PLAQUE MORPHOLOGY OF CERTAIN STREPTOMYCETE PHAGES'

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1 PLAQUE MORPHOLOGY OF CERTAIN STREPTOMYCETE PHAGES' JACQUELINE ST. CLAIR AND ELIZABETH McCOY Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication July 18, 1958 Phage host range is a useful marker in epidemiological and genetic studies of phages. Conversely, phages can often discriminate among organisms not separable serologically, thus constituting the basis of phage typing. Plaque morphology may provide identification of both host and phage still more precisely than host range or phage sensitivity. For purposes of such identification it would be valuable to know if some characteristics of s are controlled by the phage and others by the host. In the following survey, a number of freshly isolated phages were plated with several Streptomyces species and morphology of their s observed with the above question in mind. MATERIALS AND METHODS Phages and the hosts upon which they were isolated and maintained are listed in table 1. Media. The host organisms were maintained on slants of potato-dextrose agar. Phage was propagated in B-16 broth (Pittenger and McCoy, 1952) to which was added 0.05 per cent calcium chloride to provide the necessary Ca ion for phage adsorption (Van Alstyne et al., 1955). Phage filtrates were diluted in water containing 0.1 per cent peptone (Chang, 1953). All platings were done on glucose-yeast agar. Plating procedure. The usual double-layer method of plating phages is not good for differentiation among the streptomycetes, since it inhibits development of aerial hyphae and sporulation. Hence Chang's (1953) surface plating technique was used but slightly modified. RESULTS AND DISCUSSION Since most of the streptomycete phages available had been isolated and carried on Streptomyces griseus, the study began with the isolation 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. This work was supported in part by grants from Abbott Laboratories, North Chicago, Illinois, and Merck and Company, Rahway, New Jersey. of a more diverse group of phages. Soils were collected from various areas of the United States and examined for presence of phage. From these samples, nine phages, designated A through I, were isolated, purified, and tested against a number of Actinomycetales, shown in table 2. Since none of the Micromonospora or Nocardia spp. was lysed, while most of the Streptomyces spp. were attacked by one or more phages, these phages are genus specific as far as tested. Some of the phages attacked several species of Strepto- TABLE 1 Phages A to I with homologous hosts and original source Phage Homologous Host Source of Soil A Streptomyces gri- Wisconsin, sample 2 seus strain 1947 B S. griseus strain Wisconsin, sample C S. halstedii Indiana D S. griseus strain Indiana, 1949 E S. griseus strain Wisconsin, sample F S. griseus strain Utah 1947 G S. olivaceus Wyoming H S. antibioticus Wyoming I S. diastatochro- Utah mogenes myces but differentiated among strains of S. griseus; such a phenomenon, previously noted by Hoehn (1949) and others, may reflect the relative ease with which both phages and hosts may mutate or the imperfect taxonomy of Streptomyces species. Preliminary investigations revealed that certain properties were subject to variation. With the use of two test systems, phage G with Streptomyces olivaceus and phage B with S. griseus 1947, certain factors were manipulated. The results showed that: (a) Concentration of 131

2 132 ST. CLAIR AND McCOY [VOL. 77 Hosts TABLE 2 Host range of phages A to I (tested by cross streak method) Phages* A B C D E F G H Micromonospora sp. B P _ P _ Nocardia as tero ides B 970 _ - N.coeliaca B _ - - N. g loberula B N. rubra B-685 Streptomyces antibioticus S.citreus B-1264 _ S. coelicolor WAcl6a B S. cyaneus - - S. diastaticus -4_i _ i _ i S. diastatochromogenes M 248 -_ it _ t S. erythraeus - ± ± - _ S. griseus C-131 t - S. griseocarne us B _ -_ ± ± ± 1947 AC S. gypsoides _ S. halstedii _ S. lavendulae B-547 _ - - S. olivaceus ± S. microflavus - i - i S. parvus B S. rimosus _ - _ - S. rubescens B _ S. rubrireticuli B-1484 _ _ S. violaceoniger B S. v iolaceus _ _ * No lysis;, lysis; ±, partial lysis agar has relatively little effect upon size attacking Mycobacterium sp. (b) One-day-old except at the extremes of 0.8 and 2.8 per cent; poured plates allow larger and more uniform higher concentrations producing slightly smaller s than older ones. Excessively dry plates s. Mandell and Eisenstark (1953) noted a cause erratic behavior, whereas freshly poured similar stability of size with varying agar plates often contain enough surface moisture to concentrations in s formed by phages for produce irregularly shaped s. (c) Excess Xanthomonas pruni. On the other hand, Hnatko moisture introduced with the inocula likewise (1952) stated that increased agar concentrations causes irregularities and tends to inhibit optimal produce a "marked increase in the count, sporulation of the background streptomycete as well as the size of the s" with phages growthi. Excessive surface moisture in the phage I

3 1959] PLAQUE MORPHOLOGY OF STREPTOMYCETE PHAGES oi2-... Figure 1. Representative types. a. Phage A with Streptomyces halstedii at 48 hr showing halo with somewhat irregular margin. b. Phage I with Streptomyces griseus strain 1945 showing halo with small center. c. Phage A with Streptomyces olivaceus at 3 months showing s with a halo composed of spores and an outer ring of autolyzed host material (see below). d. Phage A with S. olivaceus at 48 hr. e. Phage C. with S. olivaceus at 3 days showing large s with a central growth of the host which has sporulated, surrounded by microcolonies and a peripheral ring of densely sporulated host. f. Phage A with S. griseus strain 1947 showing centered and noncentered s. g. Phage G with S. olivaceus at 48 hr showing large, irregular, turbid (see below). h. Phage G. with S. olivaceus at 48 hr, plated with excess surface moisture, showing atypical with regular margin, turbid ring and center clearing. S. G/S. olivaceus system produced a circular with a clear center area instead of the typical turbid with an irregular border (figure lg and h). (d) Thick agar allows larger s than thin agar, as shown by agar gradient plates. (e) By alternating incubation temperature, Hnatko (1952) was able to obtain as many as 16 rings of alternate clear and turbid areas surrounding one. Such a technique, however, failed to affect the type of these two systems except to slow host growth and thus formation. (f) There was no appreciable difference in form or size when 8-hr germinated, rather than ungerminated spores were used as inoculum. The hosts lysed by a majority of the phages were chosen for platings with the 9 phages, and

4 Phages TABLE 3 Descriptions of s produced by stock phages with Streptomyces hosts Hosts S. diastatochromogens strain M 248 S. griseus strain C-131 S. griscus strain 1945 A * B Clear, irregular; mm, Clear, slightly irregular; 0.3- Large halo (small center), 1.5 mm 1.0 mm, 0.6 mm; ring of round; mm, 1.5 mm dense sporulation surrounding C Clear, irregular; mm, 1.5 mm D Clear, irregular; mm, Clear, slightly irregular; 0.2- Small halo (large center), 1.5 mm 1.0 mm, 0.6 mm; ring of round; mm, 0.7 mm; dense sporulation sur- ring of slightly denser rounding sporulation surrounding E Clear, irregular; mm, Clear, slightly irregular; 0.3- Small halo (large center), 1.5 mm 1.0 mm, 0.6 mm; ring of round; mm, 2.0 mm dense sporulation surrounding F Clear, irregular; mm, Clear, round; mm, 0.9 Halo, slightly irregular; mm mm; larger s have 7.0 mm; 4.0 mm; ring of slight halo slightly denser sporulation surrounding G Clear, irregular; mm, Clear, round; mm, 0.5 Halo, slightly irregular; mm mm 1.2 mm, 0.9 mm H Inhibitory effect Inhibitory effect I Clear, irregular; mm, Clear, slightly irregular; 0.5- Large halo (small center); 2.0 mm 1.5 mm, 0.9 mm; ring of mm, 1.8 mm dense sporulation surrounding S. griseus strain 1947 S. halstedii S. olivaceus A Clear, round: mm, 1.2 Halo, round and slightly ir- Halo, round, and slightly irmm; about Y of s regular; mm, 1.3 mm regular, mm, 2.3 mm; contain center spot of turbid area of besporulated host comes sporulated with age B Clear, round and slightly ir- Clear, slightly irregular; 0.4- Inhibitory effect regular; mm, 0.7 mm 1.9 mm, 1.2 mm C Clear, round; mm, 1.5 Unclear, round; mm, mm; s quickly over- 8.0 mm; zone of host sporugrown with host sporula- lation within tion D Clear, round; mm, 0.6 Clear, round, and slightly ir- Inhibitory effect mm regular mm, 0.7 mm E Clear, round and slightly ir- Clear, round, and slightly ir- Inhibitory effect regular; mm, 0.7 mm regular; mm, 1.0 mm 134

5 1959] PLAQUE MORPHOLOGY OF STREPTOMYCETE PHAGES 135 TABLE 3-Continued S. griseus strain 1947 S. halstedii S. olivaceus F Clear, round, and slightly ir- Halo, round, and slightly ir- Clear, round; mm, regular; mm, 0.6 mm regular; mm, 1.1 mm 0.15 mm G Clear, round; mm, 0.3 Halo, round, and slightly ir- Turbid, irregular; mm regular; mm, 1.4 mm mm, 9.0 mm H Clear, round, and slightly ir- Inhibitory effect regular; mm, 1.3 mm; much host sporulation within I Clear, slightly irregular; 0.2- Clear, somewhat irregular; Inhibitory effect 1.2 mm, 0.8 mm mm, 1.1 mm; ring of denser sporulation surrounding, papillae of host growth projecting into from periphery * - = No activity. characteristics of these systems are shown in table 3. Plaque descriptions are somewhat arbitrary, for any one plate manifests a range of sizes and, to a lesser extent, of shape. Such differences, however, were phenotypic, the progeny of any extreme producing the same range. It will be noticed that no host or phage is consistently responsible for a particular characteristic. If any conclusion is warranted, it is that the host, not the phage, is the more active determinant of size and over-all morphology of the s. Thus one may characterize a phage by morphology only when the complete system is specified, i. e., the phage, host, and plating conditions. Streptomycete phages seem particularly amenable to such classification because of the variety of types they manifest. It has been reported that the size of phages and s vary inversely (see Luria, 1953). Such cannot be the case in at least some of the systems studied here where the host may be the determining factor in size. For example, phage F on S. olivaceus produces a pinpoint while G on the same host produces a very large ; yet F on S. griseus strain 1945 produces a relatively large and G a much smaller one. Such an example illustrates the danger inherent in considering only a few phages or hosts, for exceptions may be found upon extending the range of testing. The descriptions need further explanation. Some s, particularly those formed on S. griseus strain C-131, were surrounded by a ring of denser host sporulation. Although factors controlling sporulation in general are incompletely known, it is likely that a more available supply of nutrients from the medium in the aid in sporulation, abetted, perhaps, by products of the lysed host. The systems labeled "inhibitory effect" gave the following appearance: in a phage dilution series of platings, the host growth on the first plate containing 107 to 108 phage particles, either was cleared or severely inhibited, whereas the host of the second plate, with 105 to 106 particles, was uninhibited in growth and sporulated well. It was impossible to find a dilution which would give detectable s. Since some product(s) of the host upon which the phages had been maintained might have antagonized a sensitive culture, broth filtrates of 24-hr cultures of these organisms were added to the "sensitive" strains in the same manner as the phage had been. No inhibitions occurred. Likewise, cross streaking the various hosts evoked no inhibitions. Thus an antibiotic, produced by the maintenance streptomycete and active against the test host, would seem to be ruled out. The effect may be caused by a proteinaceous agent, similar to colicin, pyocin, or bacteriocin, released from the maintenance host by phage-induced lysis. This reaction is also reminiscent of a phenomenon reported by Andrewes and Elford (1932) and Whitehead et al.

6 136 ST. CLAIR AND McCOY [VOL. 77 (1952) that certain phages can adsorb onto and kill cells or inhibit growth without penetrating and multiplying in the host. Some systems produced turbid s by lysis of aerial hyphae while the vegetative mycelium remained essentially intact. The host grew and sporulated in other s, sometimes in discrete patterns (figure le and f). The latter host growths, after several serial streakings to eliminate adsorbed phage, proved nonlysogenic and specifically resistant to the phage that had produced the, but still retained the sensitivity of the parent strain to other phages. SUMMARY Nine streptomycete phages were isolated from soil and purified by serial propagation of single s. They were tested for host range and found to be specific for Streptomyces within the Actinomycetales used. Some phages, although not species specific, differentiated among strains of a species. To determine if any characteristics were controlled by the host or by the phage, each phage was plated with six Streptomyces hosts; no consistencies were found, further verifying the interdependence of the phage-host complex. Certain gross plating conditions were manipulated to determine their effect upon morphology, and a uniform procedure was adopted. REFERENCES ANDREWES, C. H. AND ELFORD, W. J The "killing" of bacteria by bacteriophage. Brit. J. Exptl. Pathol., 13, CHANG, C. H Studies on Streptomyces phages with special references to host specificity. Ph.D. Thesis, University of Wisconsin. HNATKO, S Concentric ring formation about s of M. phlei bacteriophage. Can. J. Public Health, 43, HOEHN, M. M The isolation and study of Streptomyces phages. M.S. Thesis, University of Wisconsin. LURIA, S. E General virology. John Wiley and Sons, New York. MANDELL, J. D. AND EISENSTARK, A Factors affecting formation by Xanthomonas pruni bacteriophage. Phytopathology, 43, 27. PITTENGER, R. C. AND McCoy, E Variants of Streptomyces griseus induced by ultraviolet radiations. J. Bacteriol., 65, VAN ALSTYNE, M. H., OTTO, R. H., AND MCCOY, E Characteristics of Streptomyces griseus strains resistant to phage. J. Bacteriol., 70, WHITEHEAD, H. R., HUNTER, G. J. E., AND Cox, G. A Inhibition of bacterial growth by bacteriophage as distinct from lytic action. J. Gen. Microbiol., 6,