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Population variations in chemostat cultures of Saccharomyces cerevisiae
313
FEMSMiembioioSyLetters 2 (1977) 313-316 © Copyr~ht Fedm~ttionof EuropeanMlc~robiologlcalSocieties PubllMmdby ~**vlu/North-HollandBiomedicalPre,
POPULATION VARIATIONS IN CHEMOSTAT CULTURES O F S A C C H A R O M Y C E S CEREVI$1AE
J.R. JOHNSTON and A. McKAY DelMetn~e~tof Applicd MIcroblolo&y, University o[ $trathclyde, 204 George Street, Glasgow. GI I XI¢, Scotland
Recelv~i31 August 1977
I. Introduction Despite the upsurge in recent years of the use of continuous culture techniques for studies in microbial physiology and biochemistry, there have been few corresponding genetical experiments. Yet genetical stabiliW during prolonged cultivation of yeast [ I ], and other micro-orf~nimm [2], is of considerable practical importance. Any change in genotype propagated throughout even a proportion of the cultara population may effect the character of cells (or mycelhd biomass) harvested or remit in an altered product whether the latter is under study at the laboratow level or Is being produced industrially. Many wild-type strab's of bacteria probably yield continuous culture populations which are highly pure and stable [3,4]. Even some bacterial cultures composed of several organllmls can be surprisingly ~.table, although long.term establishment of mutants~ with accompanying "enzyme evolution", has been demonstrated [5]. Neverthek~u, mutant strains of bactefla [6] and, recently, grains of the fission yeast ,q~ff,zosaceharomyces pombe [7], have shown inc~n*es in mutant frequency during chemostat growth and them have been used to measure gene mutation rates. "Periodic ,election" of new mutant types may Mac o~eut [81. Continuous cultures of diploid hybrid sttlflns ofSaccharomyces cerevislae hay8 been ~ to be prone to change by the ,election of mitotic zacomidvante [9], and in sam,,,strains ibis ]mpp,ns quickly enoIJsh to lead to stable papa. lations of homozYi~tes after 7-10 days [ 10]. At [oducad tampemtures lmtinent to brewery fermenta. Ilollt, mine I~raln~Illiv~been Ihown highly suscepti.
ble to induction of cytoplasmic respixatory-def~ient. mutants [11]. This paper reports selection of a cell line resist~mt to petite formation at sub-optimal temperatures anti results showing that different population changes may be obtained during chemostat cultures of the same strain depending on the source of the single colony isolate used for the inoculum culture.
2. Materials and Methods 2.L Strain
The diploid strain ofS. cerevisiae used in these experiments was X190, which was bred from Berkeley/Seattle laboratory stocks. Its genotype was: a ade2 o arol ,~.om2 trp4 ade8 [12] a ade2 + + + + 2.2. Continuous cultivation
Cultures were grown in the Controlled Environment Culture Apparatus (CECA) chemostat (Gallenkamp) in MYGPAD medium (0.3% malt extract, 0.3% yeast extract, 1% glucose, 0.5% peptone, 40 mg/l adenine) chosen for its rough equiwalence to brewers' weft and with addition~d adenine to counteract selection of adenine-independent ade2 revertants [13]. 1he working volume in the 5-1 vessel was 2.5 l, the pH 5, temperature 18°C, air throughout 2.5 l/vain and stirrer speed 1300 rpm. The PC2 was not moni-
314 toted due to electrode sterilization difficulties. A 0.I 7 ml vo!ume of a 50 ml/l dilution of silicone anti, foam was added in 24 sec pulses each 9 min. With a medium pump rate of 320 nd/h, giving a dilution rate of 0.13 h -t, glucose was the limiting substrate. lnocula were 10 ml shake flask cultures which were then grown as stirred batch cultures for a few hours prior to commencement of continuous cultivation. At this stage, termed to, cell densities were approx. 107/ml. 2.3. Population analysis Cells of strain XI90 produce red colonies after 3 days growth on MYGP afar plates, pigment being due to the ade2 mutation. Respiratory-deficient (p-) mutants produce smaller, white colonies on MYGP medium and, unlike respiratory-sufficient parent cells, fail to grow on GLY medium [14] in which the carbon source is glycerol. On MYGP medium, this strain produces orange colonies, similar in size to red colonies but capable of only very slow growth on GLY medium. Cells of these orange colonies are cytoplasmic mutants but are genetically unstable and revert at high frequency to respiratory. capable parent cells [ 15 ]. Adenine.independent revertants produce large ~,hite colonies, as do the rela. tively frequent [16] mitotic recombinants homozy. gous for either ads8 alone or both ads8 and trp4. The
r©~ombinants are distlnllulshed from reverttnt8 by their adenine dependence. Chemostat samples were 10 ml and, after ndcroscoplc countlnS and appropriate dilution|, cells were spread onto I0 M Y G P platesto givecolony dandtles of approx. 100 per plate.Cellphenotypk analysis, by colony colour and size,and by replica.platin8onto GLY, mh.'lud (my), my ÷ adenine and my +ade. nine + tryptophan media, was thereforebased upon totals of around 1000 colonies, All individual experiments were repeated at least once to test for reproducibility of results.
3. Remits 3.1. Isolate A !aocolum was Ipown from e Wpkal red colony of strain XI90 sampled after 7 days of continuom culture at 18°C in t stud (250 nil) iuboratow fermenter. Total ~ l l densiw in the CECA r e ~ d 13" lOS/hal after 3 days and remained eontiant for the duration (64 days) of the experiment, l~vmlts of population analysis are ~hown in FIg. 1. Tim proportion of parent cells fell to ~(}-65% within a few days, with a corresponding increase in nurabma of "oranl~" mutants, i~. those inoduelng orar4e colonist. Although there was a subsequent recreate in parent
80
\ Y, 4(
,
' zb ' '~o - - ' /o . . . . m- ; ~ ' Fig. i. Populationanalysisof maixlX190 (isolateA), o, n~l mloWj; s, onmtleoolonles;,, ~
whimoolonios.
315
ce~s to 80~ at 19 days, there was thereafter atteady decline over the next 29 days to a constant levd of 8% over the period 48-64 days, Althoush in part this was due to a second phase of selection for orange mutants, the primary cause was the selective advarttage of cells producing large white colonies. The vast majority of these were mitotic recom0inants, fi;wer th~n I% being adenine.independent revertants.
6o
.~.2. l~ok~te B Inoculum was grown from a typical red colony produced by streaking plates from the stock cu|ture of strahl XI90. Total cell density reached 9 . 107/ml after 3 days, fell t o 4 , 1 0 7 / m l after 8 days and increased again t o 2 . 1 0 S / m l after 18 days. The, remlts (Fig. 2a) show 8 rapid increase in petite mu. t&Its, which comprised the bulk of the population for about 7 days. There was then rapid reselection z~f ceils producing red colonies apparently indisting,uish. able from the original parent cells. There was a]~.o a pl'aum of selection of orange mutants to about '.i8%of the population at 15 days. 3.3. Isolate C lnoculum was [Fown from a typical red colony produced by a sample taken from a CECA culture of isolate B after 15 days growth. The cell density
'0 :s 10 ~s Fig. 3. Comparison of bevelsof parent cells (red colonies) with different cultures of strain XI90. ~,, £solateA; o, isolate B; 6. isolate C. reached 1.6" tOS/ml after 5 days and remained constant. The results (Fig. 2b) show that this inoculum produced a stable population resistant to petite formation during the first 15 days of growth, with only low levels of petite and orange mutz~=ts. Prolonged growth of this culture led to selection of mitotic recombinants in a similar manner to culture of isolate A. 4. Discussion
•
':
i'
I
"
b
'L"e>'
s to 15 Fi~. 2. Popelatk~r,=m=l}'~of '~ttslnX 190 (=, Imh,te B; b. lsokLtaC}. o, red Coienk.; e, orlt;lp colonies; 0, pe¢ltecc,lonles.
The overall patterns of these results with isolates A, B and C were entirely reproducible, although there were quantitative differences in the frequencies of component cell types ~n the population at any given time. There were also variations in the times of appearance and population levels of the observed periodic selections, e.g. in the proportions of orange mutants. Some of the~ changes in the composition of the culture population may be viewed as occurring prior to the establishment of a steady' state viz. fluctuations in cell density during the first 18 days culture of isolate B. Culture of isolate A, however, reaches a constant cell density after 3 days and most of the population variations over the initial 7-week period occur d.rLng an apparent steady state. These results show that very different populations
316 of ti, e same strain may be obtained, at least during the early weeks, depending on the source of the ~noo~lum culture. A comparison of variations in the level o f re0 cells over the initial 25 days is shown in Fig. 3. The pattern obtained with the inoculum derived from the stock culture confirms earlier remits obtained with this and other strains using simple, less sensitively controlled, laboratory chemostats [ 17]. That petite mutants are/nduced at reduced temperatures and that increase in their numbers is not due to their (mdikely) selective advantage over parent cell~ has been shown by experiments with synthesised populations [ 17 ]. Similar induction of petite mutants has been demonstrated in batch cultures [18]. The results obtained with isolate C show that red cells selected subsequent to the initial high level of petite mutants are not identical to parent (stock culture) red cells bu* represent a new line of strain XI90. This selected li;.~edoes not pl'oduce petite mutants upon exposure to reduced temperatures and, as such, is "temperature.stable". This is an example of rapid evolution in chemostat cultures [ 19,20]. Although genetic analysis is still incom. plete, preliminary results suggest that the new line of strain X190 ~arriea a dominant mutation not pres. ent in original cultures. To speculate, this may be a mutation in a regulatory gene involved in formation of mitochondr~ or synthesis of m~tochondrial DNA, with consequent alteration in the temperature sensi. tivity of its protein product. Those commercial brewing strains examined displayed genetic stability with reCOrd to petite forms. tion at reduced temperatures [: 7 ]. Presumably either these brewing strains were originally genetically insert. sitive to induction of respiratory-deficient mutants by sub-optimal temperature or, if originally sensitive, then temperature-stable lincs',','~re naturally selected during early fermentations. Nevertheless, the results repc.rted in this paper could be of significance to the Brewing Industry if other brewin 8 strains, particularly new strains produced by hybridisation, form high levels of petite mutants during continuous ferg.3entation at reduced temperatures.
Admowl~nt We are grateful to the Science Research Council for provision of a grant (B/SR/4923) to support this work.
Referenees [ ! ] Fiechter, A. (1975) In: Methods in Cell Biology,Vol. Xl (Prescott, D.M., ed.), pp. 97-130, [2] Jannuch, H.W.and Mateles, R.I. (1974) In: Advances in Microbialphyl/ology, Vol. 11 (Rose, A.H. and Tempest, D,W. ~I~), AcademicP/eu, London, [3] Hegb~t, D,, EllNVorth,R, and Telnns, R.C. (1956) J. Gen. MicmbioL 14, 601-622. 141 Powell,E.O. (1955) J. Gen. Microbiol. 18, 259-268. |51 Senior, E,, Ball, A,T. and Sitter, J,H. (1976) Nature 263,476-479 [6l Novick, A. and $ziinrd, L. (1950) i~oc. Natl. Aced. Sci. USA 36, 708-719. [71 McAthey,P. and Kilbey, B.J. llerndity (abstract), in press, |8} Atwood, K,C,, Schneider, L,K, and gyan, FJ. (195l) Cold Spring Harbor Syrup. Quant. Biol. 16, 345355. [9] Johnston, J.R. and Daniel J. (1973) Genetics 74o Jupplement, sl 2S. [tO| Johnston, J,R, (1974) Proc, 4th Int, Symp, Yusts, Part I, 173-174. i l l ] Johnston, J.R., Thornton, RJ. and McDemlott, E. (1972) Herndlty 29,130. [ 12] Mortimer, R.K. and llawthome, D.C. (197S) Jn: Methods kJ Cell Bloloig,/,Vol, XI (Ptes~tt, D,M,, ~,), p,, 221-2)3, A~tdemic l~eu, London, i13| Roman, It. (19$6) C.g. Tray. Lab. Cer|sb~l~26, 299-31,1. [14] Ogur, M. and St. JohJi. R. (1956) J. Bt.qerioL 72, 500-504. [ 15] Thornton, RJ., Ltw, E, and JohnSon, l.g, 0969) Antonio van Lc~uw©nboek35, Suppk~nt, C7~C8. [ 161 Thornton, R.J. and J(+hnston, J.R. (19',rl) C~net. Rot. 18,147-|$1. [ 17J Thornton, RJ. (1969) Ph,D. Thefts, U,~ms/t~, of $lralhclyd¢, [ 18] Ol~ur,M,, OlPLr, S, and St, John, R, (1960) General 45,189-194. [19J Silver,R,S. end Matele~,R.I. (1969) J. Dact~ol, 97, 535.-$43. [20) Vsidkamp, H, and Jerome:h,H,W, (1972) J. App), Chore, Biotechnol.22,105-1~3.
FEMSMiembioioSyLetters 2 (1977) 313-316 © Copyr~ht Fedm~ttionof EuropeanMlc~robiologlcalSocieties PubllMmdby ~**vlu/North-HollandBiomedicalPre,
POPULATION VARIATIONS IN CHEMOSTAT CULTURES O F S A C C H A R O M Y C E S CEREVI$1AE
J.R. JOHNSTON and A. McKAY DelMetn~e~tof Applicd MIcroblolo&y, University o[ $trathclyde, 204 George Street, Glasgow. GI I XI¢, Scotland
Recelv~i31 August 1977
I. Introduction Despite the upsurge in recent years of the use of continuous culture techniques for studies in microbial physiology and biochemistry, there have been few corresponding genetical experiments. Yet genetical stabiliW during prolonged cultivation of yeast [ I ], and other micro-orf~nimm [2], is of considerable practical importance. Any change in genotype propagated throughout even a proportion of the cultara population may effect the character of cells (or mycelhd biomass) harvested or remit in an altered product whether the latter is under study at the laboratow level or Is being produced industrially. Many wild-type strab's of bacteria probably yield continuous culture populations which are highly pure and stable [3,4]. Even some bacterial cultures composed of several organllmls can be surprisingly ~.table, although long.term establishment of mutants~ with accompanying "enzyme evolution", has been demonstrated [5]. Neverthek~u, mutant strains of bactefla [6] and, recently, grains of the fission yeast ,q~ff,zosaceharomyces pombe [7], have shown inc~n*es in mutant frequency during chemostat growth and them have been used to measure gene mutation rates. "Periodic ,election" of new mutant types may Mac o~eut [81. Continuous cultures of diploid hybrid sttlflns ofSaccharomyces cerevislae hay8 been ~ to be prone to change by the ,election of mitotic zacomidvante [9], and in sam,,,strains ibis ]mpp,ns quickly enoIJsh to lead to stable papa. lations of homozYi~tes after 7-10 days [ 10]. At [oducad tampemtures lmtinent to brewery fermenta. Ilollt, mine I~raln~Illiv~been Ihown highly suscepti.
ble to induction of cytoplasmic respixatory-def~ient. mutants [11]. This paper reports selection of a cell line resist~mt to petite formation at sub-optimal temperatures anti results showing that different population changes may be obtained during chemostat cultures of the same strain depending on the source of the single colony isolate used for the inoculum culture.
2. Materials and Methods 2.L Strain
The diploid strain ofS. cerevisiae used in these experiments was X190, which was bred from Berkeley/Seattle laboratory stocks. Its genotype was: a ade2 o arol ,~.om2 trp4 ade8 [12] a ade2 + + + + 2.2. Continuous cultivation
Cultures were grown in the Controlled Environment Culture Apparatus (CECA) chemostat (Gallenkamp) in MYGPAD medium (0.3% malt extract, 0.3% yeast extract, 1% glucose, 0.5% peptone, 40 mg/l adenine) chosen for its rough equiwalence to brewers' weft and with addition~d adenine to counteract selection of adenine-independent ade2 revertants [13]. 1he working volume in the 5-1 vessel was 2.5 l, the pH 5, temperature 18°C, air throughout 2.5 l/vain and stirrer speed 1300 rpm. The PC2 was not moni-
314 toted due to electrode sterilization difficulties. A 0.I 7 ml vo!ume of a 50 ml/l dilution of silicone anti, foam was added in 24 sec pulses each 9 min. With a medium pump rate of 320 nd/h, giving a dilution rate of 0.13 h -t, glucose was the limiting substrate. lnocula were 10 ml shake flask cultures which were then grown as stirred batch cultures for a few hours prior to commencement of continuous cultivation. At this stage, termed to, cell densities were approx. 107/ml. 2.3. Population analysis Cells of strain XI90 produce red colonies after 3 days growth on MYGP afar plates, pigment being due to the ade2 mutation. Respiratory-deficient (p-) mutants produce smaller, white colonies on MYGP medium and, unlike respiratory-sufficient parent cells, fail to grow on GLY medium [14] in which the carbon source is glycerol. On MYGP medium, this strain produces orange colonies, similar in size to red colonies but capable of only very slow growth on GLY medium. Cells of these orange colonies are cytoplasmic mutants but are genetically unstable and revert at high frequency to respiratory. capable parent cells [ 15 ]. Adenine.independent revertants produce large ~,hite colonies, as do the rela. tively frequent [16] mitotic recombinants homozy. gous for either ads8 alone or both ads8 and trp4. The
r©~ombinants are distlnllulshed from reverttnt8 by their adenine dependence. Chemostat samples were 10 ml and, after ndcroscoplc countlnS and appropriate dilution|, cells were spread onto I0 M Y G P platesto givecolony dandtles of approx. 100 per plate.Cellphenotypk analysis, by colony colour and size,and by replica.platin8onto GLY, mh.'lud (my), my ÷ adenine and my +ade. nine + tryptophan media, was thereforebased upon totals of around 1000 colonies, All individual experiments were repeated at least once to test for reproducibility of results.
3. Remits 3.1. Isolate A !aocolum was Ipown from e Wpkal red colony of strain XI90 sampled after 7 days of continuom culture at 18°C in t stud (250 nil) iuboratow fermenter. Total ~ l l densiw in the CECA r e ~ d 13" lOS/hal after 3 days and remained eontiant for the duration (64 days) of the experiment, l~vmlts of population analysis are ~hown in FIg. 1. Tim proportion of parent cells fell to ~(}-65% within a few days, with a corresponding increase in nurabma of "oranl~" mutants, i~. those inoduelng orar4e colonist. Although there was a subsequent recreate in parent
80
\ Y, 4(
,
' zb ' '~o - - ' /o . . . . m- ; ~ ' Fig. i. Populationanalysisof maixlX190 (isolateA), o, n~l mloWj; s, onmtleoolonles;,, ~
whimoolonios.
315
ce~s to 80~ at 19 days, there was thereafter atteady decline over the next 29 days to a constant levd of 8% over the period 48-64 days, Althoush in part this was due to a second phase of selection for orange mutants, the primary cause was the selective advarttage of cells producing large white colonies. The vast majority of these were mitotic recom0inants, fi;wer th~n I% being adenine.independent revertants.
6o
.~.2. l~ok~te B Inoculum was grown from a typical red colony produced by streaking plates from the stock cu|ture of strahl XI90. Total cell density reached 9 . 107/ml after 3 days, fell t o 4 , 1 0 7 / m l after 8 days and increased again t o 2 . 1 0 S / m l after 18 days. The, remlts (Fig. 2a) show 8 rapid increase in petite mu. t&Its, which comprised the bulk of the population for about 7 days. There was then rapid reselection z~f ceils producing red colonies apparently indisting,uish. able from the original parent cells. There was a]~.o a pl'aum of selection of orange mutants to about '.i8%of the population at 15 days. 3.3. Isolate C lnoculum was [Fown from a typical red colony produced by a sample taken from a CECA culture of isolate B after 15 days growth. The cell density
'0 :s 10 ~s Fig. 3. Comparison of bevelsof parent cells (red colonies) with different cultures of strain XI90. ~,, £solateA; o, isolate B; 6. isolate C. reached 1.6" tOS/ml after 5 days and remained constant. The results (Fig. 2b) show that this inoculum produced a stable population resistant to petite formation during the first 15 days of growth, with only low levels of petite and orange mutz~=ts. Prolonged growth of this culture led to selection of mitotic recombinants in a similar manner to culture of isolate A. 4. Discussion
•
':
i'
I
"
b
'L"e>'
s to 15 Fi~. 2. Popelatk~r,=m=l}'~of '~ttslnX 190 (=, Imh,te B; b. lsokLtaC}. o, red Coienk.; e, orlt;lp colonies; 0, pe¢ltecc,lonles.
The overall patterns of these results with isolates A, B and C were entirely reproducible, although there were quantitative differences in the frequencies of component cell types ~n the population at any given time. There were also variations in the times of appearance and population levels of the observed periodic selections, e.g. in the proportions of orange mutants. Some of the~ changes in the composition of the culture population may be viewed as occurring prior to the establishment of a steady' state viz. fluctuations in cell density during the first 18 days culture of isolate B. Culture of isolate A, however, reaches a constant cell density after 3 days and most of the population variations over the initial 7-week period occur d.rLng an apparent steady state. These results show that very different populations
316 of ti, e same strain may be obtained, at least during the early weeks, depending on the source of the ~noo~lum culture. A comparison of variations in the level o f re0 cells over the initial 25 days is shown in Fig. 3. The pattern obtained with the inoculum derived from the stock culture confirms earlier remits obtained with this and other strains using simple, less sensitively controlled, laboratory chemostats [ 17]. That petite mutants are/nduced at reduced temperatures and that increase in their numbers is not due to their (mdikely) selective advantage over parent cell~ has been shown by experiments with synthesised populations [ 17 ]. Similar induction of petite mutants has been demonstrated in batch cultures [18]. The results obtained with isolate C show that red cells selected subsequent to the initial high level of petite mutants are not identical to parent (stock culture) red cells bu* represent a new line of strain XI90. This selected li;.~edoes not pl'oduce petite mutants upon exposure to reduced temperatures and, as such, is "temperature.stable". This is an example of rapid evolution in chemostat cultures [ 19,20]. Although genetic analysis is still incom. plete, preliminary results suggest that the new line of strain X190 ~arriea a dominant mutation not pres. ent in original cultures. To speculate, this may be a mutation in a regulatory gene involved in formation of mitochondr~ or synthesis of m~tochondrial DNA, with consequent alteration in the temperature sensi. tivity of its protein product. Those commercial brewing strains examined displayed genetic stability with reCOrd to petite forms. tion at reduced temperatures [: 7 ]. Presumably either these brewing strains were originally genetically insert. sitive to induction of respiratory-deficient mutants by sub-optimal temperature or, if originally sensitive, then temperature-stable lincs',','~re naturally selected during early fermentations. Nevertheless, the results repc.rted in this paper could be of significance to the Brewing Industry if other brewin 8 strains, particularly new strains produced by hybridisation, form high levels of petite mutants during continuous ferg.3entation at reduced temperatures.
Admowl~nt We are grateful to the Science Research Council for provision of a grant (B/SR/4923) to support this work.
Referenees [ ! ] Fiechter, A. (1975) In: Methods in Cell Biology,Vol. Xl (Prescott, D.M., ed.), pp. 97-130, [2] Jannuch, H.W.and Mateles, R.I. (1974) In: Advances in Microbialphyl/ology, Vol. 11 (Rose, A.H. and Tempest, D,W. ~I~), AcademicP/eu, London, [3] Hegb~t, D,, EllNVorth,R, and Telnns, R.C. (1956) J. Gen. MicmbioL 14, 601-622. 141 Powell,E.O. (1955) J. Gen. Microbiol. 18, 259-268. |51 Senior, E,, Ball, A,T. and Sitter, J,H. (1976) Nature 263,476-479 [6l Novick, A. and $ziinrd, L. (1950) i~oc. Natl. Aced. Sci. USA 36, 708-719. [71 McAthey,P. and Kilbey, B.J. llerndity (abstract), in press, |8} Atwood, K,C,, Schneider, L,K, and gyan, FJ. (195l) Cold Spring Harbor Syrup. Quant. Biol. 16, 345355. [9] Johnston, J.R. and Daniel J. (1973) Genetics 74o Jupplement, sl 2S. [tO| Johnston, J,R, (1974) Proc, 4th Int, Symp, Yusts, Part I, 173-174. i l l ] Johnston, J.R., Thornton, RJ. and McDemlott, E. (1972) Herndlty 29,130. [ 12] Mortimer, R.K. and llawthome, D.C. (197S) Jn: Methods kJ Cell Bloloig,/,Vol, XI (Ptes~tt, D,M,, ~,), p,, 221-2)3, A~tdemic l~eu, London, i13| Roman, It. (19$6) C.g. Tray. Lab. Cer|sb~l~26, 299-31,1. [14] Ogur, M. and St. JohJi. R. (1956) J. Bt.qerioL 72, 500-504. [ 15] Thornton, RJ., Ltw, E, and JohnSon, l.g, 0969) Antonio van Lc~uw©nboek35, Suppk~nt, C7~C8. [ 161 Thornton, R.J. and J(+hnston, J.R. (19',rl) C~net. Rot. 18,147-|$1. [ 17J Thornton, RJ. (1969) Ph,D. Thefts, U,~ms/t~, of $lralhclyd¢, [ 18] Ol~ur,M,, OlPLr, S, and St, John, R, (1960) General 45,189-194. [19J Silver,R,S. end Matele~,R.I. (1969) J. Dact~ol, 97, 535.-$43. [20) Vsidkamp, H, and Jerome:h,H,W, (1972) J. App), Chore, Biotechnol.22,105-1~3.