M. W. STEWARD AND N. M. PACKTER
28c
totals/four Roux bottles), the specific activities
being 800, 1200 and 1375 counts/min./mg. respectively. Total incorporation into these metabolites
was 1.7%. Aurantiogliocladin (3-0mg.) was obtained from the thallus and had specific activity
310 counts/min./mg., a value considerably lower
than that of gliorosein found in the medium.
The 14C-labelled aurantiogliocladin derived from
[1-14C]acetate was taken up from the medium and at
least 52% ofthe initial substrate entered the thallus.
Growth was poor in this experiment. However,
14C-labelled gliorosein (8mg.) was isolated and
possessed a specific activity of 1100 counts/min./mg.
(21% incorporation). In addition, some 14C-labelled
quinol (1300 counts/min./mg.) was obtained.
Vischer (1953) has demonstrated that gliorosein
is readily converted into the quinone under alkaline
conditions with the intermediate fornmation of quinol
and quinhydrone. Further, Sheehan, Lawson &
Gaul (1958) have reported that a metabolite of
A. terreus, terreic acid (V), which contains a diketone
structure, undergoes a similar conversion into
3,6-dihydroxytoluquinone. We have shown that
gliorosein is oxidized slowly even at pH4-2. Thus
the results indicate that gliorosein is the actual
metabolite secreted by G. roseum and that the
quinol, quinhydrone and quinone are all derived
from it in the medium.
The activities determined for these substances
with [14C]acetate as substrate are consistent with
this view. Relatively highly active gliorosein is
presumably produced initially and secreted into the
medium. Gliorosein could then isomerize slowly to
1965
the quinol, which would, in turn, be partially
oxidized to quinhydrone with the same specific
activity. At later stages of growth, [14C]acetyl-CoA
would be diluted by endogenously produced
material, and gliorosein synthesized from it would
therefore have a lower activity. Accordingly, the
specific activities determined should increase in the
order: quinone (thallus), gliorosein, quinol and
quinhydrone. This pattern was, in fact, obtained,
confirming that gliorosein is probably the only
substance secreted by the thallus. Further, these
results explain the low incorporation values obtained by Bentley & Lavate (1965) since they added
[14C]acetate after 21 days' growth. At this time,
synthesis of gliorosein will have practically ceased.
Finally, we have obtained evidence consistent
with the idea that aurantiogliocladin (or its quinol)
is probably the immediate precursor of gliorosein
within the thallus.
M. W. S. acknowledges the support of a D.S.I.R. Student-
ship.
Bentley, R. & Lavate, W. V. (1965). J. biol. Chem. 240,532.
Birch, A. J., Fryer, R. I. & Smith, H. (1958). Proc. chem. Soc.
p. 343.
Brian, P. W., Curtis, P. J., Howland, J. R., Jefferys, E. G. &
Raudnitz, H. (1951). Experientia, 7, 266.
Packter, N. M. & Glover, J. (1965). Biochim. biophys. Ada,
100, 50.
Sheehan, J. C., Lawson, W. B. & Gaul, R. J. (1958). J. Amer.
chem. Soc. 80, 5536.
Vischer, E. B. (1953). J. chem. Soc. p. 815.
Biochem. J. (1965) 95, 28 c
Features of the Cell-Wall Structure of Yeast Revealed by the Action of Enzymes
from a Non-Fruiting Myxobacterium (Cytophagajohnsonii)
By J. S. D. BACON, BEATRICE D. MLNE, IRENE F. TAYLOR and D. M. WEBLEY
Macaulay Inatitute for Soil Re8earch, Aberdeen
(Received 25 March 1965)
According to current views (see Nickerson, 1963)
the yeast cell wall consists of an insoluble glucan,
with which is associated glucomannan-protein
complexes containing phosphate; glucosamine is a
minor component (2-3%) and less than one-tenth
of it can be present in insoluble chitin-like structures; lipid is also present, but no structural role has
been suggested for it. Interest in biological systems
that can attack the yeast cell wall has centred chiefly
on studies of protoplast formation, the preparations
most commonly used being those from the snail
Helix pomatia, which are known to hydrolyse the
glucan. However, snail digestive juice, in addition
to this active ,-glucosidase, contains a large number
Vol. 95
SHORT COMMUNICATIONS
29c
of other enzymes capable of hydrolysing glycosidic mineral base (Stanier, 1947) with 0.1% of Difco
linkages, including mannanase and chitinase peptone had virtually disappeared after 18-24hr.
(Holden & Tracey, 1950; Myers & Northcote, 1958), growth with shaking at 28°; the medium and walls
and so the essential nature of its action remains had been autoclaved for 20min. at 151b./in.2 before
unclear. As an alternative to snail juice some inoculation. Autoclaved whole yeast also lost its
workers have used microbial enzymes that can lyse walls under these conditions. In contrast. no action
yeast cell walls; these include preparations from was detected by microscopic examination when noncertain bacilli (Phaff, Tanaka & Higgins, 1961) and autoclaved yeast cell walls or live whole yeast cells
from strains of Streptomyces sp. (Mendoza & Villa- were incorporated in the growth medium. Other
nueva, 1962). A microbial enzyme preparation is workers have occasionally observed that micronow described that will dissolve the yeast cell wall organisms will bring about a more rapid breakdown
only when it has been subjected to certain prelimi- of autoclaved than of non-autoclaved walls (Horinary treatmnents.
koshi & Sakaguchi, 1958; Masschelein, 1959a,b).
During a recent study of the polysaccharideThe cell-wall-hydrolysing system in Cytophaga
producing bacteria in the root region of pasture johnsonii was found to be adaptive and best prograsses (Webley, Duff, Bacon & Farmer, 1965) a duced in a medium containing a minimum of organic
number of isolations were made of a non-fruiting constituents, other than the walls. The organism
myxobacterium that proved to be similar to Cyto- was therefore grown for 24hr. in the yeast cell wallphaga johnsonii (Stanier, 1947). This organism had mineral salts-peptone medium described above, and
been shown by Salton (1955) to clear agar plates the whole culture tested for its action on walls and
containing cell walls of the yeast Candida pul- whole cells. Autoclaved walls disappeared, as they
cherrima. Our strain of Cytophaga johnsonii had a also did from autoclaved whole cells, during 24hr.
similar action on autoclaved cell walls of baker's incubation at 30°. Although the enzymes in the
yeast in agar plates. Yeast cell walls (1mg. dry cultures of (ytophaga johnsonii were capable of
wt./ml.) in liquid medium consisting of dilute liberating glucose from the yeast cell walls, the
Table 1. Action of Cytophaga johnsonii culture on cell walls of yeast (Saccharomyces cerevisiae)
In each experiment 1-3ml. of a culture of Cytophagajohn8onii prepared as described in the text was incubated at
300 in a total volume of 2-0ml. in the presence of 7mM-tris-HCl buffer, pH7.5, with 0-4ml. of yeast fraction and
the other additions described. The whole cell preparation usually contained 5-5-7-0mg. of anthrone-positive
material (expressed as glucose)/ml. and the cell-wall preparation 11-14mg./ml. Samples taken after 24hr. were
centrifuged at 30000g for 30min. at 10 and the residue and supernatant fluid analysed by the anthrone procedure
of Fairbairn (1953). The incubation mixtures were also examined under the microscope.
Anthrone-positive material
Control of Thiol addition
(mg. of glucose equiv. in 1 ml. Microscopic
infection
of original cell-wall suspension) appearance
(20mm__(A, aseptic;
2-mercaptoof walls
I
Yeast
Previous
T, toluene)
ethanol)
Supernatant fluid Residue after incubation
Whole cells None
A
6-3
1-3
Present
+
Cell wallst
None
Ethyl acetate
Ethyl acetate
None
None
None
None
Preincubatedt with thiol
Preincubatedt with buffer
Heated at 750 for 30 min.
Heated at 750 for 30 min.
Cell walls incubated alone
C. johnsonii culture incubated alone
T
T
T
A
A
T
T
T
T
T
T
T
T
+
+
3.7
3-7
6-9
5.0
1-4
3-4
Present*
Absent
Present
10-9
7-5
12-1
7-5
7-8
6-3
12-1
7-8
2-8
5-4
2.1
3-7
2-5
5.2
2-5
6-4
Absent
Present
Absent
Present
Absent
Present
Absent
Present
1-2
1-9
10-6
0-6
* On continued incubation the walls disappeared.
t Prepared by the method of Northcote & Home (1952).
t Incubated for 1 hr. at 30° in 20mM-buffer solution, with or without 2-mercaptoethanol (60mM), then washed twice on
the centrifuge with buffer solution at 10.
30c
J. S. D. BACON, B. D. MILNE, I. F. TAYLOR AND D. M. WEBLEY
extent of reducing sugar formation was not a good
guide to the visible dissolution ofthe walls under the
microscope; in particular, liberation of reducing
sugar was faster at pH 5 0 than at 7 5, but the actual
disappearance of autoclaved walls was faster at the
higher pH. Paper chromatography revealed that
the principal reducing substance was glucose; some
oligosaccharide was also present, but no free mannose could be detected. A better guide to the ultimate fate of the wall was the liberation of material
reacting with the anthrone reagent (Fairbairn, 1953).
Davies & Elvin (1964) have shown that 2-mercaptoethanol facilitates protoplast formation by
snail digestive juice from suspensions of Saccharomycesfragilis, and that it is sufficient to preincubate
the cells with this compound. We therefore tested
the effect of 2-mercaptoethanol on non-autoclaved
walls and on the walls of whole yeast treated in
various ways. Some results are given in Table 1, and
show clearly that the presence of, or pretreatment
with, 2-mercaptoethanol permits the myxobacterial
culture to dissolve non-autoclaved walls. Other
experiments showed that thioglycollate could be
substituted for 2-mercaptoethanol, and that the
effect was the same at pH 5O0 in citrate-phosphate
buffer.
When living whole yeast suspensions were incubated with Cytophaga john8onii cultures in the
presence or absence of 2-mercaptoethanol, microscopic examination showed no attack on the walls
and no protoplast formation with the addition of
mannitol (final concn. 0-8M). However, if toluene
was added to non-autoclaved whole yeast, or if ethyl
acetate-treated cