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Enzyme polymorphism as an aid to identification of Rhizobium strains
5 ,,,, ~,,a,. n,<,
,,I. ,,,I
79 to 80 Pcrgamon i’ress 197~. Printed in Great Brltam
SHORT
COMMUNICATION
Enzyme poIymorphism as an aid to identification of R~i~(~~~~rnstrains I,. R. MYTTON, N. .I. MCAVAM and PATRICIA PORTLO~K Welsh Plant
Breeding
Station.
Plas Gogerddan, (Acceprerl
Aberystwyth.
SY23 3EB. Dyfed, Wales
11 JurIe 1977)
by a wick dipped in bromo-phenol blue, had migrated At present strains of R~7i=u~?if{~n may be distinguished by about 9cm towards the anode. The gel was removed and cultural characteristics. host effectiveness tests, reaction to sliced horizontally to give two thin slices, one for each various stains and nutrients. or by more strain specific tests such as serological reactions, phage typing, or the use of enzyme being assayed. mutant markers. These techniques. together with their advantages and disadvantages. are described by Vincent (i) Pho,sphoghcoisomert.se (PGI) (1970). In general. few techniques are sufficiently precise Staining solution contained 50mtvt Tris-HCI pH 8.0 to give unequivocal identification on their own. When 5 ml: fructose-6-phosphate 6 mg; 45 ml; 0.1 ‘II MgSO, taken together or in combinations their precision is imglucose-6-phosphate dehydrogenase 20 units; nicotinamide proved. but the procedure can become tedious and backadenine dinucleotide phosphate (NADP) 4mg; 3-(4. 5 ground iIlformation is still required as to the absence or dimethyl thiazolyl-2)-Z 5 diphenyl tetrazolium bromide extent of other strains able to produce similar reactions (MTT) 7.5 mg; phenazine methosulphate (PMS) 2.5 mg. to the tests. Mutant markers such as antibiotic resistance enable clear identification, but relying on selection of genetic variants from the wild type strain suffers from the disadvantage that those mutants selected may also differ The staining solution contained 0.1 M Tris-HCI pH 8.0 from the parent culture in characters other than resistance 45 ml; monosodium glucose-6-phosphate 100 mg; NADP to drugs. 15 mg; nitroblue tetrazolium (NBT) 10 mg; PMS 2 mg. The Electrophoretic separation of isozyme variants has gels were stained in the dark at 37 ‘C for about 1 h. washed recently been successfully applied to a wide range of and fixed in 509’” ethanol. macro-organisms and to some micro-organisms (e.g. Miikman. 1973). A large number of geneticalty-controlled enzyme polymorphisms have been discovered and the techFigure la shows a typical gel stained for PGI. Three nique offers promise as a rapid means of identifying genoelectrophoretically distinguishable allozymes are evident. types, populations, cultivars or lines. its usefulness for this The fast form is the most frequent but there is a slow purpose depending on the frequency and distribution of variant (strain 8a) and an intermediate variant (strain 1003) genetic variants among the populations (Almgard and present. The gel stained for G-6PDH (Fig. lb. dark bands) shows two mobility classes; in this sample of isolates the Clapham. 1975; Aimgard and Norman, 1970: Hayward fast form is again the commonest with only one slow variand McAdam. 1977: Wilkinson and Beard. 1972). Results ant (1003) present. On this gel. above the G-6-PDH bands. from a preliminary survey of RItizohium luyuminosarum are there are in addition two mobility classes for an unidentireported here. Eighteen culturally pure isolates of R. ~~~~~~~~j~~.s~~~~ fied oxidase (faint pale bands). These enzymes therefore give the possibility of 3 x 2 x 2 = 12 different mobihty collected from the Welsh Plant Breeding Station farm (ElSherbeeny. Mytton and Lawes, 1977) together with one classes, four of which occur in the present sample. Ten isolates have PC1 and G-6-PDH as fast variants with a from the Rothamsted collection (strain 1003) were used as test material. They were grown for 4 days on Yeast slow oxidase, seven individuals have the fast variant of Mannitol Agar in 9cm Petri dishes incubated at 25X’. ali the three enzymes. one isolate has slow variants of both Each isolate was harvested by scraping the growth {about PGI and the oxidase with a fast G-6-PDH. and. finally, 1 ml) from a single dish into a 50 ml glass beaker containone isolate has the intermediate PGI variant with slow ing 0.4ml of Yeast Mannitol broth. Harvested cultures variants of the other two enzymes. were maintained at 4 C for I6 h, and to each was added The small sample of isolates examined here represents 0.5 ml of Tris (hydro~ymethyl) ~~minomethane-HCI buffer a restricted genetic range. The occurrence of variants in solution (Tris HCi buffer pH 7.2, 0.1 M) containing O.l”,, all three enzymes examined suggests therefore that such mercaptoethanol. Enzymes were then released from the polymorphisms may well be common and that the techcells by ultrasonicating at 20 kHzs_’ for 2min at 2.C. nique will be useful as a recognition test. The disrupted cultures were subjected to horizontal starch Other enzyme systems which are now being examined gel electrophoresis (Scandalios, 1969). The gel used was also show electrophoretic variation. e.g. esterase is ina 1X?“,, soiutton of hydrolysed potato starch’in 9 parts fluenced by at least two loci and both show variation. The existence of these other variants increased the possible perTris-citrate (pH X.3. 50 m&r) and I part lithium borate (pH mutations and allows more strains to be characterized. For 8.3. 0.2~) buffers. the latter also being used alone as a example. even two additional enzymes each having three bridge buffer in the electrode chambers. mobility classes increases the potential number of classes The sonicated cultures were absorbed onto 2 x 6mm from the present 12 up to 108. There are still technical filter paper wicks which were placed at 2 mm intervals into a slit cut across the gel 3 cm from the cathode. Nineteen problems to be solved before certain enzyme systems can be used reliably as markers. However, even at this present wicks were placed in a single gel. Electrophoresis was carearly stage of application. the method has proved to be ried out at 4 C and 375 V for 20min, wicks were removed a useful complement to other qualitative tests, and the run contmued for 4-5 h until the front, marked 79
Short
Frg. I. Etcctrophoretrc
diifercntiatron
communications
of 19 Kirixhiw Ib) get staineci
Afk7roi~iriiyf,tric~irs--~~ thank Dr Leslie Gottlieb of the University of California. Dasis. il Visiting Research Worker at the Welsh Plant Breeding Station. for his help and guidance in applying this tcchniyuc to R~~i~ob~~~~~~.
ALMCAKU G. and CLAPHAM D. (1975) lsozyme
variation distinguishing I8 drenu culti\ar-s grown in Sweden. Swrli,\/i J. ngric. Re\. 5. 61 67. AI.&~.IGARI> G. and MRMAN T. (1970) Biochemi~l techniques as an aid to distinguishing some cultivars of barley and oats. Aqric. Hwr. Gw. 28, 117 123. EI.-SHERBEEXY M. H.. MYTTOY L. R. and LAKES D. A. (1977)Symbiotic varrability in I ‘icirr jdw I. Genetic variation in the Rhkohium /~rjun~ino.~ur,irrll population. ~[~p~i!,ri~,u 26, 149--l 56.
for
/r,y~trf~ri~o.~u,rrnlisolates. G-6-PDH.
(at Gel stained
for PCiI
:
,,I. ,,,I
79 to 80 Pcrgamon i’ress 197~. Printed in Great Brltam
SHORT
COMMUNICATION
Enzyme poIymorphism as an aid to identification of R~i~(~~~~rnstrains I,. R. MYTTON, N. .I. MCAVAM and PATRICIA PORTLO~K Welsh Plant
Breeding
Station.
Plas Gogerddan, (Acceprerl
Aberystwyth.
SY23 3EB. Dyfed, Wales
11 JurIe 1977)
by a wick dipped in bromo-phenol blue, had migrated At present strains of R~7i=u~?if{~n may be distinguished by about 9cm towards the anode. The gel was removed and cultural characteristics. host effectiveness tests, reaction to sliced horizontally to give two thin slices, one for each various stains and nutrients. or by more strain specific tests such as serological reactions, phage typing, or the use of enzyme being assayed. mutant markers. These techniques. together with their advantages and disadvantages. are described by Vincent (i) Pho,sphoghcoisomert.se (PGI) (1970). In general. few techniques are sufficiently precise Staining solution contained 50mtvt Tris-HCI pH 8.0 to give unequivocal identification on their own. When 5 ml: fructose-6-phosphate 6 mg; 45 ml; 0.1 ‘II MgSO, taken together or in combinations their precision is imglucose-6-phosphate dehydrogenase 20 units; nicotinamide proved. but the procedure can become tedious and backadenine dinucleotide phosphate (NADP) 4mg; 3-(4. 5 ground iIlformation is still required as to the absence or dimethyl thiazolyl-2)-Z 5 diphenyl tetrazolium bromide extent of other strains able to produce similar reactions (MTT) 7.5 mg; phenazine methosulphate (PMS) 2.5 mg. to the tests. Mutant markers such as antibiotic resistance enable clear identification, but relying on selection of genetic variants from the wild type strain suffers from the disadvantage that those mutants selected may also differ The staining solution contained 0.1 M Tris-HCI pH 8.0 from the parent culture in characters other than resistance 45 ml; monosodium glucose-6-phosphate 100 mg; NADP to drugs. 15 mg; nitroblue tetrazolium (NBT) 10 mg; PMS 2 mg. The Electrophoretic separation of isozyme variants has gels were stained in the dark at 37 ‘C for about 1 h. washed recently been successfully applied to a wide range of and fixed in 509’” ethanol. macro-organisms and to some micro-organisms (e.g. Miikman. 1973). A large number of geneticalty-controlled enzyme polymorphisms have been discovered and the techFigure la shows a typical gel stained for PGI. Three nique offers promise as a rapid means of identifying genoelectrophoretically distinguishable allozymes are evident. types, populations, cultivars or lines. its usefulness for this The fast form is the most frequent but there is a slow purpose depending on the frequency and distribution of variant (strain 8a) and an intermediate variant (strain 1003) genetic variants among the populations (Almgard and present. The gel stained for G-6PDH (Fig. lb. dark bands) shows two mobility classes; in this sample of isolates the Clapham. 1975; Aimgard and Norman, 1970: Hayward fast form is again the commonest with only one slow variand McAdam. 1977: Wilkinson and Beard. 1972). Results ant (1003) present. On this gel. above the G-6-PDH bands. from a preliminary survey of RItizohium luyuminosarum are there are in addition two mobility classes for an unidentireported here. Eighteen culturally pure isolates of R. ~~~~~~~~j~~.s~~~~ fied oxidase (faint pale bands). These enzymes therefore give the possibility of 3 x 2 x 2 = 12 different mobihty collected from the Welsh Plant Breeding Station farm (ElSherbeeny. Mytton and Lawes, 1977) together with one classes, four of which occur in the present sample. Ten isolates have PC1 and G-6-PDH as fast variants with a from the Rothamsted collection (strain 1003) were used as test material. They were grown for 4 days on Yeast slow oxidase, seven individuals have the fast variant of Mannitol Agar in 9cm Petri dishes incubated at 25X’. ali the three enzymes. one isolate has slow variants of both Each isolate was harvested by scraping the growth {about PGI and the oxidase with a fast G-6-PDH. and. finally, 1 ml) from a single dish into a 50 ml glass beaker containone isolate has the intermediate PGI variant with slow ing 0.4ml of Yeast Mannitol broth. Harvested cultures variants of the other two enzymes. were maintained at 4 C for I6 h, and to each was added The small sample of isolates examined here represents 0.5 ml of Tris (hydro~ymethyl) ~~minomethane-HCI buffer a restricted genetic range. The occurrence of variants in solution (Tris HCi buffer pH 7.2, 0.1 M) containing O.l”,, all three enzymes examined suggests therefore that such mercaptoethanol. Enzymes were then released from the polymorphisms may well be common and that the techcells by ultrasonicating at 20 kHzs_’ for 2min at 2.C. nique will be useful as a recognition test. The disrupted cultures were subjected to horizontal starch Other enzyme systems which are now being examined gel electrophoresis (Scandalios, 1969). The gel used was also show electrophoretic variation. e.g. esterase is ina 1X?“,, soiutton of hydrolysed potato starch’in 9 parts fluenced by at least two loci and both show variation. The existence of these other variants increased the possible perTris-citrate (pH X.3. 50 m&r) and I part lithium borate (pH mutations and allows more strains to be characterized. For 8.3. 0.2~) buffers. the latter also being used alone as a example. even two additional enzymes each having three bridge buffer in the electrode chambers. mobility classes increases the potential number of classes The sonicated cultures were absorbed onto 2 x 6mm from the present 12 up to 108. There are still technical filter paper wicks which were placed at 2 mm intervals into a slit cut across the gel 3 cm from the cathode. Nineteen problems to be solved before certain enzyme systems can be used reliably as markers. However, even at this present wicks were placed in a single gel. Electrophoresis was carearly stage of application. the method has proved to be ried out at 4 C and 375 V for 20min, wicks were removed a useful complement to other qualitative tests, and the run contmued for 4-5 h until the front, marked 79
Short
Frg. I. Etcctrophoretrc
diifercntiatron
communications
of 19 Kirixhiw Ib) get staineci
Afk7roi~iriiyf,tric~irs--~~ thank Dr Leslie Gottlieb of the University of California. Dasis. il Visiting Research Worker at the Welsh Plant Breeding Station. for his help and guidance in applying this tcchniyuc to R~~i~ob~~~~~~.
ALMCAKU G. and CLAPHAM D. (1975) lsozyme
variation distinguishing I8 drenu culti\ar-s grown in Sweden. Swrli,\/i J. ngric. Re\. 5. 61 67. AI.&~.IGARI> G. and MRMAN T. (1970) Biochemi~l techniques as an aid to distinguishing some cultivars of barley and oats. Aqric. Hwr. Gw. 28, 117 123. EI.-SHERBEEXY M. H.. MYTTOY L. R. and LAKES D. A. (1977)Symbiotic varrability in I ‘icirr jdw I. Genetic variation in the Rhkohium /~rjun~ino.~ur,irrll population. ~[~p~i!,ri~,u 26, 149--l 56.
for
/r,y~trf~ri~o.~u,rrnlisolates. G-6-PDH.
(at Gel stained
for PCiI
: