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Mutants of Staphylococcus aureus affected in the regulation of exoprotein synthesis
I:I.:MSMicrobiologyLetlers7 (1980) 203-20fi O CopyrightI:cdezationof E~trQpcanMicrobiologicalSocieties Publbhedby F.lsovier/Nozlipllolhm~iBimtttMi~aiPress
203
MUTANTS OF S T A P H Y L O C O C C U S A U R E U S AFFEC'q'ED IN THE REGULATION OF EXOPROTEIN SYNTHESIS ANDERS BJORKLIND and STAFFAN ARVIDSON Department of Bacteriology, Karol[nskaInstituter, Box 60,I00. S-t04 Ol Srockl,otm 60, Swedert
Receivedand accepted22 November1979
1. Introduction
Extracellular proteins from Staphylococcus nitrous are produced mainly after tile end of the exponential ~rowth. Under certain conditions the synthesis of exoproteins may account for as.much as 30% of the total protein synthesis [1]. Some proteins, e.g. staphylococcal serinproteinase (Protease i) [2] and alpha.toxin [1 ] seem to be produced solely during post-exponential growth, whereas others, acid phosphatase [3], staphylococcal metallo. proteinase (Pretense ill) [4], staphy|okinase [3] and DNase [5] are formed throughout the growth cycle, but mainly afterthe exponential growth. In contrast, coagulase [6] and the cell w~-a~sociated Protein A [7] axe formed preferentially during exponential growth. Several authors [8-I 1] have suggested a common regulation of the synthesis of various exo. proteins, mainly based on the isolation of pleiotropic exoprotein negative mutants. Coleman eta]. [l 2] have formulated a regulatory model for those exoproteins which are mainly synthesized during postexponential growth. Findings by Nordstr6m and Lindberg [13] suggest that Protein A, which is produced during exponential growth, and alpha-toxin which is produced during post-exponential growth, are regulated by a common mechanism. This sugges. lion is supported by the present study. Puring growth of& aureus strain V8, in continu,ous cultures, Pretense I negative mutants aocumulated rapidly in the chemostat.Thes e mutants were changed in the formation of most exoptoteins, both those produced during exponential growth and those produced during post-exponential growth. A regulatory
model based on the phenotypes of the mutants is presented. 2. Materials and Methods S, aureus strain V8 [ 14] was cultivated in a
chemostat [3] in a medium containing per litre: casein hydtolysate (Oxoid), 10 g; glycerol, 2.5 g; Nat HPO4 • 2 H~O, i 8; KHiP04, 0.4 g; (NH4)2SO4, 1 g; Dbtryptophan, 80 rag; bcystine, 10O rag; thi. amine, 2 rag; nicotinic acid, 4 roB;CaCIa • 2 !|20, 147 rag; MgSO¢ • 7 HaO, 170 rag; MnSO4. HaO, 7.6 rag; FeS04, 6.4 rag; and citric acid 6.4 rag. The pH was kept constant at 7.0 attd the culture.was suf. ficiently aerated to give an oxygen tension above 70~ saturation throughout the experiment as measured by an oxygen electrode [l 5~. Batch cultivationt were performed in baffled shake flasks in Trypticase Soy Broth (Difco) or CCY medium a~ described [4]. Mutagenesis was done using N:methyl.N*-nitronitrosoguanidine (NG) as a mutagen.
2,1, Assays Growth was determined by dry weight measurements. Proteolytic activity of the cells in growing cultures was~reened on casein afar, In order to dif, ferentiate between colonies produclng~ombinatio~z of the different protelnascs O~oteaze.! and 111)[16] it was found essential to buffer the casein atar at pH 7D with 0,1 M POebuffer, Protea~ i ~ d Proteate |ll in culture supemataMs were determined by ~ecket immunoelectrophoresis [ 16]. Alpha.toxin production
204.
was screened on rabbit blood agar and quantlfted by serial dilutions of culture supernatants using rabbit erythrocytes (1%) as substrata, Staphyloktnase, coagulase and DNase were quantified as described [3,6,17]. Protein A was released from the cells by lysostaphtn and quantified by rocket tmmunoelectrophoresls [18] using dog IgG [191.
of old stock plates tt was found that Pretense I negative mutants also accumulated on agar plates. Mutants Isolated at different occasions were analyzed for different exoproteins (Table l ) and phage type. The phage type of all mutants was Identical to that of the wild-type, As seen In the Table, loss of Prolense I was always coupled to a decrease In the formation of alpha.toxin, Pretense llI, staphyloklnase, DNase, and acid phosphatase and a simultaneous increase In the formation of coagulase and Protein A. The fact that these mutants had accumulated in continuous c~/tiures also means that they have a htgheT growth rate 1hun the wild type. This was verified in' batch cultures, the growth rate of some mutants being as much as twice that of the wild type under i:ertatn conditions (data not shown). The rela. tion between g[owth rate and exoprotein synthesis will be discussed elsewhere. All mutanls isolated could be reverted completely to the wild type in one Step.by, treatment with NG. This shows that the multiple changes of the mutants are due to single mutations. The fact that a single mutation leads to repression of some exoproteins and a simultaneous ¢lerepress'ion of other protdns and that the mutation seems to ~tluence the rate of synthesis of secreted exoprotdns, indicates that a superior/regulatory gone is involved.
3, Results and D ~ u ~ i o n
.?.1. Accumulation of mutants h~ the ehemostat Several continuous cultures at dilution rates ngJng frpm 0,1 to 0.6 were started and maintained g enou~,h to reach steady state (at least ten complete changes of medium in the fermenter). 1n none of these experiments a steady state with respeci io both bacterial growth and Pretense ! formation could be maintained for more than 48 t,. After various lengths of time, depending on th~ dilution rate, the amount of Pretense I started to decrease and reached zero levd after 7-10 generations of growth and simultaneously bacterial dry weight Increased andj reached a new steady state level at the time when Protease I was zero. By plating samples, taken during the interval when Pretense I was going down, it could be shown that the decrease in Proteaso I formation was due to the accumulation of Pretense I negative mutants. These mutants appeared in the cultures even though the inoculum was started from stocks purified by extensive cloning on casein agar. By examination
3.2. Regulatory model Since in the mutants Protein A and coagulase are derepressed while the synthesis of the other axe-
TABLE t Production of exoproteins by mutantt of Staphylococcus ~ureur Vg, ~eIect¢d dudn$ coatlnuout cultivation Each strain was grown for 5 h in the CCY-mediumin baffled shake flasks. The different exoproteins were assayed as described and the relativeamounti per rng bacterial dry weight were calculated. Strain
Pi
a-Toxin
P lit
$AK
DNate
a.pho,
V8 (wild type) K$8:12.4 K58:12-2
tO0 85 0
100 100 0
100 85 5
100 80 14
I00 40 33
1O0 117 78
coaI, 20 16 53
prot. A 0 4 66
V8 S
0
0
4
26
2t
82
53
70
KSg: t2-t KS(i: l
0 0
0 0
0 0
4 4
13 16
30 32
100 100
74 100
SAK, staphylokinale;il.pho., acid phosphatale; P I, Pretense i; P tit Pretense Ill; DNale, desoxyribonudeate; cong., coagulate; prot. A, protein A.
