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and lincomycin resistance to a l.Skb fragment and identified the region of i

DNA involved in plasmid replication. The possession of several unique

restriction endonuclease sites have made these derivatives (pSM7, pSM8, pSM9, pSMlO) attractive as cloning vehicles (Malke et al., 1981; Malke and Holm, 1982) and in addition by constructing a chimeric plasmid with pBR322, Malke and

Holm (1981) have demonstrated expression of MLS resistance in coli. Other

approaches to generating streptococcal cloning vehicles (pVA680, pVA736, pVA738) have been successfully attempted by insertion of the erythromycin resistance gene originating from pAMgl into a small cryptic plasmid from

ferus (Macrina et al., 1980; 1982). A third family of cloning vectors ;

(pGB301 and derivatives) has been obtained by spontaneous deletion of the group B plasmid pIPSOl which encodes MLS and chloramphenicol resistance

(Horodniceanu et al., 1976) and restriction endonuclease data have localised the resistance determinants, copy number control functions and replication region

(Behnke and Gilmore, 1981). pGB301 and its derivatives have been shown in 13. subtilis mini cells to produce three proteins associated with erythro­ mycin resistance, chloramphenicol resistance and a one likely to be associated with plasmid replication or maintenance respectively (Behnke et al., 1982).

Recently, detailed physical maps of faecalis MLS resistance plasmid

pAMgl (LeBlanc and Lee, 1984) and agalactiae MLS and chloramphenicol

resistance plasmid pIPSOl (Evans and Macrina, 1983) have been constructed. Analysis of deletion derivatives of pAMgl located the MLS determinant on a l.lkb fragment and the replication functions on a 2,95kb fragment (LeBlanc

and Lee, 1984). The latter functions contained in a 5kb fragment were

ligated to coli plasmid pACKCl to obtain ah coli—S. sanguis shuttle

vector which could express the _E. coli kanamycin resistance gene of pACKCl S. sanguis and a cloned chromosomal streptomycin resistance locus from

s.

functions of pIPSOl. In addition, both groups have identified regions

involved in conjugation hut neither has yet been successful in cloning these functions.

Determinants conferring inducible resistance to MLS antibiotics have been identified on a transposon, Tn917. Originally associated with the 25.9kb non-conjugative plasmid pAD2 which also encodes streptomycin and kananycin resistance (Tomich et al., 1979), the 5-lkb element was found to transpose to a conjugative haemolysin-bacteriocin plasmid pADl also harboured by S. faecalis strain DS16. Additionally, the transposon was shown to insert into pAMyl and pAMul of ^ faecalis strain DS5 (Tomich et al., 1978; 1980)

and into multiple sites of pADl (Clewell et al., 1982a). Heteroduplex

analysis has shown that the resistance determinant is flanked by homologous inverted repeat sequences of approximately 0.28kb (Tomich et al., 1980) and

I S structurally related to a family of transposons, typified by Tn3, which

have been isolated from a wide diversity of genera (Heffron, 1983). Indeed,

comparison of physical maps and heteroduplex studies have revealed extensive homology between Tn917 and the staphylococcal transposon Tn551 while DNA sequence determination has shown significant homology between the terminal inverted repeats of Tn917, Tn551 and the Gram-negative Tn3 (Perkins and

Youngman, 1984). Functional similarity between Tn917 and Tn551 or Tn3 has

also been demonstrated by the identification of a 5-base pair duplication on insertion of the transposon.

Exposure of DS16 cells to sub-inhibitory concentrations of erythromycin

was shown,not only to induce MLS resistance,but also

to

of transposition and it was thought that the stable co-integrates of pADl and pAD2 formed in recipient cells after induction represented intermediates in the transposition process which subsequently failed to resolve in the

n e w host. However, this model of the transposition process would require two copies of Tn917 to be present in the co-integrated structure and this

was shown not to be the case (Clewell et al., 1982a). The involvement of

transient co-integrate intermediates containing two copies of Tn917 never­

theless remains a possibility. Both induction of resistance and trans­

position enhancement have been found to be sensitive to protein inhibition by chloramphenicol although the isolation of mutants with normal inducible resistance phenotypes but lacking enhanced transposition response^ suggested that the two functions were under separate control (Clewell jet al., 1982a).

Tn917 has been shown to undergo transposition in subtilis and to insert

into multiple chromosomal sites indicating its value as an insertional mutagen (Youngman et al., 1983).

Preliminary work has suggested that other MLS resistance transposons

may occur in streptococci. Le Bouguenec and Horodniceanu (1982) postulated

that transposition could account for the difference in size of plasmids

isolated from parental and transconjugants strains of faecium. Banai and

LeBlanc (1983) described insertion of the faecalis MLS determinant of

pJHl into the co-resident plasmid pJH2, the size of the insertion being

similar to Tn917. They have subsequently shown that the 5.1kb segment

could insert into at least four different sites on pJH2 and was homologous to Tn917 by the criteria of DNA hybridisation and comparison of endonuclease Aval restriction patterns (Banai and LeBlanc, 1984). Like Tn917, the segment

designated Tn3871 expressed inducible MLS resistance. Characterisation of

the conjugative chromosomal insert of agalactiae B109 which encodes

resistance to chloramphenicol, tetracycline and MLS antibiotics has shown that the element is capable of transposition to multiple sites of the plasmid pADl (Smith and Guild, 1982) and results suggested that the MLS determinant

was capable of independent transposition. Interestingly, the transfer of

been shown to be inhibited and in strains containing both plPSOl and the B109 insertion element, the plasmid and insertion were found to be

incompatible, suggesting a relationship of surface exclusion and incompat­ ibility genes (Horodniceanu et al., 1981).

Several studies have demonstrated the relationship of MLS plasmids isolated from different clinical strains suggesting a common origin for

these determinants. S. faecalis pAM31 and pyogenes pACl encoding

constitutive and inducible MLS resistance respectively, were shown by ÜNA- DNA hybridisation experiments to be 95% homologous (Yagi et al., 1975) and similar results were obtained on examination of homology between plasmids

originating from groups D and B (El-Solh et al., 1978). Comparison of the

restriction endonuclease profiles of plasmids from group A, B and D isolates (Hershfield, 1979) and from groups B, C and G strains (Bougueleret e^ al., 1981; Horodniceanu et al., 1981) showed a number of digestion fragments in

common. Using complementary RNA probes prepared from pAMBl, pAM77 (a 6.8kb,

non-conjugative plasmid from S. sanguis encoding inducible MLS resistance; Yagi et al., 1978) and pl258 (a 27kb Staph, aureus plasmid containing the constitutive MLS resistance gene of Tn551; Novick et al., 19 79b), Weisblum and coworkers (1979) demonstrated sequence homology in heterologous hybrid­

isations with target DNA prepared from the above templates, pyogenes

pACl and chromosomal DNA of MLS resistant pneumoniae. Gilnwre ejt al.

(1982) investigated the relationship between MLS resistance loci and

replication function sequences of plasmids obtained from groups A, B, D and

H streptococci. Staph, aureus and

R.

of Lactobacillus easel and Streptomyces erythreus, an erythromycin producer. The DNA probes were a 1. 7kb fragment containing MLS determinants and a 1.5kb fragment containing the replication origin and copy number control region of the group A plasmid pSM19035 (Behnke and Ferretti, 19.80). Hybridisation of the resistance probe was seen with all streptococcal plasmids including

pAD2, which carries Tn917, and with StAph. àûréus pi258 CTn551) but not

with Staph, aureus pE194, fragilis plP410 or chromosomal DNA of I^. casei

or 2' erythreus. The replication origin— copy number probe hybridised with all plasmids giving positive MLS resistance homology except for pAD2, pl258 and pAM77. Lack, of homology of pAD2 and pl258 probably reflected the trans­ poson location of the determinants and suggested that the resistance gene of pAM77, an MLS plasmid which is unusual in respect of its small size, may

have originated as a transposable element. On the basis of DNA-DNA hybrid­

isation studies, Ounissi and Courvalin (1982) have identified four classes of MLS resistance determinants: class A includes all the streptococcal determinants examined as well as Staph, aureus Tn554; class B is comprised of^Staph, aureus plasmids pE194 and pE5; class C contains the chromosomally

specified locus of lichenifotmis; class D is typified by JB. fragilis plP410.

Although lack of sequence homology, as judged by DNA hybridisation experiments, has been observed, Horinouchi and Weisblum (1982b) have shown that there can be nevertheless considerable similarities in the structural

genes encoding MLS resistance. Comparison of the nucleotide sequences of the

structural genes of pAM77 and pEl94, revealed that approximately half the nucleotides were identical while analysis of the deduced amino acid sequences

showed striking conservation. Indeed, where there were differences in amino

acid sequence, the alternative corresponding residues were frequently structurally related.

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