APPLIED MICROBIOLOGYVol. 12, No. 3, p. 261-268 May, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

Rifamycin'

XXXIII. Isolation of Actinophages Active on Streptomyces mediterranei andCharacteristics of Phage-Resistant Strains

J. E. THIEMIANN, C. HENGELLER, A. VIRGILIO, 0. BUELLI, AND G. LICCIARDELLO

Lepetit S.p.A., Research Laboratories, Milan, Antibiotici Lepetit, Naples, Italy

Received for publication 16 January 1964

ABSTRACT

THIEMANN, J. E. (Lepetit S.p.A., Milan, Italy), C. HENGEL-LER, A. VIRGILIO, 0. BUELLI, AND G. LICCIARDELLO. Rifamycin.XXXIII. Isolation of actinophages active on Streptomycesmediterranei and characteristics of phage-resistant strains. Appl.Microbiol. 12:261-268. 1964.-Five actinophages highly specificfor Streptomyces mediterranei were isolated from lysed brothcultures. Studies were performed on the effect of plating condi-tions on plaque formation. The development of phage-resistantstrains of S. mediterranei not only eliminated the phage but alsosignificantly increased rifamycin yields. The phage-resistantcultures proved to be more unstable than the original sensitivestrain. Maintenance of the cultures as frozen vegetative myceliumassured culture stability and reproducibility of the results. Strictaseptic precautions throughout the laboratories and fermenta-tion areas did not eliminate the danger of phage infection;effective control was obtained only with the introduction ofresistant strains. S. mediterranei phages proved to be highlyspecific for calcium as an adsorption cofactor; addition of cal-cium-sequestering agents to sensitive mycelium completelyprevented its lysis by the phage. The resistant strains developedwere capable of adsorbing the phage and of releasing it withoutmultiplication upon aging of the mycelium. No marked morpho-logical, cultural, or biochemical differences were found amongthe various phage-resistant strains.

Actinophages active on streptomyces strains used inindustrial fermentations have been known for severalyears. Saudek and Colingsworth (1947) were among thefirst to report an actinophage outbreak in the commercialproduction of streptomycin. Similar reports, dealing al-ways with streptomycin fermentation, were published byother workers in the field (Reilly, Harris, and Waksman,1947; Smith, Kuhn, and Miesel, 1947; Carvajal, 1953a).Weindling and Kapros (1951) reported for the first timea phage outbreak in chlortetracycline fermentation by aStreptomyces aureofaciens specific actinophage (Weindling,Tresner, and Backus, 1961).

Recently, S. mediterranei, producer of rifamycin, wasfound to be lysed by a virulent and highly specific actino-phage (Thiemann et al., 1962). In the present paper, some

1 The name "rifamycin" has been adopted instead of "rifomy-cin" as in previous papers to avoid confusion with other antibi-otics.

characteristics of the actinophages isolated as well as ofthe phage-resistant colonies obtained are given.

I\IATERIALS AND METHODSThe collection consisted of five phages for which four

strains of S. mediterranei served as homologous hosts. Thephages with their host of isolation are: phages ,B and y onS. mediterranei ME/83/973, phage 17 on strain ME/R17,phage 112 on strain ME/R112, and phage 156 on theME/R156 strain. Whereas phages A and y were isolatedfrom a lysed broth culture of the original phage-sensitiveculture of S. mediterranei ME 83/973, all the other phageswere isolated from lysed broth cultures of strains previ-ously phage-resistant.The host strains were maintained in the form of frozen

vegetative mycelium. V6 medium [containing (g per liter):meat extract, 5.0; peptone, 5.0; yeast extract, 5.0; enzy-matic hydrolysate of casein, 3.0; NaCl, 1.5; glucose, 20.0;and distilled water] was inoculated and incubated on analternating shaker at 28 C for 18 to 24 hr; 20-ml samplesof the whole broth were frozen in an alcohol-Dry Ice mix-ture at -70 C and kept in a freezer at -18 C.For phage propagation, 3 ml of frozen vegetative my-

celium of the host strain were inoculated into 100 ml ofV6 medium. After overnight incubation, 1 ml of phagesuspension or an isolated plaque was added, and the flaskswere incubated for another 16 hr after which time com-plete lysis had taken place. The lysed broth was Seitz-filtered and stored at 4 C. The phage titer normally ob-tained was of the order of 109 to 1011 particles per ml.

Plaque counts were performed by serially diluting thephage either in V6 medium or in 0.1 % peptone-water towhich 0.005 M CaCl2 had been added (Van Alstyne, Otto,and McCoy, 1955). The double-layer technique outlinedby Adams (1950) was used throughout this work. As baselayer, Bennett's agar was used. Plaques were examinedafter 24 and 48 hr of incubation at 28 C. Phage-resistantcolonies were isolated by plating samples of the completelylysed broth cultures on mineral-lactose (ML) medium ofthe following composition (g per liter): lactose, 10.0;(NH4)2SO4, 2.64; K2HPO4, 5.64; KH2PO4, 2.38; MgSO4-7H20, 1.0; CuSO4-5H20, 0.0064; FeSO4*7H20, 0.001;MnSO4.4H20, 0.0079; ZnSO4r7H20, 0.0015; agar, 18.0;and distilled water.

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RESULTS AND DISCUSSION

Phage outbreak in the S. mediterranei fermentation. Dur-ing the early development work on rifamycin production,it was observed that the initially heavy mycelial growthof S. mediterranei ME 83/973 in the vegetative V6 mediumbecame suddenly and totally lysed. Confirmation of thepresence of a phage was obtained by plating serial dilu-tions of the Seitz-filtered broth onto susceptible myceliumof S. mediterranei. The possibility that S. mediterraneiwas a phage carrier or a lysogenic strain was supportedby earlier observations of MIargalith and Beretta (1960),who briefly mentioned the appearance of "actinophagelike spots" when culturing the strain on protein-rich agarmedia.From the first phage outbreak, phages f3 and -y (two

plaque-morphology variants) were isolated. Colonies re-sistant to these two phages were easily obtained by plat-ing samples of the completely lysed broth culture on MLmedium. Better results were obtained by incubating thelysed broth, prior to plating, for at least 48 hr on analternating shaker. Under these conditions a higher per-centage of fully resistant colonies was obtained.

Isolation of phage-resistant colonies directly from thesecondary growth which developed inside the plaquesafter incubation for 7 to 10 days was also tried. Most ofthe colonies obtained by this procedure were, however,not fully resistant. The increase in phage-resistant colo-nies observed by reincubating the lysed broth prior to plat-ing could be due to the total elimination of pseudo-resist-ant mycelium fragments, as well as to the developmentof secondary growth of the fully resistant mycelium, re-sulting thus in a relative increase of the resistant coloniesupon plating.

Proof of phage resistance of the isolates. The resistantcolonies obtained were transferred directly into V6 me-diumi; after abundant growth had been obtained, slantswere prepared and allowed to develop. These slants werecarefully examined for presence of plaques. Any culturethat showed plaque formation was eliminated and notfurther examined. The slants were subsequently testedfor resistance in broth by a procedure similar to that al-ready outlined by Van Alstyne et al. (1955). Triplicatebroth cultures were inoculated as follows: (i) with thetest strain alone, (ii) with the test strain and phage (108particles per ml), and (iii) with the test strain and theparent-sensitive strain. Cultures i and iii were plated outwith the sensitive host after 72 and 96 hr to detect pres-ence of phage by plaque formation. Culture ii was com-pared, for visual lysis, with the sensitive host to whichphage had been added. The true phage resistance of thenew isolates was further tested by plating samples ofyoung mycelium with high phage titers (108 to 109 par-ticles per ml). Observation of eventual plaque formationwas continued up to 96 hr of incubation. In spite of thenegative experimental results so far obtained with ultra-

violet induction of lysogenic streptomycetes (Welsch, 1959;Bradley, 1957), 10-ml samples of 18-hr-old mycelium wereirradiated for 60 sec at 20 cm from an ultraviolet lamp(Sylvania germicidal lamp, model G 15T8). After irradia-tion the suspensions were agitated in the dark for 3 hr,Seitz-filtered, and (i) plated immediatley with the sensitivehost or (ii) added to young mycelium of the host strainand plated after 24 to 48 hr. Under these conditions, ithas never been possible to obtain any indication of phageinduction with the resistant strains. Some isolated plaques,which appeared occasionally under condition i or ii, hadto be considered as air contaminants because of lack ofreproducibility of the results.None of our resistant cultures, under any of the fore-

going experimental conditions, showed evidence of phagepresence and were, therefore, considered as fully resistant.It must, however, be emphasized that the results of anyscreening work for lysogenicity are always dependent onthe chance occurrence of a suitable indicator strain amongthe various cultures included in the test. Under these con-ditions, a definite conclusion about whether a given strainis lysogenic or not can be made only if the experimentalresults are positive. Negative results, i.e., no plaque for-mation can not be considered as sufficiently convincingof the nonlysogenic character of a strain, but could sim-ply mean that the choice of the indicator strain was in-adequate. The periodic reinfections of our resistant strainscould be explained, under these conditions, if we considerS. mediterranei as lysogenic. The temperate phage liber-ated by the strain could, in rare instances, mutate to avirulent particle and thus result in the eventual lysis ofthe broth culture. A mutational origin of our variousphages (except phage 156) from a common ancestor re-ceived some support from the results of the serologicalstudies performed on them (Thiemann et al., 1964).None of the lysed broth cultures showed any evidence

of bacterial contamination, either at the time the lysis oc-curred or after further incubation of the samples. Pre-liminary experiments to detect phage presence either inthe air or in the soil gave negative results, when these de-terminations were performed some time after the intro-duction of a phage-resistant strain.To follow the eventual disappearance of phage particles

from the laboratory air after a phage outbreak had takenplace, petri dishes containing sensitive mycelium were ex-posed for 30 min at monthly intervals. The results indi-cated that, prior to any phage outbreak, no free phageparticles could be detected at any time. The appearanceof lysed shake flask cultures or jars was immediately fol-lowed by an explosive increase in the free-phage particlesin the air, which declined as soon as a new resistant strainwas introduced. The number of phage particles found inthe air was proportional to the seriousness of the infection,i.e., to the virulence of the phage (Fig. 1). Isolation offully resistant strains usually did not offer serious difficultywith the majority of the phages dealt with, except for the

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recently isolated but not further studied phage 291, which,apart from being the least virulent one of all, proved, how-ever, to be the one against which fully resistant strainswere found only after much effort. The presence of a lowgrade of infective phage 291 particles in the air for an ex-

tremely long period is a reflection of the difficulty encoun-

tered in obtaining fully resistant colonies (Fig. 1).Effect of plating conditions on plaque formation. Since

somewhat contrary results are found in the literature withrespect to the effects played by a series of environmentalconditions on plaque formation (St. Clair and McCoy,1959; Hnatko 1952), it was considered useful to definethe effects which some of the variables most frequently in-troduced in phage plating work might have on plaque for-mation of S. mediterranei phages. All the experiments were

done with phage 156 and its indicator strain ME/R156.Effect of pH. Varying the hydrogen ion concentration

of the base layer as well as of the secondary soft agar

layer from pH 5.0 to 8.0, did not influence either plaquesize or morphology; however, approximately 40% fewerplaques were formed by plating the phage-host system atpH 7.5 to 8.0.

Effect of height of agar. Increasing the thickness of theagar per plate, i.e., changing the volume of the base layerfrom 10 to 40 ml, resulted in a consistently lower plaquecount (Fig. 2).These results could be explained in the light of previous

experiments by Carvajal (1953b), who showed that theamount of lysis was dependent on the amount of oxygen

present. Undoubtedly, reducing the head space per platecould result also in a reduction in the overall amount ofoxygen available.

z0

L1.z

6001 10i 0Z00o z

400 t300

200acr~~~~~

TIME IN MONTHFIG. 1. Number of phage particles in the laboratory air after a

phage infection.

According to our results, the highest number of plaquecounts was always obtained by use of only 10 ml of baselayer per plate. However, since this small amount of agar

did not permit an extended incubation period of the plates,20 ml of agar for the base layer were always used for theroutine platings.

Effect of agar concentration. To study the effect of theagar concentration on plating efficiency and plaque size,the concentration of the agar in the secondary layer was

varied from 0.5 to 2.5 %. It was found that both the num-ber of plaques formed and their diameter were inverselyproportional to the agar concentration (Table 1).The morphological characteristics of the piaques (diffuse

border, with clear circular center, etc.) did not changewith varying agar concentrations.

In view of the marked influence that some of the vari-ables proved to have, not only on the plating efficiencybut also on the plaque size, standard plating conditionsadopted throughout this work were: pH of the media,6.0; base layer, 20 ml; agar concentration in the secondarylayer, 0.5 %.

Stability and maintenance of phage-resistant strains. Thestrain ME/R9, resistant against phages and zy and se-

lected for its higher antibiotic-producing capacity, provedto be of an extremely unstable character.

Contrary to what happened to the original culture, ME83/973, whose slants could be maintained without any no-

ticeable loss of productivity for a month and even more at 4C, the slants of ME/R9 even when maintained for 2 weeks

cn

D 120.

-J

Ua.

110.z

inn

0

0

10 20 30 40

ML AGAR PER PLATEFIG. 2. Effect of the height of agar on the number of plaques.

TABLE 1. Effect of agar concentration on number and diameter ofplaques formed

secondary layer No. of plaques Avg diam of plaques

SO mm

0.5 100 3.71.0 89 2.71.5 75 2.32.0 59 1.42.5 42 0.9

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at 4 C gave not only significantly lower antibiotic titersbut occasionally no activity at all. Parallel to the drop inactivity, an increase in myceliunm development was noticed.Whereas the original culture, ME 83/973, revealed upon

plating on A1IL agar the presence of only "typical" colo-nies, i.e., colonies with white to pink aerial mycelium andgood sporulation, strain ME/R9 yielded three main mor-

phological types: (i) typical colonies, (ii) atypical colonieswithout aerial mycelium, and (iii) intermediate-type colo-nies with only sparse aerial mycelium formation. The de-velopment on agar plates of the different colonies was alsomarkedly altered in the sense that the atypical ones showeda much more pronounced growth than did the others.Shake flask experiments performed on a number of iso-

lates of each colonial type clearly indicated that the lossof productivity noticed with culture ME/R9 was due tothe emergence of the atypical colony type (Table 2). Themore rapid growth of these colonies was also reconfirmedin the shake flask experiments, as indicated by the higherpercentage of sediment obtained.

Similar degenerative processes were also observed withresistant strains isolated from other phage infections.Under these conditions, the development of a simple,

efficient, and practical method of maintenance of the pro-duction strains became imperative. The maintenance pro-

cedure which offered the best results was the frozen vege-tative mycelium (FVM) technique already described.FVM\I, prepared from a single typical colony of high pro-

ductivity, not only shortened the tirne required for thedevelopment of the vegetative phase, but proved to behighly stable over a long period of storage at -18 C(Fig. 3).Shake flask fermnentations with phage-resistant strains.

Carvajal (1953a) already pointed out the usefulness of

phages as a mutagenic agent. Plating samples of a lysedculture of S. griseus revealed among the survivors a gamutof colony types, comparable to the mutants normally pro-

duced by the action of ultraviolet rays or other mutagenicagents. The development of phage-resistant mutants notonly ensured phage elimination but resulted also in signif-icantly increased streptomycin yields. These results were

corroborated by Welsch (1957) and Alikhanian and Jljina(1959).

Similar results were also observed in the present studywith S. mediterranei phages. Among the fully resistantcultures isolated after each phage infection, strains showingincreased antibiotic-producing capacity could be readilyisolated. As can be seen in Table 3, a significant increasein rifamycin yields was obtained with practically all ofthe resistant strains, and the yield increments obtainedafter each phage attack were different for each phage. Thiscould be a chance result of the number of isolates tested;however, experiments performed with phage 156 corrobo-rated the unusual capacity of this phage in inducing amongits survivors strains with excellent antibiotic-producingcapacity.

Morphological, cultural, and biochenmical characteristics ofthe phage-resn-stant strains. The increase in rifamycin yieldsobtained with the phage-resistant cultures prompted a

1400.

z 1200.

X41000Laoo800-

SHAKE FLASKS

'Oft1,TANKS

TABLE 2. Shake flask fermentation with typical, atypical, andintermediate-type colonies

Colony Fermentation broth atharvest

Per cent Rifa-Strain Type pH sedimyc-H mentmyi

,ug/ml

ME/R9-16 ............ Typical 6.15 25 490ME/R9-17............ Typical 6.25 28 540ME/R9-18............ Typical 6.25 26 485ME/R9-19 ............ Typical 6.15 24 545ME/R9-21 ............ Typical 6.30 24 565

ME/R9-22............ Atypical 5.35 30 0ME/R9-23 ............ Atypical 5.70 30 0ME/R9-24 ............ Atypical 6.10 32 0ME/R9-25 ............ Atypical 5.80 34 0ME/R9-26 ............ Atypical 5.75 30 0ME/R9-27 ............ Atypical 5.20 32 0ME/R9-28 ............ Atypical 5.95 38 0

ME/R9-29 ............ Intermediate 6.25 29 280ME/R9-30 ............ Intermediate 5.95 26 110

1 3 5TIME (MONTH)

7 9

FIG. 3. Rifamycin yields obtained withfrozen vegetative mycelium.

TABLE 3. Rifamycin yields obtained in shake flask with the sensitiveand various phage-resistant strains

Response against Rifa-Streptomyces phage* mycin Notemediterranci pro-

jS y 17 112 156 duced

ME 83/973. + + + + + 525 Original strainME/R 17 ...... + + + 660 Isolated after infec-

tion with phages ,Band -y

ME/R 112. -_ - + + 700 Isolated after phage17 infection

ME/R 156. - -_ - + 675 Isolated after phage112 infection

ME/R291......1,020 Isolated after phage156 infection

* Symbols: +, lysed;-, not lysed.

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ISOLATION OF S. MEDITERRANEI ACTINOPHAGES

comparative study to see whether the newly selected cul-tures differed among themselves by characters other thanphage spectra and antibiotic titer.As is evident from the results presented in Table 4,

none of the phage-resistant colonies taken for comparisonshowed any biochemical variation when compared withthe original strain. Study of the cultural characteristicsperformed in various standard media adopted for strepto-myces classification also did not reveal any marked differ-ence with respect to pigment production, color of thevegetative mycelium, or general growth behavior. Mor-phologically, apart from the "instabilization" of the cul-tures already referred to, few or no variants were found.The most striking and noticeable difference observedamong the phage-resistant colonies was the loss of aerialmycelium formation and the difference in growth charac-teristics under submerged conditions of soml-e of the mu-tants.When plated on the standard A/IL agar from the original

culture ME 83/973, the growth characteristics and mor-phology of the phage-resistant mutants ME/R17, ME/R112, and ME/R156 differed only in a lesser develop-ment of the aerial mycelium and sporulation.However, all the colonies obtained from phage 156-lysed

broth, as exemplified by isolate ME/R291, differed mark-edly from all the previous colony types in the sense thatthey were practically devoid of aerial mycelium. Undersubmerged conditions, the mycelium of strain i\IE/R291showed a pellet type of growth, whereas all the other cul-tures fragmented readily under these conditions (Fig. 4).

Calcium specificity for phage adsorption. The specific re-quirement of calcium as an adsorption cofactor for bac-teriophages is well known (Perlman, Langlykke, and Roth-berg, 1951; Cohen, 1949). Preliminary results obtainedwith the S. mediterranei phages showed that all of them

TABLE 4. Assimilation of carbohydrate and results of otherbiochemical tests performed on phage-resistant strains

MESubstrate or test* 83/973 ME/ ME/ ME/ ME/(con- R17 R112 R156 R291

trol)

Pridham agar (control) ................

Pridharn agar + lactose ............... + ++ + +Pridham agar + arabinose ............ + + + + +Pridham agar + xylose ................ + + + + +Pridham agar + glucose ............... + ± + + +Pridham agar + fructose .............. + + ± + +Pridham agar + saccharose...........± ± + + +Pridham agar + raffinose ..............

Pridham agar + mannitol ............. + ± + + +H2S production ....................... - _ _ _ _NaNO3 reduction ...................... - _ _ _ _Melanin production ................... - _ _ _ _Calcium malate .......................±+ + + + +Starch hydrolysis .....................± -- ± + +

* With all strains, gelatin liquefaction was slow, and therewas no peptonization or coagulation of litmus milk.

were highly specific for calciuim ioin, the optimal concentra-tion being of the order of 10-3 to 10-4 M. On the basis ofthese results, experiments were performed to determinewhether the addition of calcium sequestering agents tothe broth cultures would protect the mycelium from beinglysed. Sodium oxalate, sodium-- citrate, and ethylenedia-minetetraacetate (EDTA) were used as sequesteringagents.To study the effect of the calcium sequestrants on the

mycelium developmiient, 100 ml of V6 medium in 500-nlErlenmeyer flasks containing increasing amounts of se-questrants were sterilized for 20 min at 121 C, inoculatedwith 5 ml of frozen vegetative mycelium of S. mediterranei,and incubated on an alternating shaker at 28 C. Thegrowth was followed either turbidimetrically at 620 m,uwith a Lumetron colorimeter or by measuring the wetcell mass obtained by centrifuging a 10-ml sample for 10min at 3,000 rev/min. To study the effectiveness of thesequestrants as lysis inhibitors, immediately after the in-oculation of the flasks, phage was added to a concentrationof 107 particles per ml. Mycelium development wasstrongly inhibited by high concentrations of either sodiumoxalate or citrate, the former being slightly less toxic to

FIG. 4. Mycelium of Streptomyces mediterranei under submergedconditions. (A) Strain ME/IR156, 48 hr old. (B) Strain ME/IR291,72 hr old.

