PLASMID

(1992)

28,212-276

Nucleotide Sequence of the Chloramphenicol Resistance Determinant of the Streptococcal Plasmid plP501 PATRICKTRIEU-CUOT,' GILDA

DE CESPEDES,

AND THEA

HORAUD

Laboratoire des Staphylocoques et des Streptocoques. Institut Pasteur, 75724 Paris Cedex 15, France Received April 8, 1992; revised June 1, 1992 We have sequenced the chloramphenicol resistance determinant (cat) of plasmid pIPSO from Streptococcus agalactiae to investigate its relationship with other cognate cat determinants. Sequence analysis revealed that it exhibits a high degree of similarity with the cat genes of plasmids pC22 1 and pUBl12 from Staphylococcus aureus and pSCS1 from Staphylococcus intermedius. These genes, however, display several differences in their regulatory and coding regions. These results demonstrate that the cat determinant of plasmid pIP50 1 belongs to the 0 1992 Academic Press, Inc. pC22 1 subgroup of CAT variants.

Chloramphenicol resistance in bacteria of clinical importance is generally due to the synthesis of the enzyme chloramphenicol acetyltransferase (CAT),’ which exists in the native state as a trimer of identical subunits of approximately 215 amino acids each (Harding et al., 1987). Many naturally occurring variants of CATS have been described and nucleotide sequences are now available for 14 cat determinants, including 10 genes from gram-positive bacteria (Bacillus, CZo.stridium, and Staphylococcus) (for recent compilations, see Bannam and Rood, 199 1; Schwarz and Cardoso, 199 1). Comparison of the amino acid sequences of CAT proteins from gram-negative and gram-positive bacteria has revealed asignificant degree of homology among the various enzymes, and their phylogenetic relationship has been established (Bannam and Rood, 199 1; Schwarz and Cardoso, 199 1). The control of expression of CAT is generally constitutive in gramnegative bacteria, whereas it is inducible by

’ To should et des Roux,

whom correspondence and reprint requests be addressed at Laboratoire des Staphylocoques Streptocoques, Institut Pasteur, 28 rue du Dr 75724 Paris Cedex 15, France. Fax: (I) 43 06 98

35. * Abbreviation transferase. 0147-619X/92

used: CAT, chloramphenicol

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

acetyl-

subinhibitory concentrations of the drug in gram-positive bacteria (Shaw, 1983) with the remarkable exception of CATQ from Clostridium perfiingens (Bannam and Rood, 199 1). The inducible form of CAT expression is likely to be due to a post-transcriptional control via an attenuation-like mechanism (Lovett, 1990). The broad-host-range self-transferable plasmid pIPSO 1 (30.2 kb), which encodes resistance to chloramphenicol (Cm”) and to erythromycin (EmR) (macrolide-lincosamide-streptogramin B-type resistance phenotype), was originally isolated from Streptococcus agalactiae strain B96 (Horodniceanu et al., 1976). Plasmid functions (replication and transfer) and location of resistance determinants of pIP501 have been characterized (Behnke et al., 1981; Evans and Macrina, 1983; Krah and Macrina, 1989). DNA annealing studies have revealed that the cat determinant of pIPSO is widely distributed among streptococcal and enterococcal plasmids that encode resistance to chloramphenicol (Pepper et al., 1986) and is also present on the chromosome in several streptococcal strains of groups A, B, and G (Pepper et al., 1988). These experiments have also shown a close structural relationship between the cat gene of pIP50 1 and that of the straphylococcal plasmid pC22 1 (Pepper et al., 1986). Pre-

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liminary studies carried out in our laboratory have revealed that the restriction maps of the two structural genes, although similar, were not identical (data not shown). Therefore, we have determined the nucleotide sequence of the CmR determinant of the streptococcal plasmid pIPSO1 and studied its evolutionary relationships to those of the cut genes of the staphylococcal plasmids pC22 1, PUB 112, and pSCS1 which constitute the pC221 subgroup of CAT variants. The cut gene of PIP50 1 was located by molecular cloning in a 90 I-bp TuqI-Suu3A DNA fragment that was cloned in pUCl8 and sequenced in its entirety (Zhang et al., 1988) using synthetic oligonucleotides as primers. Comparison of its nucleotide sequence (Fig. 1) with those of the corresponding TaqI-Suu3A DNA fragments originating in pC221 (Shaw et al., 1985) and pUBl12 (Bruckner and Matzura, 1985) from Stuphylococcus aureus and pSCS1 (Schwarz et al., 1991) from Staphylococcus intermedius revealed that they differ at 37 positions (Table 1). Twelve differences are located in the 207bp-long region upstream from the cat structural genes (Fig. 1). Among these, only the mutations at positions 93, 130, and 20 1 are located in regions involved in CAT expression. The G to A substitution at position 93 in the nucleotide sequence of pC221 is located in the cut promoter region (Shaw et al., 1985). This transition generatesa lesscanonical -35 sequence and is therefore likely to correspond to a promoter down mutation (Moran et al., 1982). However, the effect of this mutation on the level of CAT synthesis has not been studied. The A to G transition at position 130 in pC221 occurred in the ribosome binding sequence of the leader peptide (SD1 ) and probably results in the formation of a less efficient translation signal (Moran et al., 1982; Schwarz et al., 1991). The nucleotide substitution at position 201 in the sequences of pIP501 and pC22 1 reduces the length of the base-paired regions (Fig. 1) which constitute the cut translational attenuator ( 12 bp in pIP50 1 and pC22 1 versus 13 bp in PUB 112 and pSCS1). The free energies (Tinoco et al., 1973) of formation of the

273

corresponding stem-loop structures are - 18 kcal/mol for pIPSO and pC221 and - 19.8 kcal/mol for PUB 112 and pSCS1. The differences at positions 130 and 201 have apparently no effect on the regulation of CAT expression, which, in all four plasmids, is inducible by subinhibitory concentrations of chloramphenicol (Bruckner and Matzura, 1985; Schwarz et al., 1991; Shaw et al., 1985; and our data, not shown). The structural cat genes from pIPSOl, pC22 1, pSCS1, and PUB 112 differ at 22 positions which are located in 20 codons (Table 1). Of these changes, the 12 mutations at positions 225, 273, 285, 360, 366, 441, 672, 759, 760-762, 765, and 802 are silent. The substitutions at positions 8 10 and 836 lead to isofunctional replacements: methionine (pIPSOl, pC22 1) versus isoleucine (pSCS1, pUBl12) and arginine (pIP50 1, pC22 1) versus lysine (pSCS1, PUB 112). The differences at positions 533,565,689,775, and 832-834 maintained a similarly charged residue at the same place: isoleucine (pIPSOl) versus threonine (pC22 1, pSCS1, PUB 112) leutine (pIP50 1, pC22 1) versus phenylalanine (pSCS1, pUBll2), serine (pIPSOl, pSCS1, pUB 112) versus asparagine (pC22 I), serine (pIP50 1) versus alanine (pC22 1, pSCS1, PUB 112), and histidine (pIP50 1, pC22 1) versus lysine (pSCS1, pUBl12). Only the changes at positions 226 and 525 correspond to a nonequivalent amino acid replacement: glutamic acid (pIP50 1) versus lysine (pC22 1, pSCS1, PUB 112) and lysine (pIP50 1) versus asparagine (pC221, pSCS1, pUBl12). The 45-bp-long noncoding regions downstream from the cat genesin pIP50 1, pC22 1, pSCS1, and PUB 112 differ at three positions (Table 1). These results demonstrate that the cat determinant of the streptococcal plasmid pIPSO belongs to the pC221 subgroup of CAT variants. The structural diversity of the DNA fragments encoding CmR in pIPSOl, pC22 1, pSCS1, and PUB 112 provides an additional example of microevolution under natural conditions. It is noteworthy that the TaqISau3A DNA fragment that contains the cat gene of plasmid pIPSO possessestwo differ-

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Tut/I TCQACTTTMAAGCQMC

-35

~~TTOC~~CA(~~~MC~~T~A~TTT~~~TM~TTTAT

-10

SD 1

ttgataAAAATTGCTMTTCAGtataatT~TATTTACgaggTGXATAWB3TAT 100 .

~tLYsLYBSetOllrSer*** -TCAQAQGATTATTCCTCCTMATATAAA

SD2 MetThrPheAmIleIleG1uLcuQluAmTrpAepArgLysGluTyrPheGluHirrTyrPhe MTTTllA1ATTTAgQaggMLlOTT1T1TATATAT~CT~TMTATTA~~TTA~~~TA~~TA~~CA~A~ I 200 ~snGlnGlnTh~~SerIleT~L~GluIle&pI1eThrLcuPheL~~~etIlcL~LyeLysGl~GluIleTyrPro AATCAQCAAACTACTTATAQCATl’ACTAAAGAAATTQATATTACTTTUPTTAAAQATATGATMAAM 300

