Differential Activity Staining

Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, 4914-4917, December 1975 Biochemistry

Differential activity staining: Its use in characterization of guanylyl- specific ribonuclease in the genus Ustilago (gel electrophoresis/nucleic acid-dye interaction/fungal taxonomy/agrostology/phytopathology) A. BLANK AND CHARLES A. DEKKER Department of Biochemistry, University of California, Berkeley, Calif. 94720 Communicated by Howard K. Schachman, October 8, 1975

ABSTRACT Guanylyl-specific ribonuclease can be iden- eight other members of the genus, including six parasites of tified by a novel technique employing electrophoresis in major food crops (12). We have developed a novel method polyacrylamide slabs followed by differential activity staining. The technique requires as little as 7 ng of enzyme which for identification of these enzymes in unfractionated culture may be grossly admixed with contaminants, including other media; the method, involving differential activity staining ribonucleases. following polyacrylamide gel electrophoresis, permits estab- Upon electrophoresis and activity staining, a variety of ri- lishment of their base specificity without prior purification. bonucleases can be visualized as light or clear bands in a colored background formed by toluidine blue complexed with EXPERIMENTAL PROCEDURE oligonucleotide substrate. Guanylyl-specific ribonuclease, which is detectable when using an oligonucleotide substrate Materials. Cultures of U. avenae, U. bullata, U. hordei,- of random base sequence, does not yield a band when using U. kolleri, U. nigra, U. nuda, and U. tritici were the gra- oligonucleotides bearing guanylyl residues at the 3'-termini cious gift of Dr. Jens Nielsen. U. maydis, U. halophiloides, only and containing, therefore, no susceptible internucleo- and U. cynodontis were germinated from teliospores ob- tide bonds; in contrast, a ribonuclease with a different base specificity or no base specificity yields a band with either tained from Dr. Richard Mower. U. sphaerogena was substrate. This differential activity staining method for es- cloned from a culture provided by Dr. J. B. Neilands. U. vio- tablishing guanylyl specificity permits estimation of the ex- laceae was obtained from Mr. M. Baird. RNases T1, T2, and tent of nonspecific cleavage of internucleotide linkages by a U2 were Sankyo products. RNase N1 and RNase A (5X crys- putatively guanylyl-specific enzyme and is at least as sensi- tallized) were obtained from Sigma. RNases U1 (13) and U4B tive as conventional procedures for determination of base (14) were prepared in this laboratory. Union Carbide seam- specificity. With this new technique guanyloribonuclease has been less cellulose tubing (1 inch diameter), boiled just prior to identified in the unfractionated culture medium of 10 organ- use, was employed for dialysis. isms belonging to the phytopathogenic fungal genus Ustila- Assays. Ribonuclease activity was measured in our stan- go. It is suggested that guanylyl-specific ribonuclease is dard assay (11) in which RNase U1 has a specific activity of widely distributed among Ustilago species; its electrophoret- 300,000 units/mg dry weight (13). ic properties may be revealing of phylogenetic relationships Electrophoresis. Electrophoresis was carried out in 12.5% among these plant parasites and among their hosts. The general technique of differential activity staining, de- polyacrylamide slabs at 30 mA for 90 min at 40 as described veloped for determination of the base specificity of ribonuc- by Ames (15) except that sodium dodecyl sulfate and mer- leases, may be widely applicable to analysis of enzymes cata- captoethanol were omitted from all solutions. For electro- lyzing depolymerization reactions. phoresis of RNase A only, a low pH acetate-3-alanine buffer system (16) was employed. Since discovery of the classic RNase T1, guanylyl-specific ri- Differential Activity Staining. Following electrophoresis, bonucleases have been found in bacteria, in the three classes gel slabs were sliced into a right and left half, each half of fungi, and in a slime mold (1-5). Most of these enzymes bearing identical samples. Activity staining was carried out are extracellular proteins of molecular weight about 11,000; at room temperature at pH 7.0 using 0.1 M Tris-HCl, or at the amino-acid composition of six (1-4) and the amino-acid pH 5.0 using 0.1 M sodium acetate. Gels were incubated for sequence of two of them (1, 6) have been reported. Although 5 min in buffer, for 15 min in buffer containing oligonucleo- they are most widely known as an analytical tool for deter- tide substrate (50 A260 units*/ml, about 35 ml total volume), mination of nucleotide sequence in RNA (1, 7), the guany- for 2 min in buffer, for 5 min in 0.2% Toluidine Blue 0 lyl-specific ribonucleases are highly interesting in their own (Eastman) in 0.5% acetic acid, and for 30 min in several right. On-going studies of these enzymes include analysis of changes of 0.5% acetic acid. Incubations were carried out in their mechanism of action (1) and comparison, at a basic 14 cm glass petri dishes. Gels were stored in water at 20. level, with that of ribonuclease A (8); determination of struc- Preparation of Oligonucleotide Substrates. Oligonucleo- ture-function relationships among the various G-specific tides containing guanylyl residues only at the 3'-termini RNases and the related, purine-preferring RNase U2 (9); and were generated by exhaustive enzymatic digestion of high- investigation of their distribution, biological function, and molecular-weight wheat germ RNA prepared by a modifi- regulation (10, 11). cation of the procedure of Singh and Lane (17). RNA (330 Among known guanylyl-specific ribonucleases are two ex- A260 units/ml) was incubated 42 hr at room temperature creted by members of the phytopathogenic fungal genus Us- with 12 ,g/ml of RNase U1 in sterile 0.06 M Tris-HCl, pH tilago-RNase Ui from U. sphaerogena and an enzyme 7.5; a few drops of CHC13 were included; KOH was added from the corn smut U. maydis (1, 10). We report here that extracellular guanylyl-specific ribonuclease is produced by - * One A260 unit is that amount giving an A260 of 1 when dissolved Abbreviation: G-specific, guanylyl-specific. in 1 ml and the light path is 1 cm. 4914 Downloaded by guest on September 28, 2021 Biochemistry: Blank and Dekker Proc. Nat. Acad. Sci. USA 72 (1975) 4915 as necessary to maintain pH between 7 and 8. The digest Guanyloribonuclease Digest Alkaline Hydrolysate was dialyzed versus 4 liters of distilled water at 20 for 48 hr with one change of dialysis medium. Oligonucleotides having an average chain length (n) of 19.1 were prepared by dialysis, as described above, of 20 ml of sodium ribonucleate (Schwarz Bioresearch lot NHS 6402, about 30 mg/ml, titrated to pH 7.1 with KOH). Shorter oligonucleotides (n = 2.2-9.2) were prepared by partial alkaline hydrolysis (18) of sodium ribonucleate (ICN-Nutri- tional Biochemicals lot 3990); the latter material too was oligomeric, having an average chain length after dialysis even lower than that of the Schwarz product. The starting material was dissolved, with the aid of KOH, to give a 1% solution, pH 6.5-6.9. Aliquots were made 0.3 N in KOH and incubated at 28° for 2-20 min. Following neutralization with ice- cold HCI04, or preferably with a premeasured amount of Dowex 50 X8(H+), 50-100 mesh (Bio-Rad Laboratories), hy- drolysates were Iyophilized. Fractions of various average lengths were obtained by chromatography of 5 ml of redis- FIG. 1. Differential activity staining of guanyloribonuclease. solved hydrolysate on a 2.6 X 12 cm column of Bio-Gel P2 Following electrophoresis the gel slab was sliced into a right and equilibrated with 0.1 M NH4HCO3, followed by left half bearing identical samples. Activity staining was carried (Bio-Rad) out at pH 5.0 to accommodate the acidic pH optima of RNases T2 Iyophilization. We have not determined whether the prod- and U2. The slab on the left was stained using oligonucleotides uct marketed by numerous suppliers as sodium ribonucleate from an exhaustive, dialyzed RNase U1 digest of RNA (A260 = is routinely composed of oligonucleotides; use of any partic- 49.5, average oligomer length = 5.6). The slab on the right was ular lot of such material for generation of oligomers of vari- stained using oligonucleotides generated by partial alkaline hy- ous average lengths should be initiated by determination of drolysis and gel filtration (A260 = 50.2, average oligomer length = and pilot alkaline hydrolyses (18) and fraction- 5.1). Samples are: 1, RNase T1 (10 ng); 2, RNase T1 (10 ng) and chain length, RNase T2 (0.1 units as specified by supplier); 3, RNase N1 (1 unit ation. as assayed in ref. 11); 4, RNase U1 (33 ng); 5, RNase U1 (33 ng) and For determination of average chain length, oligonucleo- RNase U2 (0.3 units as specified by supplier); 6, RNase U1 (33 ng) tide preparations (10-30 A260 units/ml in 0.05 M Tris-HCI, and RNase U4B (19 units as assayed in ref. 11). pH 9.0) were treated with bacterial alkaline phosphatase (Worthington, electrophoretically purified, 200 jg/ml) for 1 other cultures. After 5 days, activity in the U. nuda medium hr and for 2 hr. The preparations were then hydrolyzed with was too low to measure reliably (about 1 unit/ml); activity in 0.3 N KOH at 300 for 17 hr. Following neutralization with the U. tritic medium had reached 260 units/ml. Aliquots of HC04,-aliquots were chromatographed for 8 hr in 70% iso- culture medium, taken at the plateau of ribonuclease activi- propanol with 0.35 ml of NH3 per liter of air space (19) on ty, or at 5 days for U. bullata, U. nuda, and U. trtici, were Whatman 3MM paper prechromatographed in the same sol- applied to the gel shown in Fig. 2. vent. Areas containing nucleosides and nucleotides, as well as the corresponding areas in a blank lane, were cut out RESULTS AND DISCUSSION under UV light, weighed, and eluted with 0.1 N HCI. The staining Am#j due to nucleosides and to nucleotides in each sample Differential activity was determined after correction for absorbance of a blank of Activity stains for ribonuclease have employed toluidine the same weight. The average chain length of preparations blue and methylene blue, basic polyaromatic dyes which given by 1 and by 2 hr of treatment with phosphomonoest- bind to RNA, permitting visualization of ribonuclease activi- erase differed at most by 15%; an average of both determi- ty as light or clear areas in a colored background formed by nations is given throughout. /I RNA-dye complex (20). We have observed that toluidine Growth of Organisms and Production of Extracellular blue complexes with small oligonucleotides (n 2 about 4) as Ribonuclease. Log phase sporidia grown at room tempera- well as with RNA, and that oligonucleotides of average ture in minimal medium (0.1 M NH4Cl, 11.5 mM K2HPO4, chain length, n, as low as 5.1 are suitable substrates for use 59 mM sucrose, 0.52 mM citric acid, 6.0 AM thiamine, 3.2 in an activity staining procedure employing toluidine blue. mM MgSO4, 0.29 mM FeC13, 30 AM ZnSO4, 80 nM CUCI2) These observations permitted development of a novel meth- were harvested by centrifugation and resuspended at an od for determination of the base specificity of ribonucleases. OD445 of about 20 in double strength, nitrogen-free minimal The technique, which we call differential activity staining, medium. Following 3 hr of incubation on a rotary shaker, an is based on the following principle: Use in the activity stain equal volume of 1.0% oligoribonucleotides (ICN-Nutritional of an oligonucleotide substrate of random base sequence Biochemicals sodium ribonucleate lot 3990, adjusted to pH permits visualization of all those ribonucleases capable of 7.5 with KOH) was added. Extracellular ribonuclease further degrading the substrate; use of oligonucleotides gen- reached a plateau of 60-850 units/ml (except in the U. bul- erated by exhaustive digestion of RNA with a base-specific lata culture) between 30 and 100 hr after addition of oligo- ribonuclease selectively prevents visualization of ribonuc- nucleotides; activity in the U. bullata medium accumulated leases having that, same base specificity. Characterization of exponentially for 5 days to a level of 20 units/ml. U. nuda guanyloribonuclease by differential activity staining with and U. tritici were grown in 0.7% potato extract, 0.7% malt oligonucleotides of nearly random base sequence (n = 5.1) extract, 0.03% peptone, 2% sucrose; large pieces of myceli- and with oligonucleotides containing guanylyl residues only um were then transferred to nitrogen-free medium and to at the 3'-termini (n = 5.6) is illustrated in Fig. 1. Shown oligonucleotide-containing medium as described for the there is the electrophoretic and staining pattern of the well- Downloaded by guest on September 28, 2021 4916 Biochemistry: Blank and Dekker Proc. Nat. Acad. Sci. USA 72 (1975) characterized G-specific enzymes, T1, U1, and N1, the purine-preferring endonuclease U2, the nonspecific endonuclease T2, and the exonuclease U4 (1, 14). Failure of the G-specific RNases to appear in the left half of the gel is a function solely of the base sequence of the substrate, since- all other conditions of electrophoresis and activity staining are essen- tially the same. The large difference in electrophoretic mobility of RNases Ti and U1 (both acidic proteins) and the similarity in mobility of RNases U1 and N1 (the latter a neu- tral protein) presumably reflects varying fractional protona- tion of protein amino groups in the high pH buffer system. Repeated failure of RNase T2 to form well delineated bands may be due to microheterogeneity within the two known, separable entities which have been designated T2A and T2B (21). That oligonucleotides of short chain length complex with toluidine blue to form a colored background in polyacrylamide slabs is not unexpected in view of the fact that the pri- FIG. 2. Extracellular ribonuclease of Ustilago species. Activity staining was carried out at pH 7 using an oligonucleotide substrate mary interaction of the dye with nucleic acids is thought to of average chain length 19.1. Samples are: 1,. U. avenae A27-1 (1 be electrostatic (22); long-term retention of dye-oligomer unit); 2, U. bullata B3-1 (1 unit); 3, U. hordei H8-3 (1 unit); 4, U. complexes in the gel matrix may be the result of their associ- kolleri K11-4S (1 unit); 5, U. nigra Ng17-4 (1 unit); 6, U. tritici ation in large aggregates. We have observed that oligonu- T3-1 (1 unit); 7, U. nuda Nd22-2 (< about 0.03 unit); 8, U. maydis cleotide preparations of average chain length 4 to 19 give 6 (6 units); 9, U. sphaerogena F7-9 (4 units); 10, U. cynodontis (2 backgrounds of about the same intensity on carefully con- units); 11, U. halophiloides (3 units). trolled staining with toluidine blue. Thus it seems unlikely that clearing of bands by ribonucleases, particularly those ficity in the test enzyme. We can easily observe 0.01 unit of having oligomeric end products, is based solely on the de- RNase A [measured in our standard assay (11)] upon activity crease in size, per se, of fragments produced on degradation. staining at pH 7 using a guanyloribonuclease digest; in fact, Reduction in intensity of staining in areas where ribonucle- a few thousandths of a unit is detectable. Conservatively ase concentration is high may reflect, as well, enhanced dif- then, differential activity staining at pH 7 of, e.g., 1 unit of fusional mobility of enzymatic degradation products relative presumably guanylyl specific ribonuclease, permits the esti- to intact substrate. Thus, if during the period of exposure of mate that less than 1% of the total activity is directed against gel to oligonucleotide substrate nearly all the initial oligonu- internucleotide linkages that do not contain guanosine-3'- cleotide diffusing into an area of electrophoretically banded phosphoryl groups, and that therefore guanylyl preference ribonuclease undergoes degradation, i.e., if the RNase activi- of the test enzyme is 99% or greater. Obviously, application ty is sufficiently high, and if degradation products diffuse of 10 units of enzyme to gels (corresponding to 33 ng of away from the area faster than substrate diffuses in, the con- RNase U1) would suffice to estimate guanylyl specificity at centration difference alone would be expected to yield a the 99.9% level. Apparently, differential activity staining is band of reduced intensity even if end products are them- as effective in establishing base specificity as the commonly selves dye-binding oligomers. Should the clearing of bands employed procedures involving sequential ribonuclease, be in fact based on such differences in diffusional mobility, phosphomonoesterase, and base hydrolysis of RNA followed other enzymes that degrade polymeric substrates (e.g., car- by chromatographic analysis of nucleosides. bohydrases) might be visualized by an analogous procedure using a dye or stain which need not discriminate substrate Extracellular RNase of Ustilago species and product(s). We have used differential activity staining to screen for pro- Failure of base-specific ribonuclease, detectable on activi- duction of extracellular guanylyl specific ribonuclease by ty staining with oligomers of approximately random base se- twelve fungi from the genus Ustilago. The electrophoretic quence, to yield a band upon staining with oligonucleotides pattern of the ribonucleases excreted (in the presence of derived from exhaustive digestion of RNA with an RNase of RNA as a sole nitrogen source) by 11 species is shown in Fig. the same base specificity has been observed for a pyrimi- 2. To facilitate comparison, RNase(s) from the culture medi- dine-specific enzyme (pancreatic RNase A) in addition to um of all organisms is shown in a single gel stained using the guanylyl specific RNases (our unpublished data). The oligonucleotides (n = 19.1) of approximately random base general technique of differential activity staining may, in sequence. Not shown are differentially stained pairs of half fact, be applicable to analysis of a variety of enzymes that gels analogous to those of Fig. 1; in these gels the major or degrade polymers. sole band in every sample failed to appear upon staining To estimate the degree of inherent nonspecificity detect- using oligonucleotides generated by exhaustive digestion of able in a hypothetical ribonuclease having strong preference RNA with guanylyl-specific ribonuclease. Bands of lower for guanylyl residues, we determined the smallest quantity mobility in the four right-hand lanes of Fig. 2 appeared the of pancreatic ribonuclease A which could be visualized upon same in both halves of the gel pairs, as did the nonspecific ri- activity staining using oligonucleotides generated by exhaus- bonucleases used as a control. tive digestion with guanyloribonuclease. Having defined this We conclude that guanylyl-specific ribonuclease is excret- lower limit of detectable activity that is not guanylyl specif- ed by ten, and probably all, of the organisms listed in Fig. 2. ic, and knowing the total activity of any putatively G-specif- Since 1 unit or more' of guanyloribonuclease from each ic enzyme subjected to differential activity staining, we are species (except U. nuda) was applied to gels, guanylyl pref- able to estimate by proportion an upper limit for nonspeci- erence is estimated to be greater than 99%. In the case of U. Downloaded by guest on September 28, 2021 Biochemistry: Blank and Dekker Proc. Nat. Acad. Sci. USA 72 (1975) 4917 nuda RNase, whose activity in the culture medium (corre- enzyme, even at basal levels. Of possible significance in this sponding to roughly 3 ng/ml of RNase U1) was toIo' 4to regard, U. violaceae (anther smut) parasitizes nongramina- assay reliably, guanylyl preference is estimated to be at least ceous hosts, whereas the other organisms are parasites of 67%; in view of the identity of its electrophoretic mobility grasses (12). Whether the ribonucleases of these other species with that of guanyloribonuclease from closely related are excreted in host plants, perhaps contributing to viru- species, this enzyme too is probably G-specific. Though lence, or whether synthesis of extracellular RNase is limited poorly illustrated in Fig. 2, U. maydis (well 8) displays a to periods of saprophytic growth has not been definitively minor activity (traveling very slightly behind the major en- established. However, preliminary evidence (T. A. McKeon zyme) which fails to appear on staining using a guanylori- and C. A. Dekker, unpublished data) indicates that three of bonuclease digest; difficulty in determining the quantity of the extracellular RNases produced by U. maydis in axenic this satellite activity applied to gels precludes estimation of culture are present in U. maydis-infected corn. its guanylyl preference. Several of the organisms analyzed excrete ribonucleases which are not G-specific and which We thank Kathleen Franks for technical assistance in prelimi- have reduced mobility relative to the guanyloribonucleases. nary experiments and Don Harvey of Scientific Photography Labo- ratory for expert photography of gels. Tom McKeon and Rick These companion activities may be similar to the U4 RNases Mower provided continuing interest and helpful information con- of U. sphaerogena (14), seen in Fig. 2 (well 9) as slow mov- cerning the test organisms. This work was supported in part by ing bands. Grant GB-38381 from the National Science Foundation. The electrophoretic mobility of guanyloribonuclease may be revealing of phylogenetic relationships among the patho- 1. Uchida, T. & Egami, F. (1971) in The Enzymes, ed. Boyer, P. gens which excrete it and among the hosts with which the D. (Academic Press, New York), Vol. IV, pp. 205-250. pathogens have presumably coevolved. The species listed in 2. Yoshida, N., Inoue, H., Sasaki, A. & Otsuka, H. (1971) Bio- Fig. 2 are parasites of grasses (family Gramineae). The very chim. Blophys. Acta 228, 636-647. similar electrophoretic mobility of guanyloribonuclease 3. Glitz, D. G., Angel, L. & Eichler, D. C. (1972) Biochemistry from U. avenae, U. bullata, U. hordei, U. nigra, U. nuda, 11, 1746-1754. and U. tritid is noteworthy in view of the relatedness of 4. Fletcher, P. L., Jr. & Hash, J. H. (1972) Biochemistry 11, these organisms (12, 23) and of their hosts (24), which are 4274-4285. 5. Omori, A., Sato, S. & Tamiya, N. (1972) Biochim. Biophys. classified in the subfamily Festucoideae in classical and in Acta 268, 125-131. modern taxonomic systems. Interestingly, guanyloribonu- 6. Hashimoto, J. & Takahashi, K. (1974) J. Biochem. (Tokyo) 76, clease from U. halophiloides and U. cynodontis, whose 1359-1361. hosts have been moved by modern taxonomists from the 7. Paddock, G. V., Heindell, H. C. & Salser, W. (1974) Proc. Nat. Festucoideae to the newly created subfamily Eragros- Acad. Sci. USA 71,5017-5021. toideae, resembles in mobility that from U. sphaerogena, 8. Richards, F. M. & Wyckoff, H. W. (1971) in The Enzymes, whose host is placed in the subfamily Panicoideae in both ed. Boyer, P. D. (Academic Press, New York), Vol. IV, pp. traditional and new systematics. Here then may be addition- 647-806. al evidence of the putatively intermediate nature of the Era- 9. Sato, S. & Uchida, T. (1975) Biochem. J. 145, 353-360. 10. McKeon, T. A., Blank, A., Franks, K. E. & Dekker, C. A. grostoideae, which though festucoid in external morpholo- (1975) Fed. Proc. 34, 701. gy, as noted by classicists, are panicoid according to newly 11. Holloman, W. K. & Dekker, C. A. (1971) Proc. Nat. Acad. Sci. developed microanatomical, cytogenetic, and biochemical USA 68,2241-2245. criteria (24). U. maydis, whose guanyloribonuclease(s) dis- 12. Fischer, G. W. & Holton, C. W. (1957) Biology and Control plays distinctive mobility, parasitizes a panicoid host belong- of the Smut Fungi (Ronald Press, New York). ing to a different tribe than that to which U. sphaerogena's 13. Kenney, W. C. & Dekker, C. A. (1971) Biochemistry 10, host belongs. 4962-4970. The data of this study suggest that production of extracel- 14. Blank, A. & Dekker, C. A. (1972) Biochemistry 11, 3956- lular guanyloribonuclease is widespread in the genus Ustila- 3970. go. Evidently then, evolution of phytopathogenicity has not 15. Ames, G. F.-L. (1974) J. Biol. Chem. 249,634-644. 16. Reisfield, R. A., Lewis, U. J. & Williams, D. E. (1962) Nature been accompanied-at least not uniformly-by loss of ge- 195,281-283. netic information for biosynthesis of an extracellular RNase 17. Singh, H. & Lane, B. G. (1964) Can. J. Biochem. 42, 1011- common to related, free-living fungi. Variation in concen- 1021. tration of ribonuclease in the medium of the organisms ex- 18. Bock, R. M. (1967) in Methods in Enzymology, eds. Gross- amined reflects in part differences in regulatory mecha- man, L. & Moldave, K. (Academic Press, New York), Vol. XII, nisms controlling biosynthesis. Thus, U. maydis guanylori- pp. 218-221. bonuclease is repressible by inorganic phosphate (T. A. 19. Markham, R. & Smith, J. D. (1952) Biochem. J. 52,552-557. McKeon and C. A. Dekker, unpublished data), while synthe- 20. Wilson, C. W. (1969) Anal. Biochem. 31, 506-511. sis of RNases U1 and U4 of U. sphaerogena is subject to am- 21. Uchida, T. (1966) J. Biochem. (Tokyo) 60, 115-132. monia repression but not to repression by phosphate (11). 22. Swift, H. (1955) in The Nucleic Acids, eds. Chargaff, E. & Davidson, J. N. (Academic Press, New York), Vol. II, pp. 51- Our failure to observe ribonuclease in the medium of U. vio- 92. laceae, both by our standard assay and by the more sensitive 23. Bradford, L. S., Jones, R. J. & Garber, E. D. (1975) Bot. Gaz. activity staining method (which detects activity in solutions 136, 109-115. as dilute as 0.15 unit/ml) suggests that this organism, or per- 24. Gould, F. W. (1963) Grass Systematics (McGraw-Hill, New haps just this strain, lacks the capacity to synthesize active York). Downloaded by guest on September 28, 2021