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the cells (Fig. 5). Even at 0.001 and 0.0005 M concentra-tions, both sequestrants still showed a residual inhibitoryeffect on mycelium development. Sodium oxalate or sodiumcitrate, at concentrations up to 0.005 M, also effectivelyinhibited lysis (Fig. 5). With EDTA, similar results wereobtained; however, the useful concentration of this seques-trant proved to be more critical. Whereas 0.00068 M wasinsufficient to inhibit lysis, a concentration of 0.00076 Mwas already toxic. Since it is known that EDTA reactswith most metallic ions to form soluble nonionic metalchelates, its high toxicity could be due to the sequesteringof biologically important trace metals from the medium.Neither of the sequestering agents, when tested at theoptimal concentration for lysis inhibition (0.01 M for so-dium oxalate and sodium citrate and 0.00072 M forEDTA), proved to be toxic to the phage.The encouraging results obtained prompted us to study

the effects the sequestering agents might have on rifamycinyields. Some of the results of these experiments are sum-marized in Table 5. For comparative purposes, the rifa-mycin yields obtained in the control fermentation withoutsequestering agent were assigned a value of 100, and thoseobtained when the organic acid salts were added either tothe vegetative media or to the vegetative and fermenta-tive media are expressed in terms of percentage of theyields of the control. The data show that the addition of0.01 M sodium oxalate did not affect the rifamycin yieldsadversely, when the addition was done either to the vege-

3j

2-

C-z

LiJU)

1-

.1 .05 .01 .005 .001 .0005

MOLAR CONCENTRATION OF NA-OXALATE

FIG. 5. Effect of various concentrations of sodium oxatate on thegrowth of Streptomyces mediterranei strain ME/R156, with andwithout added phage (lO0 particles per ml).

tative medium alone or to both the vegetative and fer-mentative media. Because of the high concentration ofcalcium carbonate used in the fermentation media, addi-tion of any sequestering agent alone or in mixture did notsucceed in inhibiting the lysis of the mycelium. Apartfrom these negative results, the use of sequestering agentsin the vegetative medium, in working with phage-sensitivestrains, proved to be an efficient way of reducing the in-cidence of lysis in the final fermentation tanks, since anycasual infective particle would not reproduce itself duringthe inoculum buildup to a point where it could later beharmful to the fermentation. It is obvious, however, thatin any fermentative procedure threatened by a phage theuse of lysis-inhibiting additives cannot be considered asthe solution to the problem, but only as a palliative to beadopted until fully resistant strains are available.

Experiments in which mycelium development in pres-ence of sodium oxalate and phage was followed turbidi-metrically consistently showed, after the second hour ofgrowth, a drop in the optical density reading.

Preliminary experiments showed that, after a latentperiod of 2 hr, phage liberation started. It would seem,then, that even in the presence of sequestering agent someinitial phage multiplication could take place. To clarifythis point, sensitive mycelium was inoculated into V6medium containing 0.01 M sodium oxalate, and 108 phageparticles per ml were added. At hourly intervals, sampleswere withdrawn. The mycelial development was followedturbidimetrically, and the phage count was performed bystandard plating technique. As is evident from the resultspresented in Fig. 6, parallel to the drop in optical densityreading between the second and third hours, there wasalso a definite increase in the phage titer, which reachedits maximal value after 4 hr, maintaining itself constantthereafter. No second or third lytic cycles could be noticedat any time. The reason for this initial and slight lysis inthe presence of the calcium sequestrant might be due toan incomplete removal of the calcium ion from the media,or to the presence of calcium firmly bound to the cell wall.Presence of calcium-independent mutants in the phagepopulation can be excluded, because if they were present

TABLE 5. Effect of sodium oxalate on rifamycin yieldsand lysis inhibition

Vegetative FermentativeRifamycin

Addition of PMV* at Addition of PMV* at yieldssodium oxalate transfer sodium oxalate harvest

M M N

None 6.0 None 27 100None 6.0 0.01 22 1160.01 5.5 None 22 1000.01 5.5 0.01 30 1180.0lt 6.0 None Lysed0°Olt 6.0 0.01 Lysed

* PMV = per cent mycelial volume.t Phage particles (108 per ml) were added.

HgNO PHAGE 156

PHAGE (10oML)

[-B

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SENSITIVE MYCELIUMtotal or at least partial lysis after 24 hr of incubation

JEIVMEI PH +OXALATE should have occurred. To corroborate the absence of cal-E5 _u , - |

i l cium-independent mutants in the phage population, a

e!>qzl ^ + F |Aphage-sensitive S. mediterranei strain was inoculated intocc

l\ /CONTROL A V6 medium containing 0.05 M sodium oxalate and 10890iAGEj.OXALATE phage particles per ml. After 24 hr of incubation, a 10-mls sample was added to: (i) 100 ml of V6 medium containing

oxalate and (ii) to a 24-hr-old culture grown in the samemedium but without oxalate. After further incubation

1200

1iao0 /o0- SENSITIVE5~~~~0 ~~~MEfR 112

4,8-

1.000 4,6/

z

|~~~~~~~~_°o ,44-d*i> R4

ui2 4 21-2

-JME (HR) a. 4i0.

o.200- 0 RESISTANT

034 A e , * ~ME/R 47

,00 I

TIME (HR) 6I0 2 4 6 20 30 40 50

FIG. 6. Effect of 0.01 m sodium oxalate on the multiplication ofphage ,3 grown on the sen.sitive strain ME 83/973. TIME (HR)

FIG. 8. Absorption and release of phage 112 from sensitive andresistant mycelium.

50- SENSITIVE 4, 0 SENSITIVE0 ME 83/973 4,2° ME/R 156

4,60 4,02

3,8T

_j4, 3,6-

ir xo. 3,8 2

CD RESIST4,NT 0.cC ~~~~~~~~~~~~~~~~~~~~~~~ME/R156CD

0. 44 2's~~~~~~~~~~~~~ -

RESISTANTCD ME/R112 0.A

9 (!) 2,6- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~AME/R 47

00 0 ~~~~~~~~~~~~~~j2,4-00A

A

2,6- 2,0-

2,2 I ,0 2 4 20 30 40 500 2 4 6. 20 30 40 50

TIME (HR) TIME (HR)FIG. 7. Absorption and

resistant mycelium.release of phage I8 from sensitive and FIG. 9. Absorption and release of phage 156 from sensitive and

resistant mycelium.

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for 24 hr, i was used to inoculate two new flasks as de-scribed previously, and ii was used to follow the lysis byvisual examination and plating.

Presence of phage could be detected in ii only up to theseventh transfer; on further transfers, no more phage par-ticles could be detected, indicating not only that the phagedid not multiply in i but also, and most important, thatno calcium-independent mutants were present.

Phage adsorption on resistant mycelium. Van Alstyneet al. (1955) were the first to show that actinophage-re-sistant colonies, although they do not propagate the phage,are nevertheless able to adsorb it. Similar conclusions werereached by Henry and Henry (1946) working with phage-resistant staphylococci. These observations are in contrastto the general belief that resistant colonies do not adsorbthe phage (Luria, 1945; Anderson, 1949; Lieb, 1953).Our work on S. mediteiranei phages corroborated the

results obtained by Van Alstyne et al. (1955) in the sensethat phage-resistant strains of S. mediterranei are able toadsorb but not to multiply the phage. To follow the phageadsorption to resistant and sensitive mycelium, the S.mediterranei strains were grown in V6 medium for 18 hr.Phage was added to the mycelium to a final concentrationof 104 to 105 phage particles per ml. Immediately afterphage addition and at different time intervals, a 10-mlsample was withdrawn arid centrifuged for 10 min at3,000 rev/min, and a phage titer was performed on theclear supernatant in the usual way. All determinationswere made in triplicate.Under these conditions, testing the different phages

with their sensitive host as a control and with the resistantstrain(s), a marked difference was found in the capacityof the resistant host to adsorb the phage and to release itafter a certain time interval. In no instance was there no-ticed any sign of phage miultiplication by the resistantmycelia, as indicated by the absence of rise in phage titerover the initial input level (Fig. 7, 8, and 9).Whereas approximately 92 % adsorption of phage 156

on its sensitive host MIE/R156 took place in 90 min andphage release with multiplication started after 2 hr, thesame phage on the resistant mutant IE/R47 was adsorbedup to 96 % over a period of 6 hr. A positive phage releasewas noticed after 24 to 48 hr when the experiment wasinterrupted. Phage 112, on the other hand, adsorbed read-ily onto its sensitive host (MIE/R112), but only slightly(20%) on the resistant mutant MIE/R47. Similar resultswere also obtained with phage ,B on its sensitive host andtwo resistant mutants (Fig. 7).

It seems clear from these results that S. mediterraneiphages can be adsorbed to various degrees onto resistantmycelia from which they are released, without multiplica-tion, upon aging of the mycelium. The ability of the re-sistant mycelium to adsorb the phage should be takeninto consideration while working on the isolation of resist-ant colonies from lysed broth cultures, to avoid the in-troduction of phage-carrier strains in production work.

Serial transfers into liquid media containing calcium se-questrants followed by single-colony isolation should rap-idly lead to the complete elimination of any adsorbedphage.

LITERATURE CITED

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ALIKHANIAN, S. L., AND T. S. ILJINA. 1959. Mutagenic effect ofactinophage. Proc. Intern. Congr. Genet., 10th, Montreal,1958, p. 4.

ANDERSON, T. F. 1949. The reactions of bacterial viruses withtheir host cells. Botan. Rev. 15:464-505.

BRADLEY, S. G. 1957. Distribution of lysogenic Streptomyces.Science 126:558-559.

CARVAJAL, F. 1953a. Phage problems in the streptomycin fermen-tation. Mycologia 45:209-234.

CARVAJAL, F. 1953b. Host-parasite relations with a polyvalentstreptomycophage from Streptomyces griseus. Antibiot.Chemotherapy 5:28-37.

COHEN, S. S. 1949. Growth requirements of bacterial viruses.Bacteriol. Rev. 13:1-45.

HENRY, J. E., AND R. J. HENRY. 1946. Studies on the relationshipsbetween bacteriophage and bacterial host cell. I. Absorptionof phage by resistant variants of Staphylococcus. J. Bacteriol.52:481-486.

HNATKO, S. I. 1952. Concentric ring formation about plaques ofM. phlei bacteriophage. Can. J. Public Health 43:54-59.

LIEB, M. 1953. The establishment of lysogenicity in Escherichiacoli. J. Bacteriol. 65:642-651.

LURIA, S. E. 1945. Mutations of bacterial viruses affecting theirhost range. Genetics 30:84-99.

MARGALITH, P., AND G. BERETTA. 1960. Rifomycin. XI. Taxonomicstudy on Streptomyces mediterranei nov. sp. Mycopathol.Mycol. Appl. 13:321-330.

PERLMAN, D., A. F. LANGLYKKE, AND H. D. ROTHBERG, JR. 1951.Observations on the chemical inhibition of Streptomycesgriseus bacteriophage multiplication. J. Bacteriol. 61:135-143.

REILLY, H. C., D. A. HARRIS, AND S. A. WAKSMAN. 1947. Anactinophage for Streptomyces griseus. J. Bacteriol. 54:451-466.

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SMITH, R. M., W. H. KUHN, AND G. R. M. MIESEL. 1947. An actino-phage in streptomycin-producing cultures of Streptomycesgriseus. J. Bacteriol. 54:545.

ST. CLAIR, J., AND E. MCCOY. 1959. Plaque morphology of certainstreptomycete phages. J. Bacteriol. 77:131-136.

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G. LICCIARDELLO. 1964. Rifamycin. XXXIV. Physicochemicalcharacterization of actinophages active on Streptomycesmediterranei. Appl. Microbiol. 12:269-272.

VAN ALSTYNE, M. H., R. H. OTTO, AND E. McCoY. 1955. Charac-teristics of Streptomyces griseus strains resistant to phage.J. Bacteriol. 70:113-119.

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WEINDLING, R., H. D. TRESNER, AND E. J. BACKUS. 1961. Thehost-range of a Streptomyces aureofaciens actinophage. Nature189:603.

WELSCH, M. 1957. The behaviour towards actinophage of mutantssurviving its lytic action. Antonie van Leeuwenhoek J. Micro-biol. Serol. 23:5980.

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