OAUQQATATGMATTTATCCC

LysLeulenProLauTyrThrValPhe~~L~GlnThrGluLysPhcT~~nIleTr~hrGluSer~pL~~AsnPheI1eSerPhc MQTTAAATCCTTTGTATACAQFPTTTMTMQCAAACT GAAAMTTTACTMCATTTCCTQMTCTTT 500

FIG. 1. Nucleotide sequence of the 90 1-bp TuqI-Suu3A DNA fragment containing the cut determinant of plasmid pIPSO1. Numbering begins at the first bp in the sequence.The -35, - 10, and Shine-Dalgamo (SD) sequencesare written in lowercase letters. The deduced amino acid sequencesof the leader peptide (positions 144-170) and of the CAT protein (positions 208-852) are presented above the DNA sequence. The convergent horizontal arrows indicate the inverted repeat structures in the regulatory region. The EMBL accession number for the nucleotide sequence presented here is X65462.

ent evolutionary lineages, The mutated positions of the cut regulatory region of pIP501 are more related to those of pSCS1 and PUB 112 (50% identity) than to that of pC22 1 (17% identity); on the contrary, the mutated positions of the cat structural gene from pIP501 are more similar to those of pC221 (30% identity) than to those of pSCS1 and PUB 112 (8% identity). The staphylococcal plasmids pC22 1, PUB 112, and pSCS1 belong to a family of small (~5 kb), interrelated plasmids (Gillespie and Skurray, 1988; Schwarz et al., 1991) that replicate by using a singlestranded DNA intermediate (Gruss and Ehrlich, 1989). As a consequence of their mode of replication, these replicons are highly re-

combinogenic and it is thus likely that the pIP50 1-type cat gene has evolved following homologous recombination between pC22 1 and PUB 112/pSCS1-type cat genes. Numerous broad-host-range streptococcal and enterococcal EmR plasmids are highly structurally related to pIP50 1, as well as to other streptococcal plasmids encoding CmR and EmR (Horaud et al., 1985). It is therefore tempting to speculate that genesis of those plasmids encoding CmR and EmR, such as pIPSOl, is due to acquisition by EmR plasmids of a DNA fragment bearing a pC22 1-related cut gene. The apparent ability of the cut and replication genes of pC221 to move en bloc between unrelated replicons (Gillespie

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DIFFERENCES BETWEEN THE 901-bp TuqI-Suu3A DNA FRAGMENTSCONTAININGTHE cut DETERMINANTS OFPLASMIDSpIP50 1 FROMStreptococcusagalactiae, pC22 1 AND pUB 112 FROM

Staphylococcus aureus, AND pSCS1 FROMStaphylococcus intermedius Base or codor@ in Base No.” 14 18 24 36 66 93 106 130 136

177 181 201 225 226 273 285 360 366 441 525 533 565 672 689 159 760-762 765 775 802 810 832-834 836 869 870 893

Amino acid in the CAT protein of

PIP50 1

pC22 1

pUB112

pscs 1

C C A G G G A A C T A G atT Gaa aaT acT ccc ttA i%T aaA aTt ctt aaT aGc gcT ca caA Tct Cta atG CaT a(% A A T

A T G A A T G G C T G A atC Aaa aaC acG ccT ttG .%T ZUC act ctt aaC WAC gcT TtG caG Get Tta atG CaT a% G G C

C C A A G T A A T C G T atC Aaa aaC acG ccT ttG gPC Zii3C act Ttt aaC aGc fs TtA caG Get Tta atA AaT a‘% G G T

C C A A G T A A T C C T atC Aaa F& acG ccT ttG &SC SK act Ttt XC aGc gee TtA caG Get Tta atA AaG a& G G T

PIP50 1

pc22 1

IIe GIu Asn Thr Pro

IIe LYS Asn Thr Pro Leu GIY Asn Thr

LeU GIY LYS

IIe Leu Asn Ser Ala LeU

Gin Ser Leu Met His A%

pUBI

pscs 1

Be LYS Asn Thr Pro

IIe LYS Asn Thr Pro

LA% GIY

JXU GIY

Asn Asn Ala Leu Gin Ala

Asn Thr Phe Asn Ser Ala Leu Gin Ala

Asn Thr Phe Asn Ser Ala

LeU

LeU

Met His LYS

IIe LYS LYS

LeU

Leu

Gin Ala Leu IIe LYS LYS

’ The numbering refers to the first bp of the Tuql restriction site and indicates the position of nonidentical bases. * The coding regions span from positions 208 to 852. ’ Identical and nonidentical nucleotides in the codons are written in lowercase and uppercase letters, respectively.

and Skurray, 1988) is consistent with this hypothesis.

tut National de Ia Sante et de Ia Recherche MMicaIe (to T.H.).

ACKNOWLEDGMENTS We thank K. Pepper for her participation in initial cloning experiments. This work was supported by Grant 9003 12 (Molecular Genetics and Physiopathology of Streptococci, Enterococci and Staphylococci) from Inti-

REFERENCES BANNAM, T. L., AND ROOD, J. I. (1991). Relationship between the Clostridium perfringens catQ gene product and the chloramphenicol acetyltransferases from

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other bacteria. Antimicrob. Agents Chemother. 35, 411-416.

BEHNKE, D., GILMORE, M. S., AND FERRETTI, J. J. (1981). Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant. Mot. Gen. Genet. 182,4 14-42 1. BR~~CKNER, R., AND MATZURA, H. (1985). Regulation of the inducible chloramphenicol acetyltransferase gene of the Staphylococcus plasmid PUB 112. EMBO J. 4,2295-2300. EVANS,R. P., AND MACRINA, F. L. (1983). Streptococcal R plasmid pIPSO : Endonuclease site map, resistance determinant location and construction of novel derivatives. J. Bacterial. 154, 1347-1355. GILLESPIE,M. T., AND SKURRAY,R. A. (1988). Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus. FEMS Microbial. Lett. 51,205-2 10. GRUSS,A., AND EHRLICH, S. D. (1989). The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbial. Rev. 53, 23 l-24 1. HARDING, S. E., ROWE,A. J., AND SHAW,W. V. (1987). The molecular mass and trimeric nature of chloramphenicol transacetylase. Biochem. Sot. Trans. 15,5 13. HORAUD, T., LE BOUGUENEC,C., AND PEPPER,K. (1985). Molecular genetics of resistanceto macrolides, lincosamides and streptogramin B (MLS) in streptococci. J. Antimicrob. Chemother. lC(Supp1. A), 11l135. HORODNICEANU,T., BOUANCHAUD,D. H., BIETH, G., AND CHABBERT,Y. A. (1976). R plasmids in Streptococcus agalactiae (group B). Antimicrob. Agents Chemother. 10, 795-80 1. KRAH, E. R., III, AND MACRINA, F. L. (1989). Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIPSOl. J. Bacterial. 171,6005-6012.

Lov~rr, P. S. (1990). Translational attenuation as the regulator of inducible cat genes.J. Bacterial. 172, l-6. MORAN, C. P., JR., LANG, N., LEGRICE,S. F. J., LEE,G., STEPHENS, M., SONENSHEIN, A. L., PERO,J., AND LoSICK, R. (1982). Nucleotide sequencesthat signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186, 339-346. PEPPER,K., DE CESPEDES, G., AND HORAUD,T. (1988). Heterogeneity of chromosomal genes encoding chloramphenicol resistance in streptococci. Plasmid 19, 71-74. PEPPER,K., LE BOUGUENEC,C., DE CESPEDES, G., AND HORAUD, T. (1986). Dispersal of a plasmid borne chloramphenicol resistance gene in streptococcal and enterococcal plasmids. Plasmid 16, 195-203. SCHWARZ,S., AND CARDOS~,M. (199 1). Nucleotide sequence and phylogeny of a chloramphenicol acetyltransferaseencoded by the plasmid pSCS7from Staphylococcus aureus. Antimicrob. Agents Chemother. 35, 1551-1556. SCHWARZ,S., SPIES,U., AND CARDOSO,M. (199 I). Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferasegenefrom Staphylococcus intermedius. J. Gen. Microbial. 137,977-98 1. SHAW, W. V. (1983). Chloramphenicol acetyltransferase: Enzymology and molecular biology. CRC Crit. Rev. Biochem. 14, l-47. SHAW,W. V., BRENNER,D. G., LEGRICE,S. F. J., SKINNER, S. E., AND HAWKINS, A. R. (1985). Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221. FEBS Lett. 179, 101-106. TINOCO,I., BORER,P. N., DENGLER,B., LEVINE,M. D., UHLENBECK,O., CROTHERS,D. M., AND GRALLA, J. (1973). Improved estimation of secondary structure in ribonucleic acids. Nature New Biol. 246,40-4 1. ZHANG, H., SCHOLL,R., BROWSE,J., AND SOMERVILLE, C. (1988). Double-stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 16, 1220. Communicated by F. Macrina