); 8, 5'-TGGTCTGAGACGTAGAT-3'(positions 953 to 969).

Cloning of the streptogramin A group resistance determi-nant. Plasmid DNA from E. faecium BM4145 and pBR322DNA digested with HindIII were mixed, ligated, and intro-duced by transformation into E. coli DB10, a mutant sus-ceptible to macrolide-lincosamide-streptogramin A antibiot-ics (MIC of pristinamycin IIA, 2 ,ug/ml) (6). Transformantsselected on ampicillin (100 p,g/ml) and pristinamycin IIA (4p,g/ml) were screened for their plasmid contents. The small-est hybrid plasmid, pAT15, conferring pristinamycin IIAresistance (MIC of pristinamycin IIA for E. coli DB10/pAT15, 64 p,g/ml) was found to contain a 3.9-kb HindIIIfragment. Southern hybridization experiments performed onundigested plasmid DNA from BM4145 by using pAT15 as aprobe showed that the determinant responsible for pris-tinamycin IIA resistance was located on a plasmid of ap-proximately 27 kb designated pIP815 (data not shown). Theprobe did not hybridize to pIP524 from S. aureus BM3002(13) (data not shown). Plasmid pIP524 encodes an acetyl-transferase which inactivates streptogramin A components(20). For subcloning experiments, the insert of pAT15 waspartially digested with Sau3AI and was cloned into pUC18cleaved with BamHI or BamHI plus HindIII (Fig. 1). Plas-mid pAT421 conferred resistance to pristinamycin IIA in E.coli DB1O and contained a 1.6-kb BamHI-HindIII insert.Plasmids pAT422 and pAT423, which did not confer resis-tance to pristinamycin IIA, contained a 953-bp BamHI-HindIhI and a 193-bp BamHI fragment, respectively.

Sequence of the insert of pAT421 and identification of thesatA gene. The nucleotide sequence of the 1,618-bp insert ofpAT421 was determined on both strands of DNA by thestrategy displayed in Fig. 1. A single open reading frame(ORF) was found between stop codons TAA and TGAlocated at positions 114 and 789, respectively. The putativetranslation start codon ATG (position 162) was preceded at10 bp by a ribosome-binding site sequence AAAGGACGcomplementary for 8 bases (underlined) to the 3'-OH termi-nus UCU UCCUCC sequence of Bacillus subtilis 16SrRNA (23). No -35 and -10 consensus promoter sequences

VOL. 37, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.

2122 RENDE-FOURNIER ET AL.

100GATCGGTAAACGGTTTATAAAATATGCAATAGATGTTCTTAATATAAAATTTGTACAGGAAACATTAATTTATTTGTGAACAACCAGATGCTGTTTCCTGSau3AI

M G P N P M K M Y P I E GTTTTAACCCGCGCTAAGTTATTGTAAAGGTAACCATTGTACTAAAAGGAGGTATTAATTTTATGGGTCCGAATCCTATGAAAATGTATCCTATAGAAGGA 200

N K S V Q F I K P I L E K L E N V E V G E Y S Y Y D S K N G E T F DAACAAATCAGTACAATTTATCAAACCTATTTTAGAAAAATTAGAAAATGTTGAGGTTGGAGAATACTCATATTATGATTCTAAGAATGGAGAAACTTTTG 300

K Q I L Y H Y P I L N D K L K I G K F C S I G P G V T I I M N G AATAAGCAAATTTTATATCATTATCCAATCTTAAACGATAAGTTAAAAATAGGTAAATTTTGCTCAATAGGACCAGGTGTAACTATTATTATGAATGGAGC 400

N H R M D G S T Y P F N L F G N G W E K H M P K L D Q L P I K G DAAATCATAGAATGGATGGCTCAACATATCCATTTAATTTATTTGGTAATGGATGGGAGAAACATATGCCAAAATTAGATCAACTACCTATTAAGGGGGAT 500

Sau3AI

T I I G N D V W I G K D V V I M P G V K I G D G A I V A A N S V V VACAATAATAGGTAATGATGTATGGATAGGAAAAGATGTTGTAATTATGCCAGGAGTAAAAATCGGGGATGGTGCAATAGTAGCTGCTAATTCTGTTGTTG 600

K D I A P Y M L A G G N P A N E I K Q R F D Q D T I N Q L L D I KTAAAAGATATAGCGCCATACATGTTAGCTGGAGGAAATCCTGCTAACGAAATAAAACAAAGATTTGATCAAGATACAATAAATCAGCTGCTTGATATAAA 700

Sau3AI

W W N W P I D I I N E N I D K I L D N S I I R E V I W K K *ATGGTGGAATTGGCCAATAGACATTATTAATGAGAATATAGATAAAATTCTTGATAATAGCATCATTAGAGAAGTCATATGGAAAAAATGATAAATATAC 800

TAAAAACAGAATAAGGAATAAATTGCTTGTCTGTTATCCCTCAAAAGGGTGGATGGTCAGCTTTGACATATAAAGTGTGTGATGGTGTGAAAAATTATTT 900

CTTGAAGGCTTATGAAATAAATCGGGCTTCTACTCCTAAGTTAACATCCATTTGGTCTGAGACGTAGATAAATCAAGCTACTTTTGACATAATTTAATTA 1000HincII

GGAAAATTCAGAGGCGTTGAGATAAAAAATCTTGATGCCTCTGTTTTATATTAAAAACAAATATAATTTAATAAATTTGCCATAGGGCAAAACAACCATG 1100

AGGTTATGTTCTTCGTTACTGAAAAACATAGCCTCTTTTTTTGTTGCCTGATTTGCAGAAAAAGAAAGGACTGATTAAAGTGATTACAAGATTTTTGAGA 1200

GATACACCTCTCGGTGCCATAGAAATAGAAATGCAACTTGTTCCAGGCTTTACTCCTCGTGCTTGCGGAACATTCCGAAGCCAGCAAAAAATAGGCTATA 1300

AATCTCCTCAAAGTTAGGTAGTTTTTCATAAGTCAAGAATCTATAAGATTCATCTTTGTTAAACAACACCAGCTGAGATTCTAAAAGTTTCAAGAACAAG 1400

ATTCTATTTATCCATACAACTGTTAGTTGAATAGCAATATCTTCCTGCTTATCTTTGGGGACATCTTTCATATCTAATTTATCAATGATATTCTCAACAA 1500

AAGAATACCGTTGTCGTTTAGCAGGTTTCAAACGTGAAATAATTTTTGAAGTCCCTGACTTCATTTCTTCCAGCCCCATCAAATAAAGCAATTCATCATA 1600

GAAATTCTTATTAAGCTTHindIII

FIG. 2. Nucleotide and deduced amino acid sequence of the 1,618-bp BamHI-HindIII insert of pAT421 containing the satA4 gene. Theputative ribosome-binding site is underlined. *, putative TGA stop codon. Only the restriction sites shown in Fig. 1 are indicated.

similar to those of B. subtilis were found. The 627-bpsequence, which extended from the ATG codon at coordi-nate 162 to the TGA codon at coordinate 789, encoded aprotein of 209 amino acid residues with a calculated molec-ular mass of 23,634 Da; it was designated SatA (for strepto-gramin A acetyltransferase) and the corresponding gene wasdesignated satA (Fig. 1 and 2).

Plasmid pAT424, which was constructed by cloning the966-bp HincII-EcoRI fragment of pAT421 into pUC19 (Fig. 1and 2), conferred resistance to pristinamycin IIA whenintroduced into E. coli DB10. The DNA of this plasmid wasused as a template in a cell-free, coupled transcription-translation system (39). One band of approximately 22,700Da, which was in close agreement with the translatedproduct ofsatA, was encoded by pAT424 but not by pAT422or pUC18 (Fig. 3).Amino acid sequence homology between SatA and acetyl-

transferases. A computer-assisted search of the SWISS-PROT data base revealed amino acid sequence similaritybetween SatA and four novel chloramphenicol acetyltrans-ferases found in Agrobacterium tumefaciens C58 (33),Tn2424 of E. coli W677 (24), Pseudomonas aeruginosa (24),and S. aureus BM3041 (21) (Fig. 4). Similarity with apolypeptide encoded by a partially sequenced ORF locatedupstream from the chromosomal ermG resistance gene ofBacillus sphaericus 33 (22) was also found. The similaritybetween SatA and the related amino acid sequences rangedfrom 54 to 72% (Table 2). This range was similar to that (54to 83%) observed within the family of novel Cat amino acidsequences.The C-terminal half of SatA also showed significant simi-

larity with four proteins: the thiogalactoside acetyltrans-

ferase (LacA) from E. coli (60% similarity) (15), the serineacetyltransferase (CysE) from E. coli (47% similarity) (8), aputative acetyltransferase (NodL) from Rhizobium legumi-nosarum (51% similarity) (32), and the functionally unknown

a~

Mr

46

3-pre -bcta TEM-1

-4ctamae T1M-i

.0 'Sat

14

FIG. 3. Autoradiogram of L-[35S]methionine-labeled polypep-tides specified in vitro by pUC18, pAT422, and pAT424 (satA). Theproteins were electrophoresed in a 15% polyacrylamide gel contain-ing SDS (16). Molecular masses are expressed in kilodaltons. Thepositions of SatA, 1-lactamase, and pre-13-lactamase are indicated.

ANTiMICROB. AGENTS CHEMOTHER.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.

STREPTOGRAMIN A RESISTANCE IN E. FAECIUM 2123

Ef SatASa CatBs ORFAt CatPa CatEc Cat

Ef SatASa CatBs ORFAt CatPa CatEc Cat

M GPNPMKMYPI EGN.KSVQFI KPILEKLENV EVGEYSYYDSMSLLNLNNDH GPDPENILPI KGN.RNLQFI KPTITN.ENI LVGEYSYYDS

DHMENYFESPF RGITLDKQVK SP......NL VVGKYSYYSGMGNYFESPF RGKLLSEQVS NP ...... NI RVGRYSYYSGMTNYFESPF KGKLLTEQVK NP ...... NI KVGRYSYYSGC IC CI C IC I IC II IIII

KNG.ETFDKQ ILYHYP. .IL NDKLKIGKFC SIGPGVTIIM NGAN.HRMDGKRG.ESFEDQ VLYHY.E.VI GDKLIIGRFC SIGPGTTFIM NGAN.HRMDGKNSPEKFYDN IEHHY.E.FI GDKLIIGKFC AIAEGVKFIM NGAN.HRMDGYYHGHSFEDC ARYLLPD.EG ADRLVIGSFC SIGSGAAFIM AGNQGHRNEWYYHGHSFDDC ARYLMPDRDD VDKLVIGSFC SIGSGAAFIM AGNQGHRAEWYYHGHSFDDC ARYLLPDRDD VDQLIIGSFC SIGSGARFIM AGNQGHRYDW

CICIC I C ICICII II IIC I III I C II CC

40482

333333

869449828383

Ef SatA .STYPFNLFG NGWEKHMPKL DQLPIKGDTI IGNDVWIGKD VVIMPGVKIG 135Sa Cat STYPFHLFR MGWEKYMPKL KDLPLKGDIE IGNDVWIGRN VTMMPGVKIG 143Bs ORF ITTYPFNIFG CGWEKVTPTI EQLPFKGDTV IGNDVWIGQN VTIMPGVIIG 99At Cat ISTFPFFFMP EVPEFENAAN GYLP.AGDTV IGNDVWIGSE AIIMPGITVG 131Pa Cat ASTFPFHFMH EEPVFAGAVN GYQP.AGDTL IGHDVWIGTE AMFMPGVRVG 132Ec Cat VSSFPFFYMN EEPAFAKSVD AFQR.AGDTV IGSDVWIGSE AMIMPGIKIG 132

II II CC I IIIC IICIIIII CIIIC CI

Ef SatA DGAIVAANSV VVKDIAPYML AGGNPANEIK QRFDQDTINQ LLDIKWWNWP 185Sa Cat DGAIIAAEAV VTKDVAPYSI VGGDPLKFIR KRFSDGVIEE WLALQWWNLD 193Bs ORF DGAIIAANST VVKSVEPYSI YSGNPAKFIK KRFSDEKIEF LLKLEWWNWS 149At Cat DGAVIGTRAL VTKDVEPYAI VGGNPAKTIR KRFDDDSIAL LLEMKWWGWP 181Pa Cat HGAIIGSRAL VTGDVEPYAI VGGNPARTIR KRFSDGDIQN LLEMAWWDWL 182Ec Cat HGAVIGSRAL VAKDVEPYTI VGGNPAKSIR KRFSEEEISM LLDMAWWDWP 183

IICIC C I III II I IIIIIC IC III C I II C II I

Ef SatA IDIINENIDK ILDNSIIREV IWKK 209Sa Cat MKIINENLPF IIN .. DIEM LKRKRKLLDD T 222Bs ORF GEEIFDNLEI LTSEAGLEEL MNKYSKRDAI N 180At Cat AERLKAAMPL MTS. .GNVAA LYRFWRSDSL 209Pa Cat LADIEAAMPL LCT. .GDIPA LYRHWQRQAT A 211Ec Cat LEQIKEAMPF LCS. .SGIAS LYRRWQGTSA 210

I C C C C C C

FIG. 4. Alignment of the deduced amino acid sequences ofstreptogramin A acetyltransferase from E. faecium (Ef SatA), ofchloramphenicol acetyltransferases from S. aureus (Sa Cat) (21), A.tumefaciens (At Cat) (33), P. aeruginosa (Pa Cat) (24), and Tn2424ofE. coli (Ec Cat) (24) and of the partial ORF from B. sphaericus (BsORF) (22). Identical (I) and conserved (C) amino acids were presentin at least five of the six sequences. The portion of the amino acidsequence of SatA homologous with the conserved region of theNodL-LacA-CysE-Urfl acetyltransferase family (Fig. 5) (10, 35) isoverlined. Points represent gaps introduced to optimize similarity.For classification as conservative substitutions, the amino acidswere grouped as follows: RK, LFPMVI, STQNC, AGW, H, ED,and Y.

protein Eco URF-1 (Urfl) from E. coli (53% similarity) (35)(Fig. 5).

Streptogramin A group acetylation assays. Crude extractsof E. faecium BM4145, E. coli DB10/pAT424, and E. coliDB10/pUC18 were assayed for pristinamycin IIA acetyla-tion. Pristinamycin-dependent and acetyl coenzyme A-de-pendent formation of 5-thio-2-nitrobenzoate was observedonly with SlOOs from BM4145 and DB10/pAT424. Thespecific activity of SatA, measured with DB10/pAT424 S100and 0.1 mM pristinamycin IIA, was 293 ± 11 nmol/min/mg.The values were 276 ± 10 nmol/min/mg for virginiamycin M

TABLE 2. Sequence similarity between the deduced amino acidsequences of SatA, the new chloramphenicol acetyltransferasefamily, and the partially sequenced ORF of B. sphaericus0

Sequence % Similarity to:compared Sa Cat Bs ORF At Cat Pa Cat Ec Cat

Ef SatA 72 68 56 54 56Sa Cat 67 56 56 54Bs ORF 57 52 53At Cat 80 79Pa Cat 83

a Similarity (conserved and identical amino acids) between pairs of se-quences is derived from the alignment in Fig. 3. Sequences are of Ef SatAfrom E. faecium BM4145, Sa Cat from S. aureus (21), Bs ORF from B.sphaericus (22), At Cat from A. tumefaciens (33), Pa Cat from P. aeruginosa(24), and Ec Cat from E. coli (24).

SatA 111 KGDTI IGNDVWIGKDVVIMPGVKIGDGAIVAANSVVVKDIAPYMLAGGNPANEIKQR

NodL 129 GRPVSIGRHAWIGGGAIILPGVTIGDHAVIGAGSVVTRDVPAGSTAMGNPARVKAGG

LacA 129 SFPITIGNNVWIGSHVVINPGVTIGDNSVIGAGSIVTKDIPPNVVAAGVPCRVIREI

CysE 191 DRHPKIREGVMIGAGAKILGNIEVGRGAKIGAGSVVLQPVPPHTTAAGVPARIVGKP

Urf 1 392 KFKTI IGDDVFVGSDTQLVAPVTVGKGATIAAGTTVTRNVGENALAISRVPQTQKEG

Consensus IG--V-IG----I---VT-G--A-I-AGS-VT--------A-G-P-R

FIG. 5. Alignment of the conserved region of SatA from E.faecium with the acetyltransferases LacA and CysE from E. coli,NodL from R. leguminosarum (10), and the putative acetyltrans-ferase Urfl from E. coli (35). *, identical residues; :, conservedresidues. Consensus amino acids, which occurred in at least four ofthe five sequences, are shown.

and 165 + 5 nmol/min/mg for RP54476. Chemical acetylationof anhydrous pristinamycin IIA (Rf, 0.5) yielded two deriv-atives (R,s, 0.65 and 0.72) detected by thin-layer chromatog-raphy (Fig. 6B, lanes 1 and 2). By contrast, enzymaticacetylation performed with [1_1 C]acetyl coenzyme A andthe crude extract of E. coli DB10/pAT424 produced a singlepristinamycin IIA derivative (Rf, 0.72) (Fig. 6A, lane 3). Thislatter Rf value was similar to that of the upper band observedfollowing chemical acetylation. These results confirm thatSatA has acetyltransferase activity and indicate that a singleacetylated product was obtained. The additional radioactivederivatives found on autoradiography after thin-layer chro-matography were also present in the controls (Fig. 6A, lanes1 and 2).

DISCUSSION

The clinical isolate E. faecium BM4145 exhibited anunusual level of resistance to the streptogramin A compo-nents as a result of inactivation. The plasmid-borne resis-tance determinant satA was expressed after cloning in themacrolide-lincosamide-streptogramin A-susceptible mutantE. coli DB10. Amino acid sequence comparison indicated

A1

B

0 Do

Rf I A A A I Rf1 2 3 1 2

FIG. 6. Thin-layer chromatography of pristinamycin IIA and ofderivatives. (A) Enzymatic acetylation by crude cell extracts de-tected by autoradiography; lane 1, extracts of E. coli DB10/pAT424and [1- 4C]acetyl coenzyme A; lane 2, pristinamycin IIA and[1-14C]acetyl coenzyme A; lane 3, extracts of E. coli DB10/pAT424,pristinamycin IIA, and [1-14C]acetyl coenzyme A. (B) Chemicalacetylation detected with ethanolic-H2SO4; lane 1, acetyl chlorideand pristinamycin IIA; lane 2, pristinamycin IIA as a control.Pristinamycin IIA (PIIA), its presumed acetylated derivatives(PIIA-Ac), and the corresponding Rfvalues are indicated. The upperband (Rf 0.72) in panel B, lane 1, is barely visible on this analysis.

VOL. 37, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.

2124 RENDE-FOURNIER ET AL.

that the SatA protein belongs to a family of acetyltrans-ferases that includes the recently described group of chlor-amphenicol acetyltransferases from A. tumefaciens C58(33), P. aeruginosa (24), and S. aureus BM3041 (21); Tn2424from E. coli W677 (24); acetyltransferases with nonantibioticsubstrates such as LacA (15) and CysE (8); and putativeacetyltransferases such as NodL (32) and Urfl (35). Similar-ity between SatA and the four latter acetyltransferases waslimited to the carboxy-terminal portion, which contains theputative active site of the enzymes (Fig. 5) (10), whereassimilarity with the Cat enzymes extended throughout thesequence (Fig. 4). This observation indicates a closer rela-tionship between SatA and the new family of Cat proteins(24). In addition, homology was deduced by observingidentity between SatA and the polypeptide inferred from thepartially sequenced ORF upstream from the chromosomalernG macrolide-lincosamide-streptogramin B resistancegene of B. sphaericus 33 (Table 2) (22).The satA gene conferred high-level resistance to the

streptogramin A components both in the original enterococ-cal host and in E. coli DB10. This is in contrast to the levelof chloramphenicol resistance determined by the novel Catproteins, which was low in A. tumefaciens C58 and E. coliW677 and undetectable in P. aeruginosa and S. aureusBM3041. In P. aeruginosa, the chromosomal cat gene over-laps the regB regulatory gene (37) and chloramphenicolresistance occurs only in P. aeruginosa PAO1, in whichregB is not expressed (31). The cat gene of S. aureusBM3041 is expressed only after cloning into E. coli (21).Hypothetically, better substrates than chloramphenicolcould exist for these "Cat" enzymes, and they remain to beidentified.The structural relationship between SatA and acetyltrans-

ferases strongly suggested that acetylation is responsible forresistance to the streptogramin A components in BM4145. Infact, inactivation experiments confirmed that acetyl coen-zyme A is a cofactor of SatA because of acetyl coenzymeA-dependent formation of 5-thio-2-nitrobenzoate during in-activation of pristinamycin IIA. Thin-layer chromatographyshowed that a single derivative is produced by SatA (Fig. 6).Acetylation of virginiamycin M and RP54476, compoundsstructurally related to pristinamycin IIA, was also observed.In addition, we detected by thin-layer chromatography,performed with [1-14C]chloramphenicol and extracts ofDB10/pAT424, acetylation of a small amount of chloram-phenicol (data not shown). However, this inactivation wasnot sufficient to confer chloramphenicol resistance, the MICof this antibiotic for strains DB10/pAT424 and DB1O re-mained unchanged (0.5 ,ug/ml). Thus, this result suggeststhat the spectrum of activity of SatA is not confined tocomponents of the streptogramin A group. As suggested forcertain Cat-like enzymes (see above), SatA could have abroader substrate specificity. The site of acetylation onpristinamycin IIA was not identified. Among the possibletargets, the hydroxyl group is the most likely. Le Goffic et al.(20) have shown that this moiety is modified by the PAC IIAacetyltransferase encoded by plasmid pIP524 from S. au-reus. The gene coding for this enzyme was distinct from satAsince pAT15 did not display sequence identity with pIP524 inhybridization experiments (data not shown). We could notcompare the pristinamycin IIA derivatives from enterococ-cal and staphylococcal strains since we were unable tomodify streptogramin A with crude extracts of resistant S.aureus.

Streptogramin antibiotics are active against gram-positivecocci. Pristinamycin has been proposed for the treatment of

infections caused by glycopeptide- and aminoglycoside-re-sistant E. faecium (34). Since this drug could be used in thissituation, a streptogramin A should be tested in vitro be-cause of the emergence of acquired resistance to this class ofcompounds in this species.

ACKNOWLEDGMENTSWe thank A. Smith for help with the chemical acetylation of

pristinamycin IIA and for critical reading of the manuscript and S.Dutka-Malen and M. Arthur for helpful discussions.

REFERENCES1. Allignet, J., V. Loncle, and N. El Solh. 1992. Sequence of a

staphylococcal plasmid gene, vga, encoding a putative ATP-binding protein involved in resistance to virginiamycin A-likeantibiotics. Gene 117:45-51.

2. Allignet, J., V. Loncle, P. Mazodier, and N. El Solh. 1988.Nucleotide sequence of a staphylococcal plasmid gene, vgb,encoding a hydrolase inactivating the B components of virgin-iamycin-like antibiotics. Plasmid 20:271-275.

3. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L.Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977.Construction and characterization of new cloning vehicles. II. Amultipurpose cloning system. Gene 2:95-113.

4. Cocito, C. 1979. Antibiotics of the virginiamycin family, inhib-itors which contain synergistic components. Microbiol. Rev.43:145-198.

5. Courvalin, P., and C. Carlier. 1986. Transposable multipleantibiotic resistance in Streptococcus pneumoniae. Mol. Gen.Genet. 205:291-297.

6. Datta, N., R. W. Hedges, D. Becker, and J. Davies. 1974.Plasmid-determined fusidic acid resistance in the Enterobacte-riaceae. J. Gen. Microbiol. 83:191-196.

7. De Meester, C., and J. Rondelet. 1976. Microbial acetylation ofM factor of virginiamycin. J. Antibiot. 29:1297-1305.

8. Denck, D., and A. Bock. 1987. L-Cysteine biosynthesis inEschenchia coli: nucleotide sequence and expression of theserine acetyltransferase (cysE) gene from the wild-type and acysteine-excreting mutant. J. Gen. Microbiol. 133:515-525.

9. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-sive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.

10. Downie, J. A. 1989. The nodL gene from Rhizobium legumi-nosarum is homologous to the acetyl transferases encoded bylacA and cysE. Mol. Microbiol. 3:1649-1651.

11. Drancourt, M., and D. Raoult. 1989. In vitro susceptibilities ofRickettsia rickettsii and Rickettsia conorii to roxithromycin andpristinamycin. Antimicrob. Agents Chemother. 33:2146-2148.

12. Duval, J. 1985. Evolution and epidemiology of MLS resistance.J. Antimicrob. Chemother. 16(Suppl. A):137-149.

13. El Solh, N., J. M. Fouace, Z. Shalita, D. H. Bouanchaud, R. P.Novick, aid Y. A. Chabbert. 1980. Epidemiological and struc-tural studies of Staphylococcus aureus R plasmids mediatingresistance to tobramycin and streptogramin. Plasmid 4:117-120.

14. Facklam, R. R, and M. D. Collins. 1989. Identification ofEnterococcus species isolated from human infections by aconventional test scheme. J. Clin. Microbiol. 27:731-734.

15. Hediger, M. A., D. F. Johnson, D. P. Nierlich, and I. Zabin.1985. DNA of the lactose operon: the lacA gene and thetranscriptional termination region. Proc. Natl. Acad. Sci. USA82:6414-6418.

16. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

17. Leclercq, R., C. Carlier, J. Duval, and P. Courvalin. 1985.Plasmid-mediated resistance to lincomycin by inactivation inStaphylococcus haemolyticus. Antimicrob. Agents Chemother.28:421-424.

18. Lefevre, J. C., and R. Bauriaut. 1989. Comparative in vitroactivities of pristinamycin and other antimicrobial agentsagainst genital pathogens. Antimicrob. Agents Chemother. 33:2152-2154.

ANTiMICROB. AGENTS CHEMOTHER.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.

STREPTOGRAMIN A RESISTANCE IN E. FAECIUM 2125

19. Le Goffic, F., M. L. Capmau, J. Abbe, C. Cerceau, A. Du-blanchet, and J. Duval. 1977. Plasmid-mediated pristinamycinresistance: PH IA, a pristinamycin IA hydrolase. Ann. Micro-biol. Inst. Pasteur 128B:471-474.

20. Le Goffic, F., M. L. Capmau, D. Bonnet, C. Cerceau, C. Soussy,A. Dublanchet, and J. Duval. 1977. Plasmid-mediated pris-tinamycin resistance PAC IIA: a new enzyme which modifiespristinamycin IIA. J. Antibiot. 30:665-669.

21. Loncle, V. 1992. Ph.D. thesis. Universite Paris XI, Paris,France.

22. Monod, M., S. Mohan, and D. Dubnau. 1987. Cloning andanalysis ofermG, a new macrolide-lincosamide-streptogramin Bresistance element from Bacillus sphaericus. J. Bacteriol. 169:340-350.

23. Moran, C. P., Jr., N. Lang, S. F. J. LeGrice, G. Lee, M.Stephens, A. L. Sonensheim, J. Pero, and R. Losick. 1982.Nucleotide sequences that signal the initiation of transcriptionand translation in Bacillus subtilis. Mol. Gen. Genet. 186:339-346.

24. Parent, R., and P. H. Roy. 1992. The chloramphenicol acetyl-transferase gene of Tn2424: a new breed of cat. J. Bacteriol.174:2891-2897.

25. Pearson, W. R., and D. J. Lipman. 1988. Improved tools forbiological sequence analysis. Proc. Natl. Acad. Sci. USA 85:2444-2448.

26. Ross, J. I., E. A. Eady, J. H. Cove, W. J. Cunliffe, S. Baumberg,and J. C. Wootton. 1990. Inducible erythromycin resistance instaphylococci is encoded by a member of the ATP-bindingtransport super-gene family. Mol. Microbiol. 4:1207-1214.

27. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

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

29. Shaw, W. V. 1975. Chloramphenicol acetyltransferase fromchloramphenicol resistant bacteria. Methods Enzymol. 43:737-755.

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

31. Storey, D. 1993. Personal communication.32. Surin, B. P., and J. A. Downie. 1988. Characterization of the

Rhizobium leguminosarum genes nodLMN involved in efficienthost-specific nodulation. Mol. Microbiol. 2:173-183.

33. Tennigkeit, J., and H. Matzura. 1991. Nucleotide sequenceanalysis of a chloramphenicol-resistance determinant fromAgrobacterium tumefaciens and identification of its gene prod-uct. Gene 98:113-116.

34. Wade, J., L. Baillie, N. Rolando, and M. Casewell. 1992.Pristinamycin for Enterococcus faecium resistant to vancomy-cin and gentamicin. Lancet 339:312-313.

35. Walker, J. E., N. J. Gay, M. Saraste, and A. N. Eberle. 1984.DNA sequence around the Escherichia coli unc operon: com-pletion of the sequence of a 17 kilobase segment containingasnA, oniC, unc, glmS and phoS. Biochem. J. 224:799-815.

36. Weisblum, B. 1985. Inducible resistance to macrolides, lincosa-mides, and streptogramin type B antibiotics: the resistancephenotype, its biological diversity, and structural elements thatregulate expression-a review. J. Antimicrob. Chemother.16(Suppl. A):63-90.

37. Wick, M. J., D. W. Frank, D. G. Storey, and B. H. Iglewski.1990. Identification of regB, a gene required for optimal exo-toxin A yields in Pseudomonas aeruginosa. Mol. Microbiol.4:489-497.

38. Yanisch-Perron, C., J. VMeira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide se-quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.

39. Zubay, G. 1973. In vitro synthesis of proteins in microbialsystems. Annu. Rev. Genet. 7:267-287.

VOL. 37, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

ac o

n 19

Oct

ober

202

1 by

218

.159

.197

.82.