Heterokaryosis as a Cause of Culture Rundown in Penicillium

Transcription

1 Heterokaryosis as a Cause of Culture Rundown in Penicillium F. L HAAS, T. A. PUGLISI, A. J. MOSES, AND J. LEIN Research Division, Bristol Laboratories Inc., Syracuse, New York Received for publication February 27, 1956 Physiological degeneration of fungus cultures has long been a problem encountered in industrial fermentations. This has been no less true of antibiotic fermentations than of the older ones. Degeneration in antibiotic-producing cultures is usually accompanied by a number of cultural and biochemical changes, some of which may be correlated in a general way with reduction in antibiotic production by the specific culture. However, a given change is usually an indicator of physiological degeneration only in the case of a specific strain cultured and fermented under a specific set of conditions. For example, Foster et al. (1943) and Foster (1949) find that physiological degeneration usually is accompanied by reduction in sporulation and advise that a minimum number of transfers from heavily sporulating isolates should be used as inoculum. On the other hand, Whiffen and Savage (1947) found that sporulation itself was a factor promoting "penicillin rundown" (the term given by them to physiological degeneration in regard to penicillin production), and that rundown did not occur over as many as 50 consecutive vegetative transfers if all sporulation was prevented. In our experiments we find that even spore-tospore transfers over more than 25 serial transfers will not promote rundown, provided a rigid system of selection is available and utilized. Other seemingly conflicting reports are quite common for penicillin and other antibiotic fermentations. It is generally thought that changes in the strain leading to physiological degeneration are of genetic origin arising as gene mutations somewhere in the history of the strain. However, the general mechanism by which these changes are disseminated and preserved in the mold cultures and the possible role of heterokaryosis in physiological degeneration, have not been considered in detail in relation to industrial fermentations. Many genetically controlled changes are scattered and preserved in most of the Fungi Imperfecti, as well as in many of those having a perfect stage, by heterokaryosis. Hyphae of the same or different strains form anastomoses to initiate this process. This is followed by nuclear migration across the anastomoses, subsequent mixing of nuclear types within the same hyphae and, finally, adjustment of nuclear numbers of the two types within the mycelia to a ratio which affords the maximum growth rate under the immediate environmental conditions. 187 Jinks (1952a, 1952b) found that most Penicillium strains isolated from natural sources (soil and decaying vegetable matter) were stable heterokaryons. He was also able clearly to demonstrate that such a stable heterokaryon had a growth rate superior to those of its homokaryotic component strains. Whenever a component strain attained a growth rate superior to that of the heterokaryon the heterokaryon would break down and at least one pure homokaryotic strain developed in mixture with the heterokaryon. Thus heterokaryon stability and breakdown appear to follow the growth rate theory of heterokaryosis advanced by Beadle and Coonradt (1944) for Neurospora. Proof that heterokaryosis occurs in Penicillium, and the mechanics of nuclear behavior (migration and mixing) at heterokaryon formation, have been presented by Baker (1944) and by Pontecorvo and Gemmell (1944a, 1944b). The mechanics are the same as given by Hansen (1938, 1942), Hansen et al. (1932, 1943), Dodge (1942), Dowding and Buller (1940), and Beadle and Coonradt (1944) for a number of other fungi. Heterokaryosis has since been demonstrated in Penicillium many times, but to our knowledge the role played by this process in influencing fermentationproduction yields has not been presented. Certain results of Foster et al. (1943) could be interpreted as showing the effect of heterokaryosis in causing penicillin rundown, but this possibility was not investigated. One of the strains studied by us was Wisconsin (see Campbell and Curtis, 1952, for origin and history). Some mutants obtained from this strain were excellent for a study of the role of heterokaryosis in penicillin rundown, since they coupled distinct cultural characters with characteristic fermentation yields of penicillin. They also proved of value in studying the variation pattern which has been observed in all of the Wisconsin series since Q-176. This paper presents the results of a series of experiments with these mutants designed to test the effects of heterokaryosis on penicillin yields, its involvement in penicillin rundown, and the origin of different strains observed in the normal population of strain W MATERIALS AND METHODS The following Penicillium strains were used in the experiments reported: W50-935, strains A, B, C, D, E-1, E-2, H, and Strain W was obtained originally as a single-colony isolate from a plating of

2 188 F. L. HAAS ET AL. [VOL. 4 nitrogen-mustard-treated W (Campbell and Curtis, 1952). A slant of this strain kindly supplied by Dr. M. P. Backus of the University of Wisconsin, Department of Botany, served as the origin of our stocks of this strain. In our hands this strain appears to be a mixture of types. Several morphologically different populations are visible when it is plated on lactose-cornsteep medium (LCS), and these can be separated from each other by single colony isolation from this medium. In shake-flask fermentations this strain produces approximately 900 Oxford units of penicillin per ml of broth under our conditions. Strains A, B, C, D, E-1, E-2, and H are all isolates established from a plating of ultraviolet-irradiated W spores. All of these isolates were olive green, heavily sporulated, and seemed to be composed of the same colony type. In shake-flask fermentations they all produced approximately 3000 oxford units per ml of penicillin. In some experiments, series of substrains were established from these strains by selecting isolated colonies, single spores, or single hyphal tips from platings. Strain 2158 was previously obtained at Bristol Laboratories' Research Department as a selection from an ultraviolet-irradiated spore suspension of Wisconsin strain W (see Backus and Stauffer, 1955, for history of W48-701). Soil-tube cultures were made of each of the strains, and the same soil tube of each strain was used throughout this study to establish primary cultures. To determine which of the above strains would undergo penicillin rundown, serial transfers of each strain were made on LCS agar slants. Transfers were made by suspending the spores and mycelia from a slant, or spores from a soil tube, in 0.01 per cent Wetanol.1 This mass inoculum was used to inoculate fresh agar slants. Slant growth from each culture was transferred in the same manner to fresh slants after 6, 14 and 21 days' growth at room temperature (RT). This procedure was carried out through six serial transfers with each strain. The spore-mycelia suspension was also used to inoculate triplicate fermentation shake flasks at the time of inoculating each slant. Each shake flask contained 100 ml of LCS liquid medium. These fermentations were incubated for 8 days at 76 F on a rotary shaker operating at 200 rpm. Five-ml aliquots were removed from each flask on the 5th, 6th, 7th, and 8th days of fermentation and analyzed for ph, penicillin production, and population changes. Penicillin assays were run by a modification of the "cylinder-plate" method (Schmidt and Moyer, 1944) on centrifuged broth samples. Population patterns, and changes in them, were observed by plating series of serial dilutions of uncentrifuged broths on LCS agar. Triplicate plates were made at each dilution by the glass-rod-spreader technique. These plates were incubated at RT for 9 days and then examined for colony I Glyco Prod'cts Inc', B'rooklyn, N. Y. types and percentages of each type calculated. The shake-flask medium used in most cases was a modification of the LCS medium of Moyer and Coghill (1946). LCS agar slants were made of the same medium with 2.5 per cent Difco agar added. In some experiments for observing population changes and heterokaryon breakdown, honey-peptone (Churchill, 1947) and modified Czapek-Dox (Jinks, 1952a) media were used. All inocula from slants, colonies, or fermentation broths used in experiments reported here were macerated for 30 sec. in a Waring blendor before use. Inoculum for small tank fermentors was grown on LCS agar flats in 10-L wide-mouth bottles. These cultures were grown for 7 days at 76 F and then harvested aseptically in 0.01 per cent Wetanol. Samples of the harvested tank inocula were tested for penicillin production in shake-flask fermentations, and for colony types by the plating technique at the same time that they were tested in the tank fermentors. Single-spore isolations were made from most of the cultures with a de Fonbrune micromanipulator, using a modified Zelle technique (Zelle, 1951). Hyphal tips were isolated with a Chambers micromanipulator under a dissecting microscope. Heterokaryon formation between pairs of all strains was checked microscopically, using the method of Lindegren (1934). In general, this method was not successful because of a decided reluctance of the strains to form hyphal anastomoses on the solid medium used. RESULTS AND DISCUSSION Penicillin Rundown in Isolates of W During a routine investigation of strain W numbers of isolates were obtained from a screening of ultraviolet-irradiated spores which gave penicillin yields considerably higher than those obtained under the same fermentation conditions with the parent strain. These isolates were maintained on LCS agar slants and in soil-tube cultures. Several of them were tested for penicillin rundown by serial transfer of mass inoculum on LCS agar slants after various periods of growth. One series of transfers was started from soil tubes and another from slants inoculated with the primary colony at the time it was isolated from the original plates. At the time of each transfer the slant was tested in shake-flask fermentations for penicillin production and was plated out in serial dilutions on LCS agar for cultural examination. All of the strains tested in this manner exhibited rapid rundown and after three serial transfers produced only 20 to 50 per cent as much penicillin as the original isolate. At the same time, growth became heavier and the fermentation cycle shorter. The parent W strain was carried as a control throughout these experiments and did not rundown or change growth patterns through a total of nine serial transfers. However, its penicillin production

3 1956] HETEROKARYOSIS IN PENICILLIUM 189 was only approximately 25 per cent that of the isolates before they underwent rundown. Platings of the inocula at each transfer showed that certain cultural changes paralleling pencillin rundown occurred in the cultures. The original isolates and first slants of the isolates revealed only a single dark-green, heavily sporulating colony type (G-type) whenplated on LCS agar. However, at various stages of transfer, usually after the second but sometimes after only one transplanting, a few white colonies (W-type) and white-sectored dark-green colonies (S-type) would appear in the platings. The W-type strain is apparently a pure albino type. It sporulates almost as well as the G-type and its spores, as well as mycelium, have no pigment. The parent strain shows all three of these types, among others, when it is plated on LCS agar. In further transfers of the isolates, the percentages of W- and S-type colonies increased. Within two transfers after the first appearance of W- or S-type colonies the isolate had the same population pattern and penicillin production as W Transfer experiments were carried out in three series: one with a 7-day growth period, a second with a 12-day growth period, and a third with a 21-day growth period between transfers. Rundown was much slower, and cultural changes appeared later in the 7-day-old transfer series. Usually, when 12-day- and 21-day-old slants were used, rundown and cultural variants were pronounced after the first transfer. The age at which sectoring occurred, and its manner of appearance, remind one of similar sectoring observed by Churchill (1947) in earlier ancestors of W He found that flesh-colored, nonsporulating variants of Q-176 would produce heavily sporulating green sectors after 7 days' growth. He further found that this sectoring could be prevented indefinitely by transferring hyphal tips from colonies less than a week old, or by growing the colonies continuously on fresh medium. Whiffen and Savage (1947) found that penicillin rundown in Penicillium notatum NRRL 1249 B21 could be delayed by any process that decreased the percentage of spores in the inoculum. Using vegetative inocula grown under conditions that permitted almost no sporulation, they could prevent rundown indefinitely. To determine if rundown in our strains could be controlled by similar methods, experiments were run in which the cultures were kept in the vegetative state. This was accomplished by serial transfers of shake-flask growth at 24-hr intervals to fresh shake flasks of LCS medium. At each transfer aliquots were used to inoculate other shake-flask fermentations for penicillin production analysis. They were also plated out in serial dilutions on LCS agar for observation of population changes. The results showed that rundown and population changes occurred much earlier under these conditions than when 7-day-old slant transfers were made. Using vegetative transfers, rundowrn usually started with the second transfer. Plating Studies on White, Green and Sectoring Substrains of Strain A Studies on the colony types appearing in strain A immediately before and after the start of rundown were carried out on LCS agar by serial hyphal tip transfers and by mass inocula platings from single colonies of the G-, W-, and S- types. These studies showed that the W-type was very stable. No changes appeared in these substrains through as many as five transfers. Moreover, when platings were grown for as long as 25 days no sectoring appeared on any W-type isolate. The G-type was relatively stable and little or no sectoring or white colonies occurred throughout the series of transfers when they were made at 7-day intervals. In many cases sectors would appear in small numbers in platings when they were held for longer periods of time; however, a few G-type substrains were established which would not sector regardless of the length of time grown. These latter strains showed no indication of rundown through as many as five serial transfers of mass inoculum. S-type colonies were unstable and mass inocula platings always produced all three colony types, with S-type colonies accounting for 75 per cent or more of the population. Hyphal tip transfers from white sectors always produced only W-type colonies, while hyphal tip transfers from the green portions of S-type colonies almost always produced S-type colonies. In a few cases, G-type colonies were produced from hyphal tips taken from the green portion. These plating experiments were also carried out on honey-peptone agar and modified Czapek-Dox agar. On these media S-type colonies did not appear. Platings of the G-type colonies yield homogeneous light-green populations, while platings of the W-type colonies yield homogeneous white populations. Mass inocula platings of S-type colonies from LCS agar plates onto honey-peptone or Czapek-Dox agar plates yield only colonies typical of the W- and G-types. However, when the G-type colonies produced by plating S-type colonies on honey-peptone or Czakep-Dox are plated back onto LCS agar they are found to be of two types, those yielding only G-type, and those yielding G-, W-, and S-types. Therefore, while sectoring is prevented phenotypically on Czapek-Dox and honeypeptone media the factors responsible for the sectoring on LCS remain present in the mycelium and must be of a genetic nature. The most logical mechanism which could explain this behavior would be heterokaryosis in which two types of nuclei are present in the mycelium. This heterokaryon would necessarily remain balanced on Czapek-Dox and honey-peptone media, but would break down on LCS medium. Such a system would be very similar to that observed by Jinks (1952b) and Rees and Jinks (1952), who found that Penicillium

4 190 TABLE 1. Fermentation and population characteristics of substrains isolated from platings of vegetative growth of strain A after 8 days' growth in shake flasks Substrain (Colony Type)* Penicillin Production Population Pattern on Lactose-Cornsteep Agar Oxford units/ml G-type G-type colonies ' G-type G-type colonies S-type G-, W-, and S-type colonies S-type G-, W-, and S-type colonies W-type W-type colonies W-type W-type colonies * G-type = heavily sporulating colony type; S-type = white-sectored, dark-green colony type; and W-type = white colonies. heterokaryons isolated from natural sources were stable only on media containing large amounts of apple pulp but broke down on simpler media. Penicillin rundown was not studied in cultures grown on honey-peptone or Czapek-Dox media, since slants grown on these media gave very low penicillin yields when used as inocula in our shake-flask fermentation medium. Effect on Penicillin Production of the G-, S-, and W-type Colonies Since S- and W-type variants appeared more rapidly in vegetative liquid cultures than on solid media, it was probable that they would also arise in fermentation shake flasks and in the various stages of tank fermentations. If this occurred, penicillin yields would probably be reduced proportionate to the time of appearance of the variants and to the extent of their growth, since W- and S-types were always found to give low penicillin yields. A study of this possibility was carried out using slants of a G-type isolate of W The slants of this strain (strain A) were inoculated from a soil tube which had previously been tested and found to contain a homogeneous G-type population giving good penicillin yields in shake-flask fermentations. The slants were used to inoculate shake-flask fermentations which were run for 8 days. Samples of the shake-flask growth were plated out in serial dilutions at 2-day intervals, and the fermentation broths were assayed for penicillin potency on the 6th, 7th, and 8th days of fermentation. The platings revealed that S-type colonies started to appear on the 4th and 5th days of fermentation. By the 8th day approximately 40 per cent of the colonies were S-type, and a few of the W-type were also noted. Whether or not these changes interfered with penicillin production in these fermentations could not be determined. Average maximum potencies of 2850 Oxford units per ml were attained, and this was about the same as had been obtained in previous experiments with strain A. The possibility exists, however, that the yields F. L. HAAS ET AL. [VOL. 4 would have been higher if such variants could have been prevented from appearing at all. The experiments were repeated using different variations of the LCS medium. None of these variations had any noticeable effect on the cultural changes or their time of appearance. Single-colony isolations were made of each of the three colony types appearing in this fermentation. These isolates were then used as inocula for similar fermentations which were investigated by the same plating and penicillin potency tests. The results of these experiments are given in table 1. They show that certain isolates (W- and S-type colonies) taken from the normal fermentations of the G-type strain give fermentation potencies similar to those of rundown cultures. In the case of the S-type isolate, platings of 7-day-old fermentation flask growth show approximately the same ratios of G-, S-, and W-type colonies as are found in platings of rundown cultures. Results of only a few substrains tested in one experiment are given, but in all observed cases penicillin potencies of fermentations with G-, S-, and W-type substrains agree with those given in table 1. Investigations of all steps in submerged fermentation through small tank fermentors revealed the same pattern. Whenever white or sectoring colonies appeared in platings of a culture to be used, the penicillin potencies of the next stage and all subsequent stages were low. For further studies, a series of 10 LCS agar slants was made from the same soil tube of strain A. After 7 days' growth these slants were used to grow small tank fermentor inocula. At the same time they were plated out in serial dilutions on LCS agar and also used to inoculate shake-flask fermentations. The platings were examined after 10 days' growth and the fermentations were assayed for penicillin potency after 6, 7, and 8 days. The same tests were also run on the tank fermentor inocula when they were prepared. Results of these experiments are given in table 2. In this table, the slants are divided into groups based on the stage of appearance of W- or S-type colonies. In group I these colony types did not appear in either the slants or in platings of the tank inocula. In group II they did not appear in the slants but appeared to some extent in platings of the tank inocula. Group III is composed of one slant which showed a small number of W-type in the slant population and a large number in the tank fermentor inoculum. These results indicate that the W- and S-type colonies appear at random in the cultures, at least on solid media. In all groups, penicillin potencies agreed with that expected on the basis of the stage of appearance of W- and S-type colonies in platings. Similar results were obtained when small fermentation tanks were inoculated with the tank inocula given in table 2. Group I inocula produced tank fermentations with high-potency broths; those inoculated with group II or group III inocula gave low-potency broths. Platings of growth from tanks inoculated with

5 19561 HETEROKARYOSIS IN PENICILLIUM 191 TABLE 2. Random appearance of W-type and S-type colonies in a pure culture of strain At Lactose-Cornsteep Agar Slant Tank Fermentor Inoculum Grown from Lactose-Cornsteep Agar Slant Spores Slant No.* W- or S- Shake- W- or S-t3Tpe Shake- type flask colonies in flask -Colonies yil opating yed in plating yield of slantt yield of inoculumt Oxford Per cent Oxford units/ml units/mi Per cent Group 1. (No W- or S- type colonies) Group 2. (No W- or S- type colonies in slants but in platings of tank inocula) Group 3. (Small numbers, of W- type in slant and large no. in tank fermentor inoculum) * All slants were inoculated at the same time from the same soil tube of Strain A. t W-type = white colonies; S-type = white-sectored, darkgreen, colony type. group I cultures occasionally showed a large percentage of W- and S-type colonies, however, usually only a few were found and these appeared late in the fermentation cycle. Tanks inoculated with group II or III always showed a large percentage of W- and S-type colonies, and these were found early in tank fermentations. All three colony types were isolated at some stage of the fermentation from all small tank fermentors tested. These isolates, according to strain type, all gave the expected penicillin potencies when tested in shake flask fermentations. When tank inoculum which showed no variants in plating tests was used, W- and S-type colonies appeared relatively late in the tank fermentation. Such tanks were always of high penicillin potency. When the variants appeared early, penicillin potencies were low. The high-penicillin-yielding segment of the population was still present in the mycelia of these lowpotency tanks, and strains producing yields as high as those of the original parent strain could be reisolated from them by routine plating procedures. The W- and S-type variants did not produce substances which destroy penicillin already produced in the fermentation, nor any material which inhibited penicillin production of the G-type strain in the first place. This was shown by adding Seitz-filtered broths from W-type strain fermentations at intervals during G-type fermentations, and also to completed G-type fermentation broths which were then allowed to stand for 16 hr. Neither treatment reduced the yields of the G-type fermentations beyond that which could be accounted for by dilution. Strains apparently identical with the original in penicillin-producing abilities and morphology could easily be reobtained from rundown cultures by serial singlecolony reisolation using shake-flask cultures, slants, or soils as starting points. All of the rundown strains derived from W examined by us had large percentages of W- and S-type variants in their populations. In serial reisolation procedures a suitable source of the rundown culture was plated on LCS agar. From this plating a number of well-isolated typical G-type colonies were inoculated onto LCS agar slants, using a mass inoculum prepared by macerating entire colonies in separate tubes of 0.01 per cent Wetanol solution. After 7 days' growth, these slants were plated again on LCS agar and G-type colonies picked to slants again after 7 days. This procedure was repeated a third time. At each of the three platings, the slants were also tested for penicillin production in shake-flask fermentations. The results of this reisolation procedure on a number of W derivative strains are given in table 3. Note that culture E-2 was apparently pure G-type after the second reisolation, but the W-type variant reappeared in the third reisolation. This does not occur often, provided growth on the platings and slants is not over 9 days old when used or transferred. This technique has been used on rundown or impure strains of Streptomyces as well as other lines of Penicillium, and strains identical in appearance and yields with the original isolate are readily reobtained. Foster et al. (1943) state that large numbers of transfers are conducive to penicillin rundown and recommend an absolute minimum of transfers between the soil tube and ultimate use of the culture in production. They also found that rundown was accompanied by sectoring and white variants. However, we find that when carried out in TABLE 3. Response of rundown cultures to serial reisolation After After 1st After 2nd After 3rd Peni- Rundown Reisolation Reisolation Reisolation cillin StanYield W- w- w- w Strain Beifeor Peni- type Peni- type Peni- type Peni- type Run- Peni-n and n and.-and -ili and down i tllin cl S_type cilli cillin S-ype yieldc00olo- oo yedcolonies nies nies nies Oxford Oxford o Oxford on Oxford Oxford units/ml units/mi units/ml 0 units/ml units/mli B < C D E < E <

6 192 F. L. HAAS ET AL. [VOL. 4 conjunction with a selective procedure such as the above, most of the cultures are just as potent penicillin producers as the original isolate after as many as 10 transfers. Heterokaryon Formation Between Different Penicillium Strains and the Effect on Penicillin Yields A study of possible causes of rundown in Penicillium heterokaryons was made by testing artificially synthesized heterokaryons in shake flasks. Shake-flask fermentations were inoculated with mass inocula prepared from slants of strains A, H, 2158, W50-935, and White # 3. All of these slants with the exception of W had been established from single spores. Fermentations were run simultaneously on each strain separately and in all possible combinations with the other single strains. These fermentations were assayed for penicillin potencies and plated for colony types. Results of this experiment are given in table 4. Such a technique allows homokaryons and the heterokaryon type formed from the two homokaryons to exert their effects during the fermentation. There is little doubt that the heterokaryon is formed under these circumstances, since all combinations in which a white strain is mixed with a green strain give a very high percentage of whitesectoring G-type colonies and comparatively low percentages of the presumably homokaryotic W- and G-types of nonsectoring colonies. Sectoring colonies do not appear to any appreciable extent in any of the fermentations where pure W-type or pure G-type strains were the only inocula. The fact that the great majority of colonies are sectored also shows that the heterokaryon is balanced (or stable) in liquid shakeflask medium, at least throughout the greater part of the fermentation, and that it breaks down on the solid medium used as indicated by sectoring. Attempts to synthesize heterokaryons of the same strain combinations were made, using the microscope slide-culture method of Lindegren and Andrews (1945). However, these attempts were unsuccessful. All combinations, and even mycelia of the same strain, were most reluctant to form anastomoses on the agar-slide culture. Possibly the same factors responsibility for heterokaryon breakdown and sectoring are also responsible for the scarcity of anastomoses on solid medium in the slide-culture tests. Another approach to this same problem was more successful. Single hyphal tips were isolated from the white sectors and from the green portions of a number of S-type colonies obtained from the platings given in table 4. In all cases, the hyphal tips taken from white sectors gave rise only to W-type strains which gave poor penicillin yields. Most of the tips isolated from the green portion of S-type colonies produced S-type cultures which gave low to intermediate penicillin yields. However, a few cultures established in this manner were apparently pure G-type and gave high penicillin yields. The fact that a single hyphal strand gave rise to a colony which produced spores of both parental types is believed to be strong evidence that the strain was TABLE 4. Effect of W-type strains on colonial morphology and penicillin production of other strains 6th Day of 7th Day of 8th Day of Peak Assay and Fermentation Fermentation Fermentation Highest Per Cent of W-Type Colonies W-and W- and W-and W-and Yield S-type* Yield S-type Yield S-type Yield S-type colonies colonies colonies colonies Oxford o% Oxford e Oxford % Oxford % units/ml units/ml o units/ml units/ml W-type strain # W W W W Strain H (G-type strain)* 2640 O W W W W OS <0.1S <1 S <1.OS W (parent strain) W W W W 70S 67 S 75 S 75 S Strain X (from different line than W50-935) W W W W 0S 0 S 0 S 0 S W-type 3 + strain H W W W W 50 S 77 S 72 S 77 S W-type #3 + W W W W W 68S 68 S 70 S 70 S W-type 3 + strain X W W W W 20 S 15 S 13 S 13 S Strain H + W O W W W 25S 68 S 68 S Strain H + strain X 1590 O W W W W 0 S <0.1 S <0.1 S 0.1 S W strain X W W W W 40S 54 S 60 S 60 S * G = green-type; S = sectored-type; and W = white-type.

7 1956] HETEROKARYOSIS IN PENICILLIUM 193 TABLE 5. Penicillin yields and population patterns of cultures established from single hyphal tips taken from white and green sectors of the same S-type colony Source and Isolate Shake-Flask Population Pattem Yield Pplto atr Oxford units/ml White sector of S-type colony 1-S-W % W-type 1-S-W % W-type Green sector of S-type colony 1-S-G W-, S-, and G- types 1-S-G W-, S-, and G- types 1-S-G % G-type heterokaryotic. The results of this experiment are given in table 5. Only results for tests of colonies isolated from the W-type # 3 x strain A fermentation are given. The same tests were made for several other combinations and essentially the same results were obtained. The fact that the majority of colonies are S-type in the mixed strain experiments (table 4), and that the greentype growth in these sectoring colonies will yield both pure G-type, high-penicillin-producing strains and lowyielding S-type strains indicates that heterokaryons are probably fairly stable in liquid medium, but that they break down immediately on plating on solid medium. Origin of the W- and S-Type Colonies Most Penicillium species which have been studied have been found to readily form heterokaryons (Baker, 1944; Pontecorvo and Gemmell, 1944a, 1944b; Lindegren and Andrews, 1945; Jinks, 1952a, 1952b). Jinks in his studies also found that balanced Penicillium heterokaryons often break up into the component strains when they are removed to artificial media or unnatural growth conditions. Our observations on the appearance of W- and S-type variants suggested that heterokaryosis was the main factor involved here in penicillin rundown or physiological degeneration. There are several mechanisms which could lead to heterokaryosis in these strains. In one such mechanism W-type-producing nuclei, arising by mutation in the mycelium, and having a selective growth advantage in the culture, would rapidly increase in relative number. Hyphal anastomoses would spread the mutant nuclei throughout the mycelia. As their number increased relative to the normal type, a rundown culture would result, especially if they contribute little to penicillin production and use much more nutritive material for growth than does the normal strain. A second possibility is that the original isolates of the strain may be composed of more than one genotype. This heterogenic condition may have arisen from heterokaryotic mycelial fragments, polynucleate spores, or spores having heterozygous diploid nuclei (Pontecorvo and Sermonti, 1954). In any of the above situations, both W- and S-type genotypes would have to be present. In such a case, the W-type component would necessarily have little effect on the penicillin producing ability of the strain as long as it was in the balanced heterokaryotic state (or if in the same nucleus it was recessive to the G-type). Such a condition could be even more desirable than a pure strain due to hybrid vigor or heterosis contributed by the dual system (Beadle and Coonradt, 1944; Baker, 1944; Pontecorvo, 1946; Jinks, 1952a, 1952b). It is possible that with certain nuclear ratios, and under certain conditions, the heterokaryon could be expected to have the high penicillin producing ability of the G-type strain and the high growth rate of the W-type strain. However, in a mixture of homokaryotic (W- and G-types) and heterokaryotic (S-type) strains, the rapidly growing W-type would have a detrimental effect on the penicillin producing abilities of the heterokaryotic strain or the G-type strain. Thus, if for any reason the nuclear division rates should change in favor of the W-nuclear type so as to allow it to segregate from the heterokaryon, it would sporulate, pure W-type substrains would develop from those spores containing only W-type nuclei and rundown would result. Several factors indicate that the second mechanism, or possibly a combination of both mechanisms, is the more likely cause of rundown in these strains. The white sectors in S-type colonies always appear on solid media at approximately the same time in individual experiments, and they usually appear in relatively large numbers. Very seldom do they appear before the 9th or 10th day of growth. If the sectors arise as the result of mutations one would expect them to make random appearances during this time. More sectors would be expected in the later stages of growth when the nuclei are more numerous, but some would be expected to appear as early as the second or third day and on all days thereafter unless some unusual selective factors are operative. Instead, the white sectors seem to make their appearance in response to an unknown stimulus occurring at a definite time during clone growth. Moreover, the sectors are nearly always white. Only occasionally were single, dark-green or light-green sectors observed. Some medium change, such as an accumulation of metabolic products or the exhaustion of some nutrient needed for maximum growth of the G-type component, could easily be the causative factor. This is suggested since sectoring can usually be prevented, and always retarded, by transferring colonies to fresh media at 7-day intervals. This behavior would be expected of a balanced heterokaryon in which the W-type nuclear component had a slower growth rate on fresh medium than either the

8 194 F. L. HAAS ET AL. [VOL. 4 heterokaryon or the G-type nuclear component. Under these conditions, mycelia containing only W-type nuclei probably would not develop. If the W-type nuclei could multiply at a faster rate on old media, or if multiplication of G-type nuclei slowed down on it, production of W-type would exceed that of the G-type. When this occurred, the heterokaryon would be out of balance. More and more of the mycelial strands would become homokaryotic for W-type, and white sectors would develop (see Jinks 1952a, 1952b for theory). While growth rates have not been measured by precise methods, it is obvious by inspection that the white colonies and sectors grow at a faster rate than G-type colonies. The white sectors developing in G-type colonies are fan-shaped and Pontecorvo (1946) offers conclusive evidence that mycelia in fan-shaped sectors have a faster growth rate than that of the parent colony. Also white colonies are usually considerably larger than the G-type colonies, and they produce visibly more mycelia in shake flasks than the G-type strains. Occasional G-type cultures have been obtained from the serial reisolation procedures which sector very infrequently, even when they are incubated for 30 days. These cultures have never shown any evidence of rundown. All of these factors indicate that sectors and white colonies arise by breakdown of an established heterokaryon, regardless of the origin of the two nuclear types involved. A number of factors indicate that the W-type nuclei could have arisen in, or prior to, the W strain. Low-yielding W-type and S-type strains and highyielding G-type strains can be isolated in considerable numbers from platings of W Microscopic examination of spores of a number of the ancestral strains indicates the possibility that at least some of the spores are multinucleate. Pontecorvo and Sermonti (1954), using strains derived from Q-176 (an ancestral strain of W50-935), have found that some of the spores of this strain are diploid. Either or both of these conditions, that is, multinucleate spores having nuclei of two or more different types and diploids having two different sets of chromosomes, can produce polymorphous strains. Regardless of the origin of the mixture of strains or whether they are perpetuated by a heterozygous diploid, polynuclear spores, or by a series of highly mutable genes, the experiments demonstrate how heterokaryosis and heterokaryon breakdown operate to bring apout penicillin rundown. They show as well the value of serial reisolation in controlling or eliminating rundown. Many of the experiments reported here have been performed with several strains of Streptomyces with the same results. For example, heterokaryon breakdown has been observed to immediately precede rundown in several different tetracycline-producing strains. In these, however, it is not clear whether stable heterokaryons are poorer producers than pure superior strains, or whether rundown only occurs after segregation of the low-yielding component. Much the same situation appears to exist in Streptomyces as in Penicillium, and for the same reasons. ACKNOWLEDGMENTS The authors wish to express appreciation to Dr. A. B. Hatch and Mr. W. McGhee of Bristol Laboratories Department of Fermentation Development for the tank fermentor runs, and for making periodic samples of these fermentations available to us. We also wish to thank Miss E. Lively of Bristol Laboratories Department of Microbiology Research for making most of the single-spore isolations, and Dr. A. Gourevitch of the same department for many valuable suggestions and criticizing the manuscript. SUMMARY Strain rundown and culture change has been an everpresent problem in industrial fermentations. Studies on these phenomena occurring in Penicillium chrysogenum strains derived from Wisconsin are reported here. Rundown was found to be caused here by the appearance of a low-penicillin-producing white strain in the parent culture. This white derivative, which was very stable, readily formed heterokaryons with pure highpenicillin-producing strains of the green type, and in the balanced heterokaryotic state it did not appear to depress penicillin yields. However, under certain conditions the heterokaryons broke down and the pure white strain segregated out. After this a mixed population developed containing pure white and green clones as well as the heterokaryon. In such cultures, penicillin yields and growth of the green strain were rapidly and drastically depressed. Using white and green substrains derived from the same parent cultures, artificial heterokaryons were synthesized, subjected to the various cultural conditions encountered in industrial fermentations, and heterokaryon effects studied. These experiments show that heterokaryosis and heterokaryon breakdown, following the occurrence of mutation or genetic segregation in a stable culture, can be major causes of culture rundown. Procedures for preventing this type of rundown are given. Various possibilities for the origin of the white variant are discussed. Many of the same heterokaryon experiments have been repeated with certain strains of Streptomyces with the same type of results; therefore it is to be expected that rundown in Streptomyces cultures can also be due to the same causes.

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