The genetics of mycobacteria and mycobacteriophages — A review

Tubercle (1975), 56,227 OCCASIONAL SURVEY

THE

GENETICS

OF

MYCOBACTERIA AND - A REVIEW

MYCOBACTERIOPHAGES

By J. M. GRANGE from The School of Pathology, The Middlesex Hospital Medical School, RidingHouse Street, London, Wl P 7LD.

SUMMARY The genus Mycobacterium, despite its medical importance, has so far received relatively little attention from bacterial geneticists. Nevertheless examples of the transfer of genes from one strain to another by means of transformation, phage-mediated transduction and direct cellular contact have been reported. The modification of strains by experimental or naturally occurring lysogeny has been studied in some detail and there is evidence that phage may contribute significantly to variation within species. Mycobacteriophages have been the subject of genetic analyses and have proved of value in the study of certain aspects of host-induced phage modification. In addition phages are being used to develop typing systems for use in epidemiological studies. It is evident that much of interest and value awaits discovery by means of genetic analysis of the mycobacteria and their phages and hopefully the next decade or so will bring great advances in this subject.

RESUME Le genre Mycobacterium, en dtpit de son importance en mkdecine, a jusqu’ici reCu peu d’attention de la part des back%-iologistes gCnCticiens. Cependant des exemples de transfert de gknes d’une souche B une autre, au moyen de transformation, transduction A l’aide de phages et contact cellulaire direct ont &C rapport&. Des modifications de souches par 1ysogCnie experimentale ou de survenue naturelle ont CtC6tudiCes en d&ail et il existe des preuves que les phages peuvent contribuer de faGon significative aux variations entre espkes. Les mycobacttriophages ont ttC le sujet d’anlyses gCnCtiques et se sont r&Cl& d’une certaine valeur dans l’ktude de certains aspects des modifications de 1’hBte induite par le phage. De plus les phages sont utiliks pour mettre au point des systbmes de typage utilisables dans les Ctudes tpidkmiologiques. 11 est Cvident qu’un grand in&et et de grandes conskquences dkcouleront des dkouvertes des analyses gtnktiques sur les mycobacttries et leurs phages, et l’on peut espkrer que les prochaines dkcades feront bien progresser le sujet.

RESUMEN El gCnero Mycobacteria, a pesar de su importancia mkdica, ha recibido relativamente poca atenci6n de 10s genetistas bacterianos. Sin embargo se han comunicado ejemplos de

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latransferencia de genes de una cepa a otra por medio de transformacian. transduccicin por medio de fagos y por contact0 celular. La modification de cepas por lisogenia natural o experimental ha sido estudiada en forma detallada y hay evidencia que 10s fagos pueden contribuir significativamente a la variation en las especies. Los micobacteriofagos han sido objet0 de analisis gentticos y se ha demostrado que tienen valor en el estudio de ciertos aspectos de modificacidn de 10s fagos inducida por el huesped. Ademas, 10s fagos se han usado para desarrollar sistemas de tipificacion para usos en estudios epidemiologicos. Es evidente que mucho se puede esperar de 10s descubrimientos por medio de analisis gentticos de las micobacterias y de sus fagos y se espera que la proxima decada aporte nuevos avarices en este tema.

ZUSAMMENFASSUNG Bedeutung bisher wenig BeachDas Genus Mycohacterium hat trotz seiner medizinischen tung durch die bakteriologisch orientierten Genetiker gefunden. Dessen ungeachtet sind Gentransfer von einem Stamm zum anderen auf dem Wege der Transformation, phagenbedingte Transduktion und direkter zellularer Kontakt berichtet worden. Die Modifikation von Stammen durch experimentell hervorgerufene oder natiirliche Lysogenie ist schon in gewissen Einzelheiten untersucht worden, und es gibt Anhaltspunkte dafiir, dat3 Phagen signifikant zur Varietatenbildung innerhalb der Species beitragen. Mykobakteriophagen sind das Ziel genetischer Analysen geworden und haben sich als wertvoll fiir Studien gewisser Aspekte der wirts-induzierten Phagenmodifikation erwiesen. ijberdies werden Phagen zur Entwicklung von Typisierungssystemen fur epidemiologische Zwecke verwendet. Offensichtlich sind in der nachsten Dekade wichtige Ergebnisse auf Grund der genetischen Analyse von Mykobakterien und ihrer Phagen zu erwarten.

Introduction Since the discovery by Griffith in 1928 that genetic markers could be transferred from one bacterial cell to another, phenomenal advances have been made in the understanding of the structure and function of the genomes of bacteria and bacteriophages. However, studies on the genetic analysis of the genus Mycobacterium are still in their infancy. Redmond (1970a) commented that ‘we appear to be at about the point in mycobacterial genetics that the workers on E. coli were in 1946. Dare we hope that in a mere 23 years we shall know as much about mycobacterial genetics as is now known about E. coli’? In this survey the progress made so far in the fulfillment of Redmond’s hope is reviewed.

Mutation The ultimate mechanism of variation and evolution in mycobacteria, as in all forms of life, is mutation. Spontaneous mutation as a cause of resistance to antituberculosis agents is too well

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known to warrant detailed description here. It has been found that often several different phenotypes of resistance to a given antibacterial agent may be derived from any one strain (Tsukamura, 1961) implying that a number of different mutations, or combinations of mutations, are involved in the development of resistance. In the case of aminoglycoside resistance, mutations may affect the ribosomes (Yamada and others, 1972) or the cell wall, preventing the antibiotics from reaching the interior of the cell (Biinicke and Biinicke, 1970; Grange and Nordstrom, 1974). Another commonly observed class of spontaneous mutations is that causing alterations in colony morphology. Various different colonial types are observable in the mycobacteria and may be used as an aid to the identification of strains (Fregnan and Smith, 1962). In addition there have been many accounts of colonial variation within species, especially M. avium. The literature on this subject is briefly reviewed by Schaefer, Davis and Cohn (1970) and by Pattyn and HermansBoveroulle (1970). These authors draw particular attention to changes in virulence accompanying variation in colonial morphology in M. avium, observing that rough opaque forms are considerably less virulent for chickens than the smoother, transparent form. Pattyn and Hermans-Boveroulle (1970) emphasised the importance of the study of single colony isolates in mycobacteriology. The occurrence of irreversible smooth to rough variation in M. fortuitum has been described (Grange, 1973). In most cases such variation did not affect the biotype or immunodiffusion serotype of the strain but in a few cases modification of colony morphology was one of a number of changes almost certainly due to a major deletion in the genome. The role of such deletional mutation in mycobacterial variation has been summarised by Stanford and Grange (1974) in a discussion on the nature of the mycobacterial species. In addition to studies on naturally occurring variation, several authors have observed the effects of mutagenic agents on mycobacteria. Konicek and Malek (1970) obtained a large number of auxotrophic and streptomycin-resistant mutants of M. phlei by treatment with nitrosoguanidine and Konickova-Radochova, Konicek and Malek (1970) obtained similar mutants by using a number of different mutagens. Auxotrophic mutants have also been obtained from M. .rmegmatis and a technique for the selection and isolation of specific mutants has been described (Holland and Ratledge, 197 I).

DNA-mediated transformation The first successful attempt at genetic exchange in mycobacteria was published in 1954 by Katanuma and Nakasato who claimed that streptomycin resistance could be transferred to streptomycin-sensitive strains by means of DNA extracted from strains resistant to this antibiotic. Subsequently, Tsukamura, Hasimoto and Noda (1960) were able to transform resistance to isoniazid and streptomycin in M. avium by exposing sensitive strains to high concentrations of transforming DNA for five days. Juhasz, Gelbart and DeSalle (1971) found that xylose utilisation could be transferred from xyZ+ to xyl- strains of M. phlei by transformation as well as by transduction (see below). In contrast, Bloch, Walter and Yamamura (1959) were unsuccessful in their attempts to transfer resistance to straptomycin and isoniazid to strains of M. smegmatis, M. phlei and M. tuberculosis by means of DNA extracted from resistant mutants of the same strains. These authors were also unable to obtain transformation of pigment production to non-pigmented saprophytic mycobacteria or virulence from M. tuberculosis H37Rv to the rabbit-avirulent strain H37Ra. Likewise Bradley (1970) reported the inability to transform resistance to cycloserine, streptomycin, isoniazid and bacteriophage lysis in M. kansasii, M. smegmatis and M. avium intracellulare. In the same year Tarnock and Bbnicke (1970) also reported on the failure to obtain transformation in mycobacteria and stressed the need for studies on factors influencing the uptake

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of DNA into the bacterial cells. In the same study the binding of 32p-labelled DNA to M. .sI~I~~mutis was investigaed. Surprisingly, strontium ions caused strong binding of DNA to the bacterial cell wall but no competence for transformation could be demonstrated.

Transfection In contrast to the apparent lack of competence of mycobacteria for the uptake of bacterial DNA, competence for the uptake of extracted bacteriophage DNA (transfection) has been repeatedly demonstrated (Tokunaga and Sellers, 1964; Tokunaga and Nakamura, 1968). Although only a very small number of bacterial cells (less than 10s5) were susceptible to transfection, various factors were found to increase competence. Thus Sellers and Tokunaga (I 966) found that the presence of calcium ions caused a significant increase in the plague count; Tokunaga and Sellers (1970) noted that certain amino-acids especially D-serine and D-threonine caused an increased rate of transfection and Nakamura (1970) observed a marked increase in competence in a minimal medium at pH 3.8 to 4-O (standard acid medium). Tokunaga and Nakamura (1968) demonstrated that competence of mycobacteria for transfection is maximal at the late log phase of the growth of a culture, at which phase the transfection rate increases IOO-fold. However, attempts to obtain transformation with bacterial DNA under the same conditions failed. Thus mycobacteria appear to differ from Bacillus subtilis in which there is no significant difference in the factors governing development of competence for the uptake of bacterial or phage DNA (Bott and Wilson, 1967). Nevertheless, studies on transfection may enable the optimum conditions for obtaining successful DNA-mediated transformation to be determined.

Phage-mediated

transduction

As in the case of transformation, studies on transduction in mycobacteria are few in number and are accompanied by several reports of failed experiments. In 1960 Juhasz found that streptomycin resistance was acquired by M. phki when grown in a culture filtrate of streptomycin-resistant M. bovis BCG. This acquisition of streptomycin resistance was not inhibited by deoxyribonuclease and, although positive proof was lacking, transduction was considered the most likely mechanism. A claim that a host-range variant of phage Bo2 (Bo2h) could mediate the in toto conversion of M. phlei to M. smegmatis was made by Juhasz (1967). However it was subsequently found that the so-called M. smegmatis was in fact a mixture of M. phki lysogenic for phage Bo2h and an organism with the properties of M. vaccae, probably a contaminant (Juhasz, 1970). Using phage Bo2, Gelbart and Juhasz (1970) were able to transduce xylose utilisation from xyl+ to xyl- strains of M. phlei. Transduction of the xylf marker in this host-phage system was repeatable whereas attempts to transduce resistance to streptomycin, ethambutol, cycloserine and rifampicin consistently failed (Gelbart and Juhasz, 1973). Transduction of genetic markers to auxotrophic strains of rapidly growing mycobacteria have been reported in two studies. Redmond (1970 a) investigated a number of auxotrophic strains and was able, in one case, to obtain repeated transduction of adenine and guanine synthesis to a strain unable to grow on media lacking these compounds. Phage 13, isolated from soil in India, was shown to transduce various markers to auxotrophic mutants of M. smegmatis SN2 (Sundar Raj and Ramakrishnan, 1970). This phage is the only mycobacteriophage so far found to possess a contractile tail (Kozloff and others, 1972). Jones and David (1972) described transduction of streptomycin resistance in M. smegmatis. Evidence was presented that, in this case, the genetic determinant involved was situated on an

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episome and mediated enzymic inactivation of streptomycin. In this context, it has been found that serotypes III and IV of M. ,fortuitum enzymically inactivate neomycin and kanamycin (Grange and Nordstrom, 1974) but in this example the transducibility of the genes concerned was not investigated. In contrast to these successful reports of transduction Russell, Jann and Froman (1963) failed to repair auxotrophic mutants or obtain streptomycin resistant variants of M. smegmatis by transduction. Bradley (1970) attempted to alter colonial morphology, biochemical characteristics and antibiotic resistance in M. ,fortuitum. M. smegmatis and M. phlei by transduction without success.

Conjugation In recent years reports of genetic exchange in mycobacteria by direct cellular contact have appeared in the literature. Redmond (1970a) described recombination between a smooth, auxotrophic, non-chromogenic mycobacterum (referred to as strain 34Fi) and a rough, prototrophic, chromogenic strain (strain RYD). When these strains were mixed the 34 Fi strain disappeared from the culture whilst many smooth, pale yellow colonies appeared. It was considered that recombination, possibly by some form of conjugation, had occurred. Mizuguchi and Tokunaga (1971) obtained prototrophic recombinants between auxotrophic strains of M. smegmatis. These auxotrophs were obtained from two different strains of this species (strains Jucho and Lacticola). Recombination was obtained when the strains were cultured together on solid media but not in liquid media. The technique has been used to determine the chromosomal location of loci determining glycine and leucine synthesis, antibiotic resistance and resistance to lysis by phage Rabinowitchi. (Mizuguchi, 1972; Suga and Mizuguchi, 1974). In order to obtain pairs of strains that would recombine both Redmond and Mizuguchi had to test large numbers of strains. Therefore this technique of genetic analysis will be limited to those few pairs of strains able to recombine. Recombination by direct cell contact has been more thoroughly investigated in some nocardialike organisms referred to as ‘Nocardia’ erythropolis and ‘Nocardia’ (Jensenia) canicruria (Adams and Bradley, 1963, Adams, 1964). Conjugation in these organisms, termed syncytial recombination, has enabled the sequence of several genetic loci to be determined (Adams, 1970). As in the case of the mycobacteria, only a very limited number of pairs of nocardiae are capable of recombination and it has been shown that mating compatibility is determined by two alleles designated C and E. In order for recombination to occur one strain must possess allele C and the other allele E (Brownell and Kelly, 1967). Mizuguchi (1972) postulated that a similar system of genetic determinants controlled mating in M. smegmatis. Subsequently Tokunaga, Mizuguchi and Suga (1973) produced evidence suggesting that a one-way transfer of genetic material occurred from M. smegmatis strain Rabinowitchi to strain Jucho. Further evidence for such a polar transfer of genes has been obtained by the use of recombination deficient (ret-) mutants (Mizuguchi, 1974). In addition to determining the sequence of loci on the chromosome by genetic recombination, Mizuguchi (1970, in Japanese, quoted by Mizuguchi and Tokunaga, 1971) also used a technique for mapping which does not involve genetic exchange. Nitrosoguanidine causes mutations in the chromosome at the point at which the DNA helix is replicating. If nitrosoguanidine is added at timed intervals to a culture of bacteria in which the cell division, and therefore the DNA replication, is in synchrony, the incidence of various point mutations at the different times will depend on the region of the chromosome replicating at those times. As DNA replicates in a linear manner the incidence of mutants at different times give an indication of the sequence of the genes on the chromosome (Altenbern, 1968).

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<; KA N c; I Phage conversion

In contrast to, and largely as a result of, the paucity of successful systems for the genetic analysib of mycobacteria, considerable attention has been given to the establishment of lysogeny as a cause ofvariation within this genus. Indeed Redmond (197Ob), referring to mycobacteria, stated that any’ discussion of bacterial variation would be incomplete without serious consideration being given to the part played by bacteriophage. Early studies on the establishment of lysogeny in mycobacteria showed that lysogenic variants were frequently of smoother colonial form than the non-lysogenic parent forms (Russell, Jann and Froman, 1960; White, Foster and Lyon, 1962). Subsequently extensive studies on the nature and mechanism of phage conversion in M. smegmatk were initiated by Dr. Rudolf Bonicke of the Forschungsinstitut fur Experimentelle Biologie und Medizin, Borstel, Germany. A preliminary study (Biinicke, 1967) showed that strains of M. .smegmafis rendered lysogenic for phages Bol or Bol3 possessed a markedly increased malachite green reductase activity and a decreased rate of nicotinic acid hydrolysis. In further studies on M. smegmrrris lysogenic for phage Bol, the enzymic activities of ten lysogenic clones were compared with those of ten non-lysogenic clones (Biinicke and Saito, 1970). Nine lysogenic clones produced smooth colonies whereas one lysogenic and all the non-lysogenic clones produced rough colonies. The malachite green reductase activity was markedly increased in all lysogenic clones, there was considerable reduction in nitrite reductase. degradation of histidine, urocanic acid, nicotinjc acid and catechol derivatives and diamine oxidase activity in the nine smooth lysogenic clones. Reduction of these enzymic activities also occurred in the single rough lysogenic clone with the exception of nitrite reductase which was markedly enhanced. Wenzel(l972), in a doctoral thesis, continued these studies after the death of Bbnicke. Lysogenic variants of a single strain of M. smegmatis were prepared using six different mycobacteriophages. Although the establishment of lysogeny caused the modification of a large number of enzymic activities, there was little difference in the modifications induced by the six different phages. In contrast, Juhasz, Gelbart and Harize (1969) infected three different strains of M. smegmatis with a single phage, Bol. Two of these strains were able to hydrolyse salicylamide (S& ). Several lysogenic clones from each strain were examined for variation in the activity of nitrate reductase. arylsulphatase and a number of amidases. The lysogenic clones from the .~a/ 1 strains were all very similar but the clones from the sal- strain varied considerably amongst themselves. These studies suggest that the expression of phage conversion is, in a large part, determined by the genotype of the host rather than that of the phage. It was considered that the nature of phage conversion in M. smegmatis depends on the site of insertion of the bacteriophage genome into the host chromosome. It was postulated that the usual site of phage insertion is at or near the .~a/ locus but that if this region of the genome is absent the phage may insert at one of a number of alternative sites. each site being associated with a different form of host modification. Although further studies are required to substantiate this hypothesis it is noteworthy that in the srrl +~ strains the presence of phage caused a complete supression of the activity of this enzyme, possibly due to mechanical disruption of the locus by the inserting phage genome. Whatever the mechanism of the loss of the salicylamidase activity the effect of the phage is superficially similar to that of a mutagen. Similarly an alteration of nutritional requirements in M. phlei on establishment of lysogeny with phage B2h has been described (Juhasz, 1968). Such changes occurring in M. phfei were compared with the mutagen-like effect of phage MUI on Escherichia co/i as described by Taylor (1963). As experimental lysogeny in M. .smegmatis causes considerable modification of the properties of this species, it is likely that naturally occurring lysogeny in this and other species may lead to difficulties in identification of isolates. Thus for example M. ,fbrruitum NCTC 8573 was found to have a greatly diminished allantoinase activity relative to other strains of this species (Grange and Stanford, 1974) and was found to be lysogenic (Nordstiim and Grange, 1974). As referred to above, mycobacteriophages cause variation in colonial morphology, the lysogenic

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variants frequently being smoother than tht non-lysogenic forms. Wenzel (1972) observed that lysogenic strains of M. smegmatis contained large amounts of deoxyribonucleic acid in the cell walls whereas the non-lysogenic strains contained small amounts only. This could account for the changes in colony morphology. In addition to causing changes in the colonial form, the lysogenic state may modify the antigenic nature of the cell walls as detected by agglutination serology, different phages causing different modifications (Jones and Beam, 1969). Several authors have reported the use of cell wall agglutination techniques for the typing of mycobacteria - either for the subdivision of M. avium (Schefer, 1965) or the identification of rapidly growing species (Jenkins, Marks and Schaefer, 1971). The possibility of lysogeny leading to anomalous results or even being responsible for some subspecific serotypes must be considered when using these techniques. The fact that there are several examples of pathogenic bacteria owing their virulence to the lysogenic state (Hayes, 1968) raises the question as to whether bacteriophage may modify mycobacterial pathogenicity. Juhasz (1970) found that M. smegmatis made lysogenic for phage Bo7 (propagated on M.fortuitum) was able to remain viable in the mouse peritoneum for longer than the non-lysogenic parent strains. M. smegmatis is normally a harmless saprophyte but a pure growth of this species was obtained from the lung and caseous hilar lymph nodes of a patient who died of aspiration pneumonia. This strain was found to be lysogenic for phage Bo6 (Bonicke and Juhasz, 1970). Subsequently it was shown that this strain, unlike other strains of this species, caused kidney tubercles in mice and rabbits (Juhasz, 1970). It is obvious that the association between phage and pathogenicity in the mycobacteria is far from clear and requires further elucidation. Naturally occurring lysogeny in the mycobacteria In contrast to extensive data on the experimental establishment of lysogeny, little information is available on the nature and incidence of naturally occurring lysogenic mycobacteria. In 1969 Bonicke found only seven reports on the detection of lysogenic mycobacteria in the literature. It was considered that this paucity of reports on naturally occurring lysogenic strains was due to the lack of suitable techniques for their detection. A valuable advance was made in this subject by Mankiewicz and Tamari (1972) who showed that some lysogenic mycobacteria produced readily detectable amounts of deoxyribonuclease. Subsequently Grange and Bird (1975) found a relatively high incidence of lysogeny due to complete and defective phages in M. fort&urn by the observation of anomalous cultural and biochemical properties, resistance to super-infection, phage recombination and deoxyribonuclease production. The presence of phage was confirmed by the use of electron microscopy. In addition to the occurrence of true lysogeny, examples of pseudolysogeny have been reported. In pseudolysogeny of the so-called ‘carrier-state’ type the phage genome does not integrate with the host chromosome and is not transmitted to all daughter cells. When, from time to time, the phages enter the lytic state they are able to infect and lyse surrounding non-lysogenic cells. Thus this type of lysogeny is readily detectable by the observation of plaques appearing spontaneously on cultures of the strains. A detailed description of a pseudolysogenic strain of M. fortuitum has been given by Baess (1971) together with a review of the literature on pseudolysogeny. Two further examples of pseudolysogeny in M. jiortuitum have been described by Nordstrom and Grange (1974) and the phenomenon has been observed in a strain of M. diernhoferi (unpublished observations). Genetic studies on mycobacteriophages Although members of the genus Mycobacterium have proved to be surprisingly resistant to attempts at genetic analysis, it appears that their phages may be examined from the genetic standpoint as readily as phages from other bacterial genera. Thus successful genetic mapping of a

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mycobacteriophage was performed by Mizuguchi and Sellers (1970) who isolated a number OI stable temperature sensitive (ts) mutants of phage D29 propagated on M. .smegmatis. By means of recombination between these ts mutants a map showing the linear arrangement of the mutational sites with the distances between them was produced. Sivcev and Bijnicke (1970), Sellers (1970) and Juhasz and Biinicke (1970) described changex occurring in host range mutants of mycobacteriophages. Phage Bo2 is lytic for a strain of M. phlei. On adaptation onto another strain of the same species, the phage showed changes in electron microscope appearance, plaque morphology, heat and ultra-violet sensitivity and antigenic structure (Juhasz and BGnicke, 1970). As the changes were retained when the modified phage was propagated on the original host it was possible to rule out host mediated modification as the mechanism of the observed changes. Mutation was considered improbable on account of the multiplicity of changes and the most likely explanation was the occurrence of recombination of the phage genome with DNA in the new host. Grange and Bird (1975) observed that phage 13 was adaptable onto immunodiffusion serotype Ill strains of M..fi?rtuitum. Phage 13, propagated on M. smegmatis SN2. has a short contractile tail whereas the adapted variant possessed a longer, non-contractile tail. It was found that the serotype I11 strains of M.,ftirtuitum liberated structures resembling phage tails and it was considered that in this example adaptation was associated with recombination between phage 13 and defective prophage in the alternative host. The role of cryptic or defective phage in the intraspecific variation of bacteria and in the evolution of new species is poorly understood. It has been suggested (Luria, 1953) that every bacterium might contain DNA derived from phage and that any part of the bacterial genome may have the potentiality to become a phage. Redmond (1956) suggested that M. tuberculosis might be lysogenic, as strains of this species were found to lyse when treated with agents known to induce phage replication in lysogenic bacteria. Further evidence for the presence of phage DNA in the genome of M. tuherculosis was provided by the use of phage LEO which was obtained from a lung biopsy taken from a patient with sarcoidosis (Mankiewicz and Redmond, 1968). These authors observed that this phage produced plaques of uniform appearance on M. .smegmutis ATCC 607 whereas three distinct types of plaque were produced on M. tuberculo.si.s H37Rv. Attempts to purify these three variants

by passage on H37Rv were unsuccessful as on each passage the three plaque types were produced but they could be purified by passage on ATCC 607. Further studies are required to determine whether this phenomenon indicates the presence of defective phages in M. tuberculosis and whether such phages contribute to the pathogenicity and other properties of this important species.

Exchange of genes between mycohacterial species In view of the difficulties encountered in obtaining transfer of genes between mycobacterial strains the occurrence of intraspecific hybridization is highly questionable. On the basis of an antigenic analysis of over one thousand mycobacterial strains, each species appeared to be a distinct entity with no intermediate forms (Stanford and Grange, 1974). In contrast to the lack of evidence for the exchange of bacterial DNA between species, the crossing of species boundaries by some bacteriophages undoubtedly occurs. The question whether this. constitutes intraspecific hybridization is a philosophical one at our present state of knowledge. The origin of mycobacteriophages is unknown, some may even have arisen in different bacterial genera. It is also unknown how much of ‘bacterial’ DNA in a given strain had its origin in phage. It is possible that defective prophage DNA can add to the size of a bacterial chromosome and can act as substrate for the creation of new genes by mutation. If, subsequently, the defective phage would be repaired by recombination with a superinfecting phage the new genes could be transferred to other species. It has been suggested that viruses play an important role in evolution of bacteria and possibly also of higher forms of life (Grange, 1974).

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Genetic aspects of bacteriophage typing Following the first description of mycobacteriophage by Penso and Ortali (1949) it was hoped that phage typing would prove of value in the classification of mycobacteria (Juhasz and Bonicke, 1965). However, it was found that the host ranges of many mycobacteriophages were not restricted to the species of the propagator strain. For example, phage BK4 has been shown to lyse strains of M. fort&urn, M. smegmatis, M. Javescens, M. chelonei and a strain of M. vaccae (Baess and Weis Bentzon, 1969; Kubica and others 1972). Likewise phage Mx2 lyses some strains of M. ,fortuitum, M. smegmatis, M. phlei, M. duvalii and M. tuberculosis (unpublished data). Phage typing is therefore of little or no taxonomic value but does offer a means of subdividing species for epidemiological purposes. Several phages able to subdivide M. tuberculosis have been described (Froman, Will and Bogen, 1954; Baess, 1966) and subsequently used in epidemiological studies (Baess, 1969; Bates and Mitchison, 1969). For further details on the phage typing of M. ,tuberculosis, Redmond and Ward, 1966 and Sula and others, 1973, should be consulted. The factors controlling the somewhat unusual host ranges of mycobacteriophages are in most instances unknown. The cell wall receptor sites for a few phages have been isolated and shown to be the peptidoglycolipids known as mycosides (Goren, 1972). However, phage attachment is only one of many genetically controlled steps leading to the eventual lysis of the bacterial cell. Grange and Nordstrom (1973) found that the ability of phage BK4 to lyse strains of M.,fortuitum correlated very closely with the ability of the strains to utilise inositol irrespective of their other biochemical and antigenic properties. It was postulated that a genetic determinant essential for the replication of phage BK4 is situated close to the inositol locus and that both loci are lost together at a high frequency by deletional mutation. Other phages showing the same property have been found and have been named the Tara phages (Nordstrom and Grange, 1974). The host range of a phage is to some extent determined by the occurrence of lysogeny in the bacterial strains under study. The ability of phage 13 to lyse serotype III strains of M. ,fortuitum and the association of this lysis with defective prophage in these strains has been referred to above. Conversely, the presence of prophage may prevent lysis by other phages by means of superinfection immunity. Thus three strains of M. fortuitum that utilised inositol but were not lysed by the Tara phages were found to be lysogenic (Grange and Bird, 1975). In most cases, however, the variations in susceptibility to phage lysis in otherwise homogeneous strains are inexplicable at the present time.

Mycobacterial genetics - present and future As the genus Mycobacterium is responsible for more chronic bacterial disease than all other genera together, the importance of a better understanding of this genus from the genetic standpoint cannot be doubted. This survey of the literature shows that exchange of DNA for the purpose of genetic analysis is not easily obtained in the mycobacteria. Nevertheless the documented examples of transduction, transfection, transformation and conjugation will, hopefully, be of use in determining the optimum conditions for genetic exchange between any selected strains. The isolation of increasing numbers of lysogenic mycobacteria and bacteriophages not only opens new approaches to the study of bacterial variation but allows the development of phage typing systems for species of epidemiological importance. Finally, the study of variation within species is already providing clues to the nature and evolutionary origin of pathogenic mycobacteria and may well lead to a greater understanding of M. leprae. Stanford and Grange (1974) stated that over the last twenty years or so the genus Mycobacterium has changed from one of the worst classified to one of the best classified of bacterial genera. One can but hope that in two decades time it will be possible to make similar claims with respect to the understanding of the mycobacterial genome.

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I wish to thank Dr. J. L. Stanford for his helpful comments and criticisms.

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