205 proteins are repressed it is assumed that Protein A and coagulase are regulate~lby. regulatoi'y genes coding for represser molecules,The regulation of Protein A by a represser molecule has been suggested by Nordstr6m and Llndberg [13 [, who also found an inversecorrelationbetween Protein A production and the production of certain other exoprotelns (alpha-toxin and beta.toxin). To explain the phenotypes of tile mutants and the wild.type we suggest that a protein (factorEX), in additionto the gNA. polymerase, is needed for the transcription of the represser genes of Protein A and coagulase and the structural genes of the other exoprotelns, Furthermore, to explain the fact that Protein A and coagulase are produced solely during exponential growth whereas the other exoproteins are preferentially produced during postexponential growth we assume that factor EX is activated by a signal substance S (intermediate metabolite, e.g, nucleotide) which is accumulated during post-exponential growth as response to nutrient Hmitation. Factor EX is supposed to be produced throughout the growth cycle. We believe that all the mutants in Table 1 are mutated in the same gene, which affects the amounts of active EX-S complex in the cells. The mutation could either be in factor EX or in some enzyme involved in the synthesis of S. The different patterns of exoprotein production in the mutants can be explained in terms of different affinity between EX-S complex or EX-SRNA polymerase complex and the individual promoter regions,i.e.promoter regionsof Protein A, and coagulase represser genes and tile structural genes of Pretense I, alpha.toxin, Pretense III etc. For example in mutant K68-12-2 and V8S (Table 1) the level of EX-S is supposed to be too low for Protein A represser and Pretense t synthesis to occur but high enough to allow a reduced synthesis of the other exoproteins, while in K68.12-I the level of EX-S is still lower, resulting in the abolishment of Pretense 111 and alpha.toxin synthesis. On the basis of the data in Table 1 it is possible to arrange the different promoter regions with respect to their affinity for EX-S or EX-S-RNA-polymerasecomplex, namely:. Pretense I ~: Alpha-toxin < Pretense I11 < Staphylokinase < DNase < Phosphatase ~ Coagulase represser < Protein A represser.
This order issupported by the order tffappearance of the different exoproteins during growth of tile wild type. Protein A isgenerally produced only during exponential growth 171. Tile same is true for all mutants described (Table I ). In the wild type (strain V8) no Protein A is produced[ This means that the level of EX-S complex ishigh enough to allow the synthesis of protein A represser. However, this level is not high enough to allow synthesis of Pretense l, Prolease illand alpha toxin which are not produced until the end of the exponential growth when the concentration of EX-S is supposed to inc,ease. Protease Ill always appears somewhat earlier than Pretense I which supports the assumption that Protease[ and alpha.toxin promoter regions has the lowest affinity for EX-S/gNA.polymerase complex. Small amounls of staphylokinase, acid phosphalase and DNase are produced during exponential growth but ti{erate of synlllesisincreasesmarkedly during post,exponenti.',lgrowth. The suggested relative affinity between EX-S complex and the promt~tor of coagulase represser gene is based on the fact that coagolase is almost completely repressed in the wild type. The similarity between the regulatory model proposed in this paper and catabolite repression is obvious. However, the cessation of the exponential growth phase and the simuhaneous appearance of Protease I is not correlated to the exhaustion of the main energy source, i.e. glucose, glycerol, glycerophosphate [23]. Furthermore the ~ddition of glucose (1 g per liter) during active Protease i synd,esis had no effect on the differential rat.eofProtease ! synthesis. These results are in agreement with dO" earlier findings that exoprotein synthesis is mainly influenced by the amino acid composition of the medium [21, which means that the co-activator S should be a substance iftfluenced by amino acid metabolism,
Aclmowledllements This work was supported by the Swedhh Medical Research Council (project no, 4513). The skilfull technical astittance of Mrs, Ingrid M6Ueg~rdand Agneta Wahlquist is gratefully acknowledged.
206
Referencei {11[ Ahbas-Al[, B, and Co[¢man, G. (1977) J. Gen. Microbiol. 99, 27"/-282. [21 Bj6Tklind,A. and Arvldson,S. (1978) J. Gem Microblol. 107, 367-375. [3] Arvtdson, S., ilJ6rkiind, A., Erik~on, R. and I lolme, T. (1976) Continuouz culture: Al~plicationand new flold~. Proceedlng,t of fir 6th i~lernational Sympo'qum on Continuous Cuhurcs of Mlc~'oorganbms. pp. 238250. EIIts llorwood, Chlchestcr [4] Arvidson, S., IIolme. T. and Li[ldholm. B. (1972) Acta Palhol. Mlcrobiol. Stand. B80,~835-844. [5] Carpenter, D, and Cbcsbro, W,/(19"/4) Can. J. Micro° biol. 20. 337-345. 16l Engels, W,. Kamps, M. and va~ Boven, C,P.A, (1978) J, Gcn, Microbiol. 109.237-24~1. [71 Movltz. J. 0974) Eur. J. Blocpeq~.48, 131-136. [8 ] I:orsgren, A,, Nordsu~m, K.,~hllip.~on, L. and Sj~quist, L (1971) J. Bactcri61.10"/, 245~-250.
[9] Omenn, G.S, and Friedman, J, (1970) J, Bacterlol, 101, 921-924, [ 10] Yodkawa, M., Malsuda, I:,, Naka, M., Murofoshi, E. and Tsunomatsu, Y. (1974) J. IlacterloL ] 19, 117-122. [ I 1 | Duvai-lflah, Y,, van liefJenoort, |., Rou~oau, M, and Ralbaud, P. (19"/7) ,I, Bnc~erlol, 130, 1281-1291. [12] Coleman, G., Brown, S, and Stormonth, D,A. (1975) J. Theor. BIol. 52, 143-148. [131 Nordltr~m, K. and Llndl~rg, M. (1978) J. Bacwriol. 133,614-620. [14| Gladstone, G,P, and van lleynlgen, W,E. (1957) br, J, Exp. Pathol, 38, 123-137. [15] Bozkowskl, LD, and Johnson, MJ. (1967) Btoteeh. Bioeng, 9, 635-639. [16] Bj~rklind, A. and Arvidspn, S. (1977) Acta Pathot. Mlcroblol. Stand. B85,277-280, 1171 Arvldson, S. and llolme~T. (1971) Aola Pathol. Microbiol. Stand. B79, 406-,413. [ 18] Laur¢il, C.-B. (1966) Anal. Blochem.f15, 45-52. [19] Lind, 1., Live, L and Mansa, B. (1970) Acta PathoL Microbiol. Stand. B78~673-682.
203
MUTANTS OF S T A P H Y L O C O C C U S A U R E U S AFFEC'q'ED IN THE REGULATION OF EXOPROTEIN SYNTHESIS ANDERS BJORKLIND and STAFFAN ARVIDSON Department of Bacteriology, Karol[nskaInstituter, Box 60,I00. S-t04 Ol Srockl,otm 60, Swedert
Receivedand accepted22 November1979
1. Introduction
Extracellular proteins from Staphylococcus nitrous are produced mainly after tile end of the exponential ~rowth. Under certain conditions the synthesis of exoproteins may account for as.much as 30% of the total protein synthesis [1]. Some proteins, e.g. staphylococcal serinproteinase (Protease i) [2] and alpha.toxin [1 ] seem to be produced solely during post-exponential growth, whereas others, acid phosphatase [3], staphylococcal metallo. proteinase (Pretense ill) [4], staphy|okinase [3] and DNase [5] are formed throughout the growth cycle, but mainly afterthe exponential growth. In contrast, coagulase [6] and the cell w~-a~sociated Protein A [7] axe formed preferentially during exponential growth. Several authors [8-I 1] have suggested a common regulation of the synthesis of various exo. proteins, mainly based on the isolation of pleiotropic exoprotein negative mutants. Coleman eta]. [l 2] have formulated a regulatory model for those exoproteins which are mainly synthesized during postexponential growth. Findings by Nordstr6m and Lindberg [13] suggest that Protein A, which is produced during exponential growth, and alpha-toxin which is produced during post-exponential growth, are regulated by a common mechanism. This sugges. lion is supported by the present study. Puring growth of& aureus strain V8, in continu,ous cultures, Pretense I negative mutants aocumulated rapidly in the chemostat.Thes e mutants were changed in the formation of most exoptoteins, both those produced during exponential growth and those produced during post-exponential growth. A regulatory
model based on the phenotypes of the mutants is presented. 2. Materials and Methods S, aureus strain V8 [ 14] was cultivated in a
chemostat [3] in a medium containing per litre: casein hydtolysate (Oxoid), 10 g; glycerol, 2.5 g; Nat HPO4 • 2 H~O, i 8; KHiP04, 0.4 g; (NH4)2SO4, 1 g; Dbtryptophan, 80 rag; bcystine, 10O rag; thi. amine, 2 rag; nicotinic acid, 4 roB;CaCIa • 2 !|20, 147 rag; MgSO¢ • 7 HaO, 170 rag; MnSO4. HaO, 7.6 rag; FeS04, 6.4 rag; and citric acid 6.4 rag. The pH was kept constant at 7.0 attd the culture.was suf. ficiently aerated to give an oxygen tension above 70~ saturation throughout the experiment as measured by an oxygen electrode [l 5~. Batch cultivationt were performed in baffled shake flasks in Trypticase Soy Broth (Difco) or CCY medium a~ described [4]. Mutagenesis was done using N:methyl.N*-nitronitrosoguanidine (NG) as a mutagen.
2,1, Assays Growth was determined by dry weight measurements. Proteolytic activity of the cells in growing cultures was~reened on casein afar, In order to dif, ferentiate between colonies produclng~ombinatio~z of the different protelnascs O~oteaze.! and 111)[16] it was found essential to buffer the casein atar at pH 7D with 0,1 M POebuffer, Protea~ i ~ d Proteate |ll in culture supemataMs were determined by ~ecket immunoelectrophoresis [ 16]. Alpha.toxin production
204.
was screened on rabbit blood agar and quantlfted by serial dilutions of culture supernatants using rabbit erythrocytes (1%) as substrata, Staphyloktnase, coagulase and DNase were quantified as described [3,6,17]. Protein A was released from the cells by lysostaphtn and quantified by rocket tmmunoelectrophoresls [18] using dog IgG [191.
of old stock plates tt was found that Pretense I negative mutants also accumulated on agar plates. Mutants Isolated at different occasions were analyzed for different exoproteins (Table l ) and phage type. The phage type of all mutants was Identical to that of the wild-type, As seen In the Table, loss of Prolense I was always coupled to a decrease In the formation of alpha.toxin, Pretense llI, staphyloklnase, DNase, and acid phosphatase and a simultaneous increase In the formation of coagulase and Protein A. The fact that these mutants had accumulated in continuous c~/tiures also means that they have a htgheT growth rate 1hun the wild type. This was verified in' batch cultures, the growth rate of some mutants being as much as twice that of the wild type under i:ertatn conditions (data not shown). The rela. tion between g[owth rate and exoprotein synthesis will be discussed elsewhere. All mutanls isolated could be reverted completely to the wild type in one Step.by, treatment with NG. This shows that the multiple changes of the mutants are due to single mutations. The fact that a single mutation leads to repression of some exoproteins and a simultaneous ¢lerepress'ion of other protdns and that the mutation seems to ~tluence the rate of synthesis of secreted exoprotdns, indicates that a superior/regulatory gone is involved.
3, Results and D ~ u ~ i o n
.?.1. Accumulation of mutants h~ the ehemostat Several continuous cultures at dilution rates ngJng frpm 0,1 to 0.6 were started and maintained g enou~,h to reach steady state (at least ten complete changes of medium in the fermenter). 1n none of these experiments a steady state with respeci io both bacterial growth and Pretense ! formation could be maintained for more than 48 t,. After various lengths of time, depending on th~ dilution rate, the amount of Pretense I started to decrease and reached zero levd after 7-10 generations of growth and simultaneously bacterial dry weight Increased andj reached a new steady state level at the time when Protease I was zero. By plating samples, taken during the interval when Pretense I was going down, it could be shown that the decrease in Proteaso I formation was due to the accumulation of Pretense I negative mutants. These mutants appeared in the cultures even though the inoculum was started from stocks purified by extensive cloning on casein agar. By examination
3.2. Regulatory model Since in the mutants Protein A and coagulase are derepressed while the synthesis of the other axe-
TABLE t Production of exoproteins by mutantt of Staphylococcus ~ureur Vg, ~eIect¢d dudn$ coatlnuout cultivation Each strain was grown for 5 h in the CCY-mediumin baffled shake flasks. The different exoproteins were assayed as described and the relativeamounti per rng bacterial dry weight were calculated. Strain
Pi
a-Toxin
P lit
$AK
DNate
a.pho,
V8 (wild type) K$8:12.4 K58:12-2
tO0 85 0
100 100 0
100 85 5
100 80 14
I00 40 33
1O0 117 78
coaI, 20 16 53
prot. A 0 4 66
V8 S
0
0
4
26
2t
82
53
70
KSg: t2-t KS(i: l
0 0
0 0
0 0
4 4
13 16
30 32
100 100
74 100
SAK, staphylokinale;il.pho., acid phosphatale; P I, Pretense i; P tit Pretense Ill; DNale, desoxyribonudeate; cong., coagulate; prot. A, protein A.
205 proteins are repressed it is assumed that Protein A and coagulase are regulate~lby. regulatoi'y genes coding for represser molecules,The regulation of Protein A by a represser molecule has been suggested by Nordstr6m and Llndberg [13 [, who also found an inversecorrelationbetween Protein A production and the production of certain other exoprotelns (alpha-toxin and beta.toxin). To explain the phenotypes of tile mutants and the wild.type we suggest that a protein (factorEX), in additionto the gNA. polymerase, is needed for the transcription of the represser genes of Protein A and coagulase and the structural genes of the other exoprotelns, Furthermore, to explain the fact that Protein A and coagulase are produced solely during exponential growth whereas the other exoproteins are preferentially produced during postexponential growth we assume that factor EX is activated by a signal substance S (intermediate metabolite, e.g, nucleotide) which is accumulated during post-exponential growth as response to nutrient Hmitation. Factor EX is supposed to be produced throughout the growth cycle. We believe that all the mutants in Table 1 are mutated in the same gene, which affects the amounts of active EX-S complex in the cells. The mutation could either be in factor EX or in some enzyme involved in the synthesis of S. The different patterns of exoprotein production in the mutants can be explained in terms of different affinity between EX-S complex or EX-SRNA polymerase complex and the individual promoter regions,i.e.promoter regionsof Protein A, and coagulase represser genes and tile structural genes of Pretense I, alpha.toxin, Pretense III etc. For example in mutant K68-12-2 and V8S (Table 1) the level of EX-S is supposed to be too low for Protein A represser and Pretense t synthesis to occur but high enough to allow a reduced synthesis of the other exoproteins, while in K68.12-I the level of EX-S is still lower, resulting in the abolishment of Pretense 111 and alpha.toxin synthesis. On the basis of the data in Table 1 it is possible to arrange the different promoter regions with respect to their affinity for EX-S or EX-S-RNA-polymerasecomplex, namely:. Pretense I ~: Alpha-toxin < Pretense I11 < Staphylokinase < DNase < Phosphatase ~ Coagulase represser < Protein A represser.
This order issupported by the order tffappearance of the different exoproteins during growth of tile wild type. Protein A isgenerally produced only during exponential growth 171. Tile same is true for all mutants described (Table I ). In the wild type (strain V8) no Protein A is produced[ This means that the level of EX-S complex ishigh enough to allow the synthesis of protein A represser. However, this level is not high enough to allow synthesis of Pretense l, Prolease illand alpha toxin which are not produced until the end of the exponential growth when the concentration of EX-S is supposed to inc,ease. Protease Ill always appears somewhat earlier than Pretense I which supports the assumption that Protease[ and alpha.toxin promoter regions has the lowest affinity for EX-S/gNA.polymerase complex. Small amounls of staphylokinase, acid phosphalase and DNase are produced during exponential growth but ti{erate of synlllesisincreasesmarkedly during post,exponenti.',lgrowth. The suggested relative affinity between EX-S complex and the promt~tor of coagulase represser gene is based on the fact that coagolase is almost completely repressed in the wild type. The similarity between the regulatory model proposed in this paper and catabolite repression is obvious. However, the cessation of the exponential growth phase and the simuhaneous appearance of Protease I is not correlated to the exhaustion of the main energy source, i.e. glucose, glycerol, glycerophosphate [23]. Furthermore the ~ddition of glucose (1 g per liter) during active Protease i synd,esis had no effect on the differential rat.eofProtease ! synthesis. These results are in agreement with dO" earlier findings that exoprotein synthesis is mainly influenced by the amino acid composition of the medium [21, which means that the co-activator S should be a substance iftfluenced by amino acid metabolism,
Aclmowledllements This work was supported by the Swedhh Medical Research Council (project no, 4513). The skilfull technical astittance of Mrs, Ingrid M6Ueg~rdand Agneta Wahlquist is gratefully acknowledged.
206
Referencei {11[ Ahbas-Al[, B, and Co[¢man, G. (1977) J. Gen. Microbiol. 99, 27"/-282. [21 Bj6Tklind,A. and Arvldson,S. (1978) J. Gem Microblol. 107, 367-375. [3] Arvtdson, S., ilJ6rkiind, A., Erik~on, R. and I lolme, T. (1976) Continuouz culture: Al~plicationand new flold~. Proceedlng,t of fir 6th i~lernational Sympo'qum on Continuous Cuhurcs of Mlc~'oorganbms. pp. 238250. EIIts llorwood, Chlchestcr [4] Arvidson, S., IIolme. T. and Li[ldholm. B. (1972) Acta Palhol. Mlcrobiol. Stand. B80,~835-844. [5] Carpenter, D, and Cbcsbro, W,/(19"/4) Can. J. Micro° biol. 20. 337-345. 16l Engels, W,. Kamps, M. and va~ Boven, C,P.A, (1978) J, Gcn, Microbiol. 109.237-24~1. [71 Movltz. J. 0974) Eur. J. Blocpeq~.48, 131-136. [8 ] I:orsgren, A,, Nordsu~m, K.,~hllip.~on, L. and Sj~quist, L (1971) J. Bactcri61.10"/, 245~-250.
[9] Omenn, G.S, and Friedman, J, (1970) J, Bacterlol, 101, 921-924, [ 10] Yodkawa, M., Malsuda, I:,, Naka, M., Murofoshi, E. and Tsunomatsu, Y. (1974) J. IlacterloL ] 19, 117-122. [ I 1 | Duvai-lflah, Y,, van liefJenoort, |., Rou~oau, M, and Ralbaud, P. (19"/7) ,I, Bnc~erlol, 130, 1281-1291. [12] Coleman, G., Brown, S, and Stormonth, D,A. (1975) J. Theor. BIol. 52, 143-148. [131 Nordltr~m, K. and Llndl~rg, M. (1978) J. Bacwriol. 133,614-620. [14| Gladstone, G,P, and van lleynlgen, W,E. (1957) br, J, Exp. Pathol, 38, 123-137. [15] Bozkowskl, LD, and Johnson, MJ. (1967) Btoteeh. Bioeng, 9, 635-639. [16] Bj~rklind, A. and Arvidspn, S. (1977) Acta Pathot. Mlcroblol. Stand. B85,277-280, 1171 Arvldson, S. and llolme~T. (1971) Aola Pathol. Microbiol. Stand. B79, 406-,413. [ 18] Laur¢il, C.-B. (1966) Anal. Blochem.f15, 45-52. [19] Lind, 1., Live, L and Mansa, B. (1970) Acta PathoL Microbiol. Stand. B78~673-682.