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The ability to produce haemolysins and bacteriocins is widely distributed among Gram-negative and Gram-positive bacteria and indeed, production of g- haemolysin has been used as a major criterion in the classification of pyogenic streptococci and is the criterion for the sub-classification of group D

2' faecalis subsp. zymogenes. All the determinants for production of 3-

haemolysin in faecalis subsp. zymogenes, which have so far been examined,

are carried by plasmids of similar size, 50 to 60kb (Clewell, 1981; Borderon

je^ al., 1982). The production of haemolysin in these strains is always

associated with bacteriocin production and it has been shown that the two activities are attributable to the same molecule (Brock and Davie, 1963; Dunny and Clewell, 1975; Yagi e^ al., 1983).

Evidence for the plasmid linkage of haemolysin-bacteriocin was first reported by Tomura et al., (1973) who described both the transfer of these traits to 2» faecalis recipients by a mechanism which was probably conjugation

and the loss of the traits on irradiation with the ultraviolet light. Sub­

sequently Jacob and coworkers (1975) identified, in strains of faecalis

subsp. zymogenes, plasmids which encoded haemolysin-bacteriocin production and which were transmissible by conjugation in broth culture to a plasmid free

recipient. Plasmids pJH2 and pJH3 were shown to be of similar size (57.5kb),

present as a single copy and in addition to haemolysin-bacteriocin, encoded

resistance to the corresponding bacteriocin. On studying the loss of the

pJH2 associated haemolysin trait, some strains could be isolated which were non-haemolytic and non-bacteriocinogenic but retained bacteriocin resistance and pJH2. This observation was presumably due to a point mutation or small

deletion in the haemolysin-bacteriocin determinant of pJH2, faecalis

haemolysin has been shown to he composed of an activator A and a catalytic molecule L (Granato and Jackson, 1971a,b) and, on cross—streaking of the

were observed suggesting that mutations had occurred producing either defective A or L.

Reports describing the haemolysin-bacteriocin plasmid pAMyl of faecalis '

strain DS5 (Clewell et al., 1974) not only showed that the plasmid was capable of transfer by conjugation in broth culture but that it could also mobilise the non-conjugative, co-resident tetracycline resistance plasmid pAMal (Dunny and Clewell, 1975). However, certain discrepancies in the phenotypes of derivatives were observed which led to the suggestion that there were in fact

two bacteriocin activities encoded by pAMyl. It has been shown since that

in fact strain DS5 contains three plasmids - pAMyl (60kb) which encodes haemolysin-bacteriocin activity and bacteriocin resistance, pAMyZ (52.8kb)

which encodes a second bacteriocin and bacteriocin resistance, and pAMy3 (45.8kb) the presence of which reduces the production of the pheromone cPDl (Clewell

et al., 1982b; LeBlanc e_t aJL., 1983). All three plasmids have been shown to be capable of mobilising pAMal.

Similar conjugative plasmids encoding haemolysin-bacteriocin, including

pADl (Clewell al., 1982a) pOBl (Oliver et al., 1977), pX-14 (Frazier and

Zimmermann, 1977) and pFD5 (Yagi ef al., 1983) have been described in other

strains of faecalis although in some cases, notably pOBl and pPD5, there

has originally been confusion as to the activities encoded by these'plasmids because it has subsequently been shown that their hosts in fact contained

two plasmids, one of which encoded haemolysin-bacteriocin activity and the

other which encoded a different bacteriocin. In the case of strain 39-5

which harbours pPD5.,. the. co^rr^ident bacteriocin plasmid pPDl was found to be almost the same size and often co-transferred (Yagi et al., 1983).

A characteristic of the transfer of all the faecalis haemolysin-

bacteriocin plasmids (with the possible exception of pPD5), the bacteriocin

plasmids pPDl, pAMy2 (but not pOB2; Oliver ^ , 1977) and pAMy3 is the

transfer in broth culture to recipient faecalis (Clewell, 1981). A study of the host range of such plasmids found that no transfer was observed

to faecium, dur ans, bovis,

E.

et al., 1982). As described previously, acquisition of these plasmids results in the production of a proteinaceous aggregation substance on the cell surface

in response to the corresponding pheromone of faecalis recipient cells.

The location of the genes for aggregation substance is not yet known but it 1

has been shown that aggregation substances produced in response to different pheromones are inmunologically related (Kessler and Yagi, 1983; Yagi et al., 1983).

Digestion of pAMyl and pADl with the restriction endonuclease EcoRl resulted in the generation of identical fragments indicating the close structural relationship of these haemolysin-bacteriocin plasmids isolated from different sources (Clewell et al., 1982b). Moreover the two plasmids were shown to share the same pheromone system and both exhibited the property

of inhibiting the transfer of the MLS plasmid pAMgl. Similarly, restriction

endonuclease profiles of haemolysin-bacteriocin plasmids isolated from a group of six 2' faecalis strains were identical (Borderon ejt al., 1982) but comparison of the physical maps of pADl and the bacteriocin plasmid pPDl

showed no similarity (Yagi ^ al., 1983). Using Tn916 and Tn917 as^ insertional mutagens, the haemolysin-bacteriocin determinant has been located on the physical

map of pADl (Clewell al., 1982a) and has been used as a probe to investigate

homology between haemolysin and bacteriocin genes from several sources (LeBlanc et al., 1983). DNA-DNA hybridisation studies indicated that the haemolysin-

bacteriocin loci of different faecalis plasmids are very closely related

and, although restriction endonuclease profiles of pADl and pAMyl were qui-te different from pJH2, pPD5 and pOBl, there was considerable DNA sequence homology

suggestive of a common origin. There was no hybridisation observed between

pAMyZ and pPDl, or pAMy3 and likewise no homology with total cell DNA

isolated from pyogenes, £. agalactiae, ahgihosus and sanguis all of

which produce chromosomally determined 3-haemolysins.

Other species of bacteriocinogenic group D streptococci are commonly encountered (Tomura et al., 1973) but there is little information on the

nature of the genes for bacteriocin production in strains other than faecalis. -r

In 2" faecium, Keness and coworkers (1978) reported that bacteriocin determin­ ants were located on small 3.6 to 5.8kb plasmids but Le Bouguenec and

Horodniceanu (1982), although they described transferable bacteriocin production, could not relate this phenotype with the acquisition of particular plasmids.

In two strains of faecium, however, bacteriocin production and resistance

to bacteriocin was correlated with transfer of a 5.3kb plasmid and a 3.6kb or a 7.1kb plasmid respectively (Kramer et al., 1983; Reichelt et al., 1984).

In group D streptococci, it is known that antibiotic resistance, as well as haejnolysi'n and b.acteriociti production, is often encoded by genetic determinants located on plasmids, many of which are capable of self transfer by conjugation or which can be mobilised by other conjugative plasmids. Alternatively, antibiotic resistance may be specified by genes situated

in the chromosome, some of which, may be capable of transposition to other replicons and of transfer by conjugation in the absence of plasmid DMA.

Although numerous enterococcal strains which display multiple antibiotic resistance phenotypes have been described, detailed studies of the entire plasmid complement of such strains and the location of the resistance genes have been limited to only three Streptococcus faecalis strains, namely DS5

(Clewell et al., 1974; 1982b), DS16 (Clewell et al., 1982a; Franke and Clewell, 1981), and JHl (Jacob and Hobbs, 1974; Banai and LeBlanc, 1983;

1984a,b). Group D streptococcal strains in this study were chosen on

the basis that they had shown resistance to two or more antibiotics in a

previous investigation (Blankson, 1981). The principle aims of the research

were 1. to extend the information on the phenotypes and ascertain the mode