NOTES
309
Comparison of the cell envelope proteins of Micrococcus cryophilus with those of Neisseria and Branhamella species'J
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
R. R. B. RUSSELL~ AND I. J. MCDONALD Divisiorl of Biologiccil Scierlces, Notiorlcll Reserrrch Corrricil of Ccmoda, Ottci,tlcr, Ccir~cidcrKIA OR6 Accepted November 18. 1975
RUSSELL, R. R. B., and I. J. MCDONALD. 1976. Comparison of the cell envelope proteins of Micrococc~rscryophillrs with those of Neisseria and Brat~hcimrlla species. Can. J . Microbiol. 22: 309-3 12. In an attempt to elucidate the relation between Mioococcirs cryopl~ilrrs,Neissrricr cci~,inr, Neisserirr o ~ f i and s , Brcit~l~ritnellr~ catrrrr/~rilis,fractions derived from outer membranes of a strain of each organism were examined for protein composition by SDS - polyacrylamide gel electrophoresis. Micrococcrrs cryoplrilrrs outer membrane protein showed extensive similarities to that of N . 012;s and contained a heat-modifiable protein which behaved almost identically with the corresponding bands previously shown to exist in N. cmririe and N . o~jis.Brrrnl~rrrnellric.rrtnrrhrrlis protein was distinctly different from those of M. cryophilrrs and the two 'false neisserias' N . cci~,irreand N. o ~ , i s . RUSSELL, R. R. B., et I. J . MCDONALD. 1976. Comparison of the cell envelope proteins of Micrococcrrs cryoplrilrrs with those of Neisserirr and Brrit~hrimellrrspecies. Can. J . Microbiol. 22: 309-3 12. Afin de determiner la relation qui peut exister entre Micrococcrrs cryoplliliis. Neissoicl ccrvirrr, Neissrrirr ovis et Brrrtihrrmelln crrtrirrhcilis,nous avons examine par electrophorese sur gel SDS polyacrylamide les fractions provenant des membranes exterieures d'une souche de chaque organisme. Les proteines de la membrane exttrieure de M . cryopl~ilrtssont fortement similaires i celles de N . ovis et contiennent une prottine sensible i la chaleur qui se comporte d'une f a ~ o n presque identique aux bandes correspondantes qui ont ete observkes anterieurement chez N . crrviae et N . ovis. La proteine de B. catcirrl~cilisest tres difftrente de celle de M . cryopllilris ainsi que des deux 'pseudoneisseria' N . cavirre et N . ovis. [Traduit par le journal]
The organism Micrococcus cryophilus, first described by McLean et al. in 1951 (8), has uncertain taxonomic status, but on the basis of its deoxyribonucleic acid (DNA) base composition (l), peptidoglycan composition (ll), and cell wall structure (6, 13), it should clearly be excluded from the genus Micrococcus. Sleytr and Kocur (13) published electron micrographs showing that M . cryophilus possessed a cell envelope typical of that for gram-negative bacteria, and on the basis of the similarities in morphology and in DNA base composition proposed that M. cryophilus should be classified among the non-pigmented asaccharolytic Neisseria (the 'false Neisseria'). Recently we reported some data on the cell envelope proteins of various species of Neisseria 'Received July 18, 1975. 'NRCC No. 15093. 3Present address: Roval College of Surgeons of England, Dental ~ e s e a r c hUnit, Downe, K&, England BR6 7JJ.
and here compare envelope proteins of (9, Neisseria caviae NRCC 31003 (ATCC 14659), Neisseria ovis NRCC 31020 (KC 987 obtained from M. A. S. Lambert, Center for Disease Control, Atlanta, Georgia), and Branhamella (formerly Neisseria) catarrhalis NRCC 3 1002 (ATCC 8176), with those of M . cryophilus NRCC 14020 (ATCC 12226). Bacteria were cultivated with shaking at 37 "C ( M , cryophilus at 18 "C) in tryptic soy broth (Difco) containing 0.5% yeast extract (Difco). Cells were removed from log-phase cultures by centrifugation (10 000 x g for 10 min); 'free endotoxin' was sedimented from the clear supernatant fluid by ultracentrifugation (200 000 x g for 1 h). Figure 1 shows the protein composition of 'free endotoxin' of the various organisms exam4Russell, R. R. B., K. G . Johnson, and I. J. McDonald. 1975. Studies of the cell envelope proteins of Neisseria species as a possible aid to classification. Can. J. Microbiol. Submitted for publication.
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CAN. J. MICROBIOL. VOL. 22, 1976
FIG.1. SDS - polyacrylamide gel electrophoresis of free endotoxin proteins of (A) and (B) M. cryOPII~ILIS NRCC 14020; (C) N. ovis NRCC 31020; ( D ) N. covioe NRCC 3 1003; (E) B. cotorrhalis NRCC 31002. Sample (A) was solubilized at 37 "C, the others at 100 "C. Theexperimental procedure was that of Russell et 01. (10) except that a slab gel apparatus (14) was used. The growth medium was tryptic soy broth (Difco) with added yeast extract. Cells were removed from log-phase culture by centrifugation, and 'free endotoxin' was collected by ultracentrifugation. Micrococcus c r y o p l ~ i l ~was t s grown at 18 "C, and the other organisms were grown at 37 "C.
ined, as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Free endotoxin, a term introduced by Crutchley et 01. (2) for material released spontaneously from the outer membrane of gram-negative bacteria during normal growth, was selected for study. This material, which is extremely easy to pre-
pare, contains a limited complenient of proteins thus facilitating comparison o f band patterns and reducing the possibility of the position of bands corresponding merely by chance. Free endotoxin contains all the major proteins of the As has been reported for cell envelope (9, other gram-negative bacteria, several of the M.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
NOTES
31 1
cryophilrrs envelope proteins display heat-modifi- teins of related organisms will have undergone able behavior, that is, their mobility in electro- little evolutionary divergence, and so will have phoresis is dependent upon the temperature at similar electrophoretic mobilities. Two recent which they were solubilized in SDS. The relation- publications suggest possibilities for a reconciliaship of protein bands observed after solubilizing tion of our findings on envelope proteins with at 37 "C to those produced by 100 "C treatment those of Fox and McClain on soluble proteins: can be elucidated by a two-dimensional electro- Langridge (5) has presented evidence obtained phoresis technique (9) and we have found that a from mutagenesis experiments, that membrane major protein of M. cryopllilus which has an components are much more likely t o be inactiapparent molecular weight of 35 000 after solu- vated by mutation than are soluble proteins and bilization a t 37 "C has an apparent molecular so would be expected to show greater evoluweight of 50 000 after solubilization at 100 "C. tionary conservation. The net effect of such conThis interconversion of protein bands is indi- servation would be that data on the electrophorcated by the dotted line in Fig. 1. The corre- esis of membrane proteins would be capable of sponding protein bands in N. cauiae and N. ouis discovering similarities between organisms taxoafter solubilization a t 100 "C have shown almost nomically further apart than would data on identical heat-modifiable behavior (9, 10, and soluble proteins. The second report is that of unpublished result^).^ Of the dozen strongest- Schnaitman et 01. (12), who found a temperate staining protein bands from M. cryophilus, seven bacteriophage which causes production of a new correspond precisely with bands from N . ouis. outer membrane protein i n Escl~ericlliocoli by Neisseria cauiae envelope protein possesses a the process of lysogenic conversion. If such major heat-modifiable band corresponding to phages are widespread in nature, they could those of N. ouis and M. cryophil~w,whereas the result in species with otherwise distinct memB. cntarrllnlis pattern shows no correspondence brane protein profiles having common (phagewith those of N. cauiae, N . ouis, or M . ~ ~ . ~ o ~ l l i l r rdetermined) s. proteins. At the same time, of In addition to having similar cell envelope course, the bacterial species would have to be proteins, M. cryopllilus resembles N . ouis in being sufficiently closely related to be able to harbor oxidase-positive and asaccharolytic (15). Fur- the same phage. thermore, studies in this laboratory have shown Thus, information on cell envelope proteins of that N . caviae and N . ovis grow in a simple deM. cryophilus indicates that the organism might fined medium containing mineral salts, glutamic be related to N . ovis K C 987 but clearly further acid, and nicotinic acid (7); M. cr)~oplzilrrsalso data are needed to clarify the relationship of M. grows on such a simple inediuni (below 23 "C), cr)~opllilusto the so-called 'false neisserias'. In with glutamate as a sole source of carbon and particular, studies on DNA-DNA hybridization nitrogen (15). Repeated attempts by us, howand inter species transformation on M. cryophilr~r and the 'false neisserias' would be of ever, have failed to get B. ratm.rlla1i.r to grow in the same medium (unpublished). value. Our findings of relatedness between M. cryopl~ilusand N . ovis are at variance with those of I. BOHACEK, J . . M. K O C U Rand , T. M A R . I I N E C1967. . Fox and McClain, who used gel electrophoresis DNA base composition and tnxonomy of some micrococci. J. Gen. Microbiol. 46: 369-376. to exaniine soluble protein patterns (3) and the 2. C R L T C H L - EM. Y ,J . , D. G. MARSH, AND J . CAMERON. mobility of various enzymes (4) from M. cry1967. Free endotoxin. Nature (London), 214: 1052. opl7ilu.s and several Neisset.in and Brat~llnmella 3. Fox. R . H..and D. E . M C C L A I N1974. . Evaluationof species. Fox and McClain concluded that M. the taxonomic relationship of ,klic.~.oc,oc.c.,,s c.ryoplrilr~s.B~~r~~rlrtr~~rcllrr cotrrr~.lrtrli.\,and Neisseriae by cr)~o/~hilu.s was unrelated to N. cauiae and N . ouis comparative polyacrylamide gel electrophoresis of (i.e. 'false neisserias'), other Neisseria, or Bransoluble proteins. Int. J . Syst. Bacterial. 24: 172-176. l~anzellabut that the 'false neisserias' were prob1. Fox, R . H.. A N D D. E. MCCLAIN.1975. Enzyme ably related to Br(~t717ntnella.The present results electrophoretog~xmsin the ;~nalysisof taxon relatedness of ~Mic~~~oc~oc~c~rrs c~~:\opIiilr~s, Brrrrrlzrc~~rclltr 1.rrrrrrshow that envelope proteins of strains of B. r1rtrli.s and atypical Neisserias. J . Gen. Microbiol. 86: catarrllalis, N. ravine, and N. oilis are distinctly 210-216. different. 5 . L.ANCRIDCE. J . 1971. Mutation spectra and the new The use of gel electrophoresis in taxonomic t~xlityof mutations. Aust. J. Biol. Sci. 27: 309-3 19. studies relies upon the assumption that the pro6. M A Z A K E CK.. , M. KOCUR,and T. M,znTlNEc. 1966.
312
7.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
8.
9.
10.
CAN. J. MICROBIOL. VOL. 22, 1976 Electron microscopy of ultrathin sections of MC rrococcrts cryopl~ilrrs.Can. J. Microbiol. 12: 465470. MCDONALD, I . J., and K. G. JOHNSON.1975. Nutritional requirements of some non-pathogenic Nci.s.seriri grown in simple synthetic media. Can. J . Microbiol. 21: 1198-1204. MCLEAN.R . A., W. L . S U L Z B A C H Eand R , S. MUDD. 1951. Microc~oc~c~ii.~ cryol)liilris, sp. nov: a large c o c c ~ l s especially suitable for cytologic study. J. Bacteriol. 62: 723-728. RUSSELL,R. R. B. 1976. Two-dimensional SDSpolyacrylamide gel electrophoresis of heat modifiable outer membrane proteins. Can. J. Microbiol. 22: 83-9 1. RUSSELL,R. R. B.. K . G . JOHNSON.and I. J. MCDONALD.1975. Envelope proteins in Nris.\rricr. Can. J . Microbiol. 21: 1519-1534.
l I. S C H L E I F E K R ,. H., and 0 . K A N D L E R1972. . Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36: 407477. 12. S C H N A I T M A N C., , D. S M I T H ,and M. FORN DE SALSAS.1975. Temperate bacteriophage whichcauses the production of a new m21jor out membrane protein by Eschcricl7irr cnli. J. Virol. 15: 1121-1 130. 13. SLEYTR,U.. and M. K o c u ~ . 1971. Structure of Micrococc~ii.~ cryoplii1ii.s after freeze-etching. Arch. Mikrobiol. 78: 353-359. 14. STUDIER,F. W . 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79: 237-248. 15. T A I ,P-C., and H. JACKSON. 1969. Mesophilicmutants of an obligate psychrophile, Mioococclis c,:vophiliis. Crrti. J . Microhiol. 15: 1145-1 150.
P-Xylosidases in the yeast Cryptococcus albidus var. aeriusl V. NOTARIO,T. G. VILLA, A N D J. R. VILLANUEVA Drptirt~nrtitc~/'Microhiology,Frrcrilty of Scici~ccsunrl C.S.I.C.. Ui~i~.o-.sity c!fSrilrr~ncii~cri,Srilrrinrri~crr,Sprrin Accepted October 17, 1975 NOTARIO,V., T . G. V I L L A . and J. R. V I L L A N U E V A 1976. . p-Xylosidases in the yeast Ci-\~ptococcri.srr1birlii.s var. oeriirs. Can. J. Microbiol. 22: 312-3 15. 0-Xylosidase activity has been detected in cell-free extracts and in culture fluids when C~-\~pfococcii,s rrll?idris var. rieriris was grown on glucose a s the sole carbon source. The enzyme appears to be constitutive. Mild acid treatment of whole cells suggested that the total activity is located in the periplasmic space and some experiments indicated that it is partially associated with the cell walls. DEAE-Sephadex A,, chromatography has shown that there are two different forms ofp-xylosidase in the cell-free extracts, but only one form is present in the supernatants of culture. NOTARIO,V.. T. G. V I L L Aet J. R. V I L L A N U E V1976. A . p-Xylosidases in the yeast Ciypfococclis ri1hidii.s var. rirrili.~.Can. J. Microbiol. 22: 312-315. On a dicele une activiti p-xylosidase dans des extraits acellulaires et dans des milieux de culture liquides lorsque Ciyptococois rrlhidris var. rrrriris est cultive avec du glucose comme seule source de carbone. L'enzyme semble Ptre constitutif. Un traitement doux i I'acide des cellules entikres suggere que I'activite totale est localisee dans I'espace periplasmique; des experiences montrent que cette activite est partiellement associie a la paroi cellulaire. La chromatographie sur DEAE-Sephadex A,, demontre qu'il existe deux formes differentes de p-xylosidase dans les extraits acellulaires, cependant. une seule forme est presente dans le surnageant des cultures. [Traduit par le journal]
P-Xylosidases (EC 3.2.1.37) commonly occur in algae (Manners and Mitchell 1967), bacteria (Howard et al. 1960; Kersters-Hilderson et al. 1970), fungi (King and Fuller 1968), invertebrates (Fisher et al. 1966), and higher animals (Fisher and Kent 1969). On the contrary, little is known 'Received August 5, 1975.
about their presence in yeasts. P-D-Xylose is a n important compound as a part of the complexes in the cell envelopes of cryptococci. The study of enzymes hydrolyzing such complexes is important in order to improve our understanding of the morphogenetic processes in this group of microorganisms. Cryptococcus albidus var. aerius, obtained
309
Comparison of the cell envelope proteins of Micrococcus cryophilus with those of Neisseria and Branhamella species'J
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
R. R. B. RUSSELL~ AND I. J. MCDONALD Divisiorl of Biologiccil Scierlces, Notiorlcll Reserrrch Corrricil of Ccmoda, Ottci,tlcr, Ccir~cidcrKIA OR6 Accepted November 18. 1975
RUSSELL, R. R. B., and I. J. MCDONALD. 1976. Comparison of the cell envelope proteins of Micrococc~rscryophillrs with those of Neisseria and Brat~hcimrlla species. Can. J . Microbiol. 22: 309-3 12. In an attempt to elucidate the relation between Mioococcirs cryopl~ilrrs,Neissrricr cci~,inr, Neisserirr o ~ f i and s , Brcit~l~ritnellr~ catrrrr/~rilis,fractions derived from outer membranes of a strain of each organism were examined for protein composition by SDS - polyacrylamide gel electrophoresis. Micrococcrrs cryoplrilrrs outer membrane protein showed extensive similarities to that of N . 012;s and contained a heat-modifiable protein which behaved almost identically with the corresponding bands previously shown to exist in N. cmririe and N . o~jis.Brrrnl~rrrnellric.rrtnrrhrrlis protein was distinctly different from those of M. cryophilrrs and the two 'false neisserias' N . cci~,irreand N. o ~ , i s . RUSSELL, R. R. B., et I. J . MCDONALD. 1976. Comparison of the cell envelope proteins of Micrococcrrs cryoplrilrrs with those of Neisserirr and Brrit~hrimellrrspecies. Can. J . Microbiol. 22: 309-3 12. Afin de determiner la relation qui peut exister entre Micrococcrrs cryoplliliis. Neissoicl ccrvirrr, Neissrrirr ovis et Brrrtihrrmelln crrtrirrhcilis,nous avons examine par electrophorese sur gel SDS polyacrylamide les fractions provenant des membranes exterieures d'une souche de chaque organisme. Les proteines de la membrane exttrieure de M . cryopl~ilrtssont fortement similaires i celles de N . ovis et contiennent une prottine sensible i la chaleur qui se comporte d'une f a ~ o n presque identique aux bandes correspondantes qui ont ete observkes anterieurement chez N . crrviae et N . ovis. La proteine de B. catcirrl~cilisest tres difftrente de celle de M . cryopllilris ainsi que des deux 'pseudoneisseria' N . cavirre et N . ovis. [Traduit par le journal]
The organism Micrococcus cryophilus, first described by McLean et al. in 1951 (8), has uncertain taxonomic status, but on the basis of its deoxyribonucleic acid (DNA) base composition (l), peptidoglycan composition (ll), and cell wall structure (6, 13), it should clearly be excluded from the genus Micrococcus. Sleytr and Kocur (13) published electron micrographs showing that M . cryophilus possessed a cell envelope typical of that for gram-negative bacteria, and on the basis of the similarities in morphology and in DNA base composition proposed that M. cryophilus should be classified among the non-pigmented asaccharolytic Neisseria (the 'false Neisseria'). Recently we reported some data on the cell envelope proteins of various species of Neisseria 'Received July 18, 1975. 'NRCC No. 15093. 3Present address: Roval College of Surgeons of England, Dental ~ e s e a r c hUnit, Downe, K&, England BR6 7JJ.
and here compare envelope proteins of (9, Neisseria caviae NRCC 31003 (ATCC 14659), Neisseria ovis NRCC 31020 (KC 987 obtained from M. A. S. Lambert, Center for Disease Control, Atlanta, Georgia), and Branhamella (formerly Neisseria) catarrhalis NRCC 3 1002 (ATCC 8176), with those of M . cryophilus NRCC 14020 (ATCC 12226). Bacteria were cultivated with shaking at 37 "C ( M , cryophilus at 18 "C) in tryptic soy broth (Difco) containing 0.5% yeast extract (Difco). Cells were removed from log-phase cultures by centrifugation (10 000 x g for 10 min); 'free endotoxin' was sedimented from the clear supernatant fluid by ultracentrifugation (200 000 x g for 1 h). Figure 1 shows the protein composition of 'free endotoxin' of the various organisms exam4Russell, R. R. B., K. G . Johnson, and I. J. McDonald. 1975. Studies of the cell envelope proteins of Neisseria species as a possible aid to classification. Can. J. Microbiol. Submitted for publication.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
CAN. J. MICROBIOL. VOL. 22, 1976
FIG.1. SDS - polyacrylamide gel electrophoresis of free endotoxin proteins of (A) and (B) M. cryOPII~ILIS NRCC 14020; (C) N. ovis NRCC 31020; ( D ) N. covioe NRCC 3 1003; (E) B. cotorrhalis NRCC 31002. Sample (A) was solubilized at 37 "C, the others at 100 "C. Theexperimental procedure was that of Russell et 01. (10) except that a slab gel apparatus (14) was used. The growth medium was tryptic soy broth (Difco) with added yeast extract. Cells were removed from log-phase culture by centrifugation, and 'free endotoxin' was collected by ultracentrifugation. Micrococcus c r y o p l ~ i l ~was t s grown at 18 "C, and the other organisms were grown at 37 "C.
ined, as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Free endotoxin, a term introduced by Crutchley et 01. (2) for material released spontaneously from the outer membrane of gram-negative bacteria during normal growth, was selected for study. This material, which is extremely easy to pre-
pare, contains a limited complenient of proteins thus facilitating comparison o f band patterns and reducing the possibility of the position of bands corresponding merely by chance. Free endotoxin contains all the major proteins of the As has been reported for cell envelope (9, other gram-negative bacteria, several of the M.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
NOTES
31 1
cryophilrrs envelope proteins display heat-modifi- teins of related organisms will have undergone able behavior, that is, their mobility in electro- little evolutionary divergence, and so will have phoresis is dependent upon the temperature at similar electrophoretic mobilities. Two recent which they were solubilized in SDS. The relation- publications suggest possibilities for a reconciliaship of protein bands observed after solubilizing tion of our findings on envelope proteins with at 37 "C to those produced by 100 "C treatment those of Fox and McClain on soluble proteins: can be elucidated by a two-dimensional electro- Langridge (5) has presented evidence obtained phoresis technique (9) and we have found that a from mutagenesis experiments, that membrane major protein of M. cryopllilus which has an components are much more likely t o be inactiapparent molecular weight of 35 000 after solu- vated by mutation than are soluble proteins and bilization a t 37 "C has an apparent molecular so would be expected to show greater evoluweight of 50 000 after solubilization at 100 "C. tionary conservation. The net effect of such conThis interconversion of protein bands is indi- servation would be that data on the electrophorcated by the dotted line in Fig. 1. The corre- esis of membrane proteins would be capable of sponding protein bands in N. cauiae and N. ouis discovering similarities between organisms taxoafter solubilization a t 100 "C have shown almost nomically further apart than would data on identical heat-modifiable behavior (9, 10, and soluble proteins. The second report is that of unpublished result^).^ Of the dozen strongest- Schnaitman et 01. (12), who found a temperate staining protein bands from M. cryophilus, seven bacteriophage which causes production of a new correspond precisely with bands from N . ouis. outer membrane protein i n Escl~ericlliocoli by Neisseria cauiae envelope protein possesses a the process of lysogenic conversion. If such major heat-modifiable band corresponding to phages are widespread in nature, they could those of N. ouis and M. cryophil~w,whereas the result in species with otherwise distinct memB. cntarrllnlis pattern shows no correspondence brane protein profiles having common (phagewith those of N. cauiae, N . ouis, or M . ~ ~ . ~ o ~ l l i l r rdetermined) s. proteins. At the same time, of In addition to having similar cell envelope course, the bacterial species would have to be proteins, M. cryopllilus resembles N . ouis in being sufficiently closely related to be able to harbor oxidase-positive and asaccharolytic (15). Fur- the same phage. thermore, studies in this laboratory have shown Thus, information on cell envelope proteins of that N . caviae and N . ovis grow in a simple deM. cryophilus indicates that the organism might fined medium containing mineral salts, glutamic be related to N . ovis K C 987 but clearly further acid, and nicotinic acid (7); M. cr)~oplzilrrsalso data are needed to clarify the relationship of M. grows on such a simple inediuni (below 23 "C), cr)~opllilusto the so-called 'false neisserias'. In with glutamate as a sole source of carbon and particular, studies on DNA-DNA hybridization nitrogen (15). Repeated attempts by us, howand inter species transformation on M. cryophilr~r and the 'false neisserias' would be of ever, have failed to get B. ratm.rlla1i.r to grow in the same medium (unpublished). value. Our findings of relatedness between M. cryopl~ilusand N . ovis are at variance with those of I. BOHACEK, J . . M. K O C U Rand , T. M A R . I I N E C1967. . Fox and McClain, who used gel electrophoresis DNA base composition and tnxonomy of some micrococci. J. Gen. Microbiol. 46: 369-376. to exaniine soluble protein patterns (3) and the 2. C R L T C H L - EM. Y ,J . , D. G. MARSH, AND J . CAMERON. mobility of various enzymes (4) from M. cry1967. Free endotoxin. Nature (London), 214: 1052. opl7ilu.s and several Neisset.in and Brat~llnmella 3. Fox. R . H..and D. E . M C C L A I N1974. . Evaluationof species. Fox and McClain concluded that M. the taxonomic relationship of ,klic.~.oc,oc.c.,,s c.ryoplrilr~s.B~~r~~rlrtr~~rcllrr cotrrr~.lrtrli.\,and Neisseriae by cr)~o/~hilu.s was unrelated to N. cauiae and N . ouis comparative polyacrylamide gel electrophoresis of (i.e. 'false neisserias'), other Neisseria, or Bransoluble proteins. Int. J . Syst. Bacterial. 24: 172-176. l~anzellabut that the 'false neisserias' were prob1. Fox, R . H.. A N D D. E. MCCLAIN.1975. Enzyme ably related to Br(~t717ntnella.The present results electrophoretog~xmsin the ;~nalysisof taxon relatedness of ~Mic~~~oc~oc~c~rrs c~~:\opIiilr~s, Brrrrrlzrc~~rclltr 1.rrrrrrshow that envelope proteins of strains of B. r1rtrli.s and atypical Neisserias. J . Gen. Microbiol. 86: catarrllalis, N. ravine, and N. oilis are distinctly 210-216. different. 5 . L.ANCRIDCE. J . 1971. Mutation spectra and the new The use of gel electrophoresis in taxonomic t~xlityof mutations. Aust. J. Biol. Sci. 27: 309-3 19. studies relies upon the assumption that the pro6. M A Z A K E CK.. , M. KOCUR,and T. M,znTlNEc. 1966.
312
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Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/13/14 For personal use only.
8.
9.
10.
CAN. J. MICROBIOL. VOL. 22, 1976 Electron microscopy of ultrathin sections of MC rrococcrts cryopl~ilrrs.Can. J. Microbiol. 12: 465470. MCDONALD, I . J., and K. G. JOHNSON.1975. Nutritional requirements of some non-pathogenic Nci.s.seriri grown in simple synthetic media. Can. J . Microbiol. 21: 1198-1204. MCLEAN.R . A., W. L . S U L Z B A C H Eand R , S. MUDD. 1951. Microc~oc~c~ii.~ cryol)liilris, sp. nov: a large c o c c ~ l s especially suitable for cytologic study. J. Bacteriol. 62: 723-728. RUSSELL,R. R. B. 1976. Two-dimensional SDSpolyacrylamide gel electrophoresis of heat modifiable outer membrane proteins. Can. J. Microbiol. 22: 83-9 1. RUSSELL,R. R. B.. K . G . JOHNSON.and I. J. MCDONALD.1975. Envelope proteins in Nris.\rricr. Can. J . Microbiol. 21: 1519-1534.
l I. S C H L E I F E K R ,. H., and 0 . K A N D L E R1972. . Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36: 407477. 12. S C H N A I T M A N C., , D. S M I T H ,and M. FORN DE SALSAS.1975. Temperate bacteriophage whichcauses the production of a new m21jor out membrane protein by Eschcricl7irr cnli. J. Virol. 15: 1121-1 130. 13. SLEYTR,U.. and M. K o c u ~ . 1971. Structure of Micrococc~ii.~ cryoplii1ii.s after freeze-etching. Arch. Mikrobiol. 78: 353-359. 14. STUDIER,F. W . 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79: 237-248. 15. T A I ,P-C., and H. JACKSON. 1969. Mesophilicmutants of an obligate psychrophile, Mioococclis c,:vophiliis. Crrti. J . Microhiol. 15: 1145-1 150.
P-Xylosidases in the yeast Cryptococcus albidus var. aeriusl V. NOTARIO,T. G. VILLA, A N D J. R. VILLANUEVA Drptirt~nrtitc~/'Microhiology,Frrcrilty of Scici~ccsunrl C.S.I.C.. Ui~i~.o-.sity c!fSrilrr~ncii~cri,Srilrrinrri~crr,Sprrin Accepted October 17, 1975 NOTARIO,V., T . G. V I L L A . and J. R. V I L L A N U E V A 1976. . p-Xylosidases in the yeast Ci-\~ptococcri.srr1birlii.s var. oeriirs. Can. J. Microbiol. 22: 312-3 15. 0-Xylosidase activity has been detected in cell-free extracts and in culture fluids when C~-\~pfococcii,s rrll?idris var. rieriris was grown on glucose a s the sole carbon source. The enzyme appears to be constitutive. Mild acid treatment of whole cells suggested that the total activity is located in the periplasmic space and some experiments indicated that it is partially associated with the cell walls. DEAE-Sephadex A,, chromatography has shown that there are two different forms ofp-xylosidase in the cell-free extracts, but only one form is present in the supernatants of culture. NOTARIO,V.. T. G. V I L L Aet J. R. V I L L A N U E V1976. A . p-Xylosidases in the yeast Ciypfococclis ri1hidii.s var. rirrili.~.Can. J. Microbiol. 22: 312-315. On a dicele une activiti p-xylosidase dans des extraits acellulaires et dans des milieux de culture liquides lorsque Ciyptococois rrlhidris var. rrrriris est cultive avec du glucose comme seule source de carbone. L'enzyme semble Ptre constitutif. Un traitement doux i I'acide des cellules entikres suggere que I'activite totale est localisee dans I'espace periplasmique; des experiences montrent que cette activite est partiellement associie a la paroi cellulaire. La chromatographie sur DEAE-Sephadex A,, demontre qu'il existe deux formes differentes de p-xylosidase dans les extraits acellulaires, cependant. une seule forme est presente dans le surnageant des cultures. [Traduit par le journal]
P-Xylosidases (EC 3.2.1.37) commonly occur in algae (Manners and Mitchell 1967), bacteria (Howard et al. 1960; Kersters-Hilderson et al. 1970), fungi (King and Fuller 1968), invertebrates (Fisher et al. 1966), and higher animals (Fisher and Kent 1969). On the contrary, little is known 'Received August 5, 1975.
about their presence in yeasts. P-D-Xylose is a n important compound as a part of the complexes in the cell envelopes of cryptococci. The study of enzymes hydrolyzing such complexes is important in order to improve our understanding of the morphogenetic processes in this group of microorganisms. Cryptococcus albidus var. aerius, obtained
