No abstract

Nucleotide sequences of the homologous tetracycline resistance (tet) determinants of plasmid RP1 and transposon Tn1721 have been determined. Two open reading frames of divergent polarity have been assigned to a regulatory gene (tetR) and a gene encoding a resistance protein (tetA). The intercistronic region contains appropriate regulatory and transcription signals. The tetR gene can code for a protein of 216 amino acids (deduced mol.wt. 23,288) and the tetA gene for a protein of 399 amino acids (deduced mol. wt. 42,205). Based on the deduced amino acid sequence, the tetA proteins of RP1/Tn1721 are 78% homologous with that of pBR322 and 45% homologous with that of Tn10. We conclude that a single tetA gene mediates resistance in each of these tet determinants.

RP1, a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline and originating in a strain of Pseudomonas aeruginosa , was freely transmissible between strains of P. aeruginosa, Escherichia coli , and Proteus mirabilis . Acquisition of the multiple drug resistance specified by RP1 by these strains was accompanied by acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid of molecular weight about 40 million daltons and of buoyant density 1.719 g/cm 3 (60% guanine plus cytosine).

The reaction of the EcoRI restriction endonuclease was studied with both the plasmid pMB9 and DNA from bacteriophage lambda as the substrates. With both circular and linear DNA molecules, the only reaction catalysed by the EcoRI restriction endonuclease was the hydrolysis of the phosphodiester bond within one strand of the recognition site on the DNA duplex. The cleavage of both strands of the duplex was achieved only after two independent reactions, each involving a single-strand scission. The reactivity of the enzyme for single-strand scissions was the same for both the first and the second cleavage within its recognition site. No differences were observed between the mechanism of action on supercoiled and linear DNA substrates. Other restriction endonucleases were tested against plasmid pMB9. The HindIII restriction endonuclease cleaved DNA in the same manner as the EcoRI enzyme. However, in contrast with EcoRI, the Sa/I and the BamHI restriction endonucleases appeared to cleave both strands of the DNA duplex almost simultaneously. The function of symmetrical DNA sequences and the conformation of the DNA involved in these DNA--protein interactions are discussed in the light of these observations. The fact that the same reactions were observed on both supercoiled and linear DNA substrates implies that these interactions do not involve the unwinding of the duplex before catalysis.

IT has already been suggested that most, if not all, methicillin-resistant strains of Staphylococcus aureus have evolved from a single origin (Lacey, 1972a) on the following evidence : that methicillin-resistance is essentially non-transferable ; that, once lost, methicillin-resistance cannot be restored in vitro; and that certain specific genetic markers (penicillinase production, resistance to streptomycin and tetracycline in addition to resistance to methicillin itself) and their location was the same in several strains. But methieillin-resistant strains isolated over the last decade give a variety of phage-typing patterns (Parker and Hewitt, 1970). Furthermore these strains also possess variable patterns of other properties, particularly resistance to erythromycin, neomycin, chloramphenicul, fusidic acid and novobiocin (e.g. Parker and Hewitt). Such variations are not necessarily inconsistent with ihe evolution of the strains from a single source: thus, changes in the lysogenic state (and therefore phage-typing pattern) of staphylococci are In this paper we attempt to strengthen the hypothesis of a single origin for all methidin-resistant strains. We have also characterised some of the genetic elements specifying antibiotic resistance in these strains and considered the way in which the antibiotic resistance has been acquired. MATERJALS AND IWIXODSMethicillin-resistant staphylococci. Strain nos. FAR1 and FAR2 (both isolated in 1971) were supplied by Dr G. A. J. AyliBe, Birmingham, strain no. 8657 was supplied by Dr Elizabeth H. Asheshov, Cross-Infection Reference Laboratory, Colindale, and strain nos. B109 and B262 were isolated in Bristol during 1969 and 1971 respectively. All the cultures had the following characteristics : heterogeneous resistance to methicillin at 37OC, homogeneous resistance at 30% or below and in the presence of 5 per cent. (w/v) NaCl both at W C and at 37OC.Strain nos. 6936 and 649 have been described previously (Lacey, 1972a; and Grinsted and Lacey, 1973a respectively).Various tests md treatments. Media, detection of loss of antibiotic resistance, recording of pigment, bacteriophage typing, mitomycin Ginduction, irradiation of lysates with ultraviolet 0 light and transduction were as described previously (Lacey, 197%).Determination of minimum inhibitory concentration ( M K ) of antibiotic was by incorporating doubling dilution of each antibiotic into nutrient agar. Generally the inoculum consisted ~~