Molecular analysis of the translational attenuator of a constitutively expressed erm(A) gene from Staphylococcus intermedius
Christiane Werckenthina,b and
Stefan Schwarza,*
a Institut für Tierzucht und Tierverhalten der Bundesforschungsanstalt für Landwirtschaft (FAL), Dörnbergstraße 2527, 29223 Celle;
b Institut für Medizinische Mikrobiologie, Infektions- und Seuchenmedizin, Tierärztliche Fakultät, Ludwig-Maximilians-Universität München, Veterinärstrasse 13, 80539 München, Germany
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Abstract
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During the course of a study on macrolide, lincosamide and streptogramin B resistance among staphylococci from animal sources, a Staphylococcus intermedius isolate was found to carry a constitutively expressed erm(A) gene on the 70 kb plasmid pSES29. The molecular basis of constitutive erm(A) expression was investigated by cloning and sequence analysis of the erm(A)-associated translational attenuator. Two point mutations in this regulatory region were detected. These mutations cause constitutive erm(A) gene expression by destabilization of mRNA secondary structures required for the inducible type of erm(A) gene expression.
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Introduction
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Resistance to macrolide, lincosamide and streptogramin B antibiotics (MLSB resistance) in staphylococci is due mainly to the dimethylation of the antibiotic target site, an adenine residue in the 23S rRNA.1 Four different erythromycin ribosome methylase (erm) genes, erm(A), erm(B), erm(C) and erm(F), are known to play a role in MLSB resistance of staphylococci.2 The erm(A) gene is part of transposon Tn5543 which usually integrates at a specific site, att554, in the Staphylococcus aureus chromosome, but may also be located on plasmids.35 Chromosomal and plasmid locations of the erm(A) gene have also been described in Staphylococcus epidermidis.4, 5 The expression of erm(A) can be either inducible or constitutive.1,3 Inducible expression is assumed to occur via translational attenuation.1, 3 All information concerning the molecular basis of constitutive erm(A) gene expression is from mutants derived from in vitro selection experiments.3 In this study we analysed the regulatory region of a constitutively expressed naturally occurring erm(A) gene from Staphylococcus intermedius.
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Materials and methods
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S. intermedius isolate CW9 was obtained from a carrier pigeon. Its MLSB resistance patterns were determined by the agar disc diffusion method with discs containing 15 µg erythromycin or 10 µg clindamycin (Becton Dickinson, Heidelberg, Germany), or tablets containing 30 µg azithromycin, 200 µg spiramycin or 150 µg tylosin (Hiss Diagnostics, Freiburg, Germany).6 Preparation of plasmid and whole-cell DNA as well as transformation of plasmids into S. aureus RN4220 protoplasts were performed as described previously.6 The erm(A) gene was detected by Southern blot hybridization. An internal 434 bp PCR fragment (primers bp 46394653 and 50725055 in the Tn554 sequence, GenBank accession no. X03216), amplified from the erm(A) structural gene of S. aureus strain 12067 served as a specific gene probe. Moreover, a PCR assay for amplification of the regulatory region was developed (primers bp 51385159 and 57315712 in the Tn554 sequence). The resulting amplicon obtained using a Pfu DNA polymerase (Stratagene, Amsterdam, The Netherlands) was cloned into pUC18 and the inserts of three independent clones were sequenced on both strands (EBI database accession no. AJ276730). The stabilities of predicted mRNA secondary structures were calculated as described previously.8 Conjugation of the erm(A)-carrying plasmid was tested by filter-mating experiments with the rifampicin- and fusidic acid-resistant S. aureus strain B111 as recipient.9
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Results and discussion
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S. intermedius CW9 harboured a single plasmid of c. 70 kb, designated pSES29, which carried an erm(A) gene as confirmed by hybridization and PCR analysis. In naturally occurring S. aureus and S. epidermidis isolates, erm(A) genes have rarely been detected on plasmids,5 and have so far not been identified in S. intermedius. The erm(A) gene is usually located on the non-conjugative transposon Tn554 which inserts preferentially at a specific site in the chromosome of S. aureus. If this site is already occupied or deleted, integration of Tn554 at secondary insertion sites in the chromosome or in plasmids is possible.3 The two erm(A)-carrying plasmids known so far, from Australian S. aureus and S. epidermidis, were structurally closely related and were able to transfer by conjugation.5 In the case of plasmid pSES29 from S. intermedius, it was not possible to demonstrate conjugative transfer. Hybridization experiments identified restriction fragment length polymorphisms of erm(A)-carrying EcoRI fragments which range in size between 4.85 and 10.5 kb.5, 7, 10 The erm(A) gene of pSES29 was located on an EcoRI fragment of 6.5 kb. Since only one EcoRI site is located in Tn554, the occurrence of different-sized erm(A)-bearing EcoRI fragments indicates the presence of variable sequences in the regions adjacent to the integrated Tn554.
The original S. intermedius CW9 as well as S. aureus RN4220:pSES29 showed an ML resistance pattern typical of constitutive erm(A) expression, namely resistance to all tested 14-, 15- and 16-membered macrolides, as well as clindamycin. The erm(A) gene of Tn554 is expressed inducibly with only 14- and 15-membered macrolides acting as inducers. Based on the close structural relatedness of the regulatory regions of erm(A) and erm(C), similar, if not identical, regulatory processes can be assumed to occur during inducible gene expression via translational attenuation. The regulatory region is located immediately 5'; of the structural gene. It consists of at least one open reading frame for a small peptide of 19 amino acids and at least two pairs of inverted repeated sequences.1, 3 In the absence of inducers, the erm(A) transcripts are believed to form stable mRNA secondary structures by pairing of IR1:2 and IR3:4 (Figure, a
), thereby burying the erm(A)-associated ribosome binding site and the first six bases of the erm(A) structural gene in the IR3:4 stemloop structure (Figure, a
). As a consequence, the erm(A)-associated ribosome binding site is not accessible to ribosomes and the transcripts cannot be translated. In the presence of inducers, however, a macrolide-bound ribosome is stalled at the ninth codon of the small peptide and in part overlaps the IR1 sequence. Thus, pairing of IR1:2 is impossible and the most stable mRNA secondary structure is formed by pairing of IR2:3 (Figure, b
). As a consequence, the erm(A)-associated ribosome binding site located in IR4 is now accessible for ribosomes, allowing the translation of the erm(A) transcripts.
PCR amplification of the regulatory region of the erm(A) gene from pSES29 yielded a product of 592 bp comprising the entire regulatory region and the 5'; end of the erm(A) structural gene. Two pairs of inverted repeated sequences (IR14) were detected in the region immediately upstream of the erm(A) structural gene. Compared with the sequence of Tn554 only two alterations (5353C
5353T and 5362G
5362C) were detected in the erm(A) translational attenuator of pSES29. These two exchanges were located within the IR1 sequence. Previous analysis of the regulatory region of a constitutively expressed erm(A) mutant selected under in vitro conditions in the presence of non-inducers also identified a point mutation in IR1.3 This point mutation, designated lin-71, involves the replacement of C by G at position 5363 in the Tn554 sequence.3 The lin-71 mutation was considered to cause constitutive erm(A) expression by destabilization of the stemloop formation between IR1 and 2.3 One of the two point mutations detected in the erm(A) regulatory region of pSES29 (5362G
5362C) was located next to the lin-71 mutation in IR1. Comparative calculation of the stabilities of the IR1:2 mRNA secondary structures in Tn554 and pSES29 showed that the two point mutations in pSES29 distinctly decreased the stability of this stemloop structure from
G 54.3 kJ/mol to
G 15.9 kJ/mol. The low stability of the IR1:2 stemloop structure favours the pairing of IR2:3 resulting in a distinctly more stable mRNA secondary structure (
G 86.9 kJ/mol). Due to the mutations in IR1, pairing of IR2:3 will occur independently of the presence or absence of inducers and thus allow constitutive expression of the erm(A) gene.
Comparison of the lin-71 mutation with those seen in the erm(A) regulatory region of pSES29 confirmed that mutations leading to constitutive erm(A) gene expression to those that arise under natural conditions can be selected under in vitro conditions. Studies of staphylococci carrying inducibly expressed erm(C) genes showed that the presence of non-inducers, such as 16-membered macrolides, lincosamides and streptogramins, favours the switch from the inducible to the constitutive type of erm gene expression. This switch is accompanied by irreversible structural alterations in the regulatory regions, including deletions, duplications and also point mutations.1, 8 Although several non-inducers, such as tylosin, spiramycin and virginiamycin, have been banned from use as feed additives in the EU, non-inducing MLS antibiotics are still widely used in human and veterinary medicine. Since the development of constitutively resistant strains will strongly reduce the efficacy of MLS antibiotics in the control of pathogenic staphylococci, prudent use of the non-inducers in both human and veterinary medicine is highly recommended.
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Acknowledgments
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The authors thank Henrik C. Westh and William C. Noble for providing S. aureus strains 1206 and B111.
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Notes
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* *Corresponding author. Tel: +49-5141-384673; Fax: +49-5141-381849; E-mail: stefan.schwarz{at}fal.de 
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Received 22 March 2000;
returned 25 June 2000; revised 12 July 2000;
accepted 7 August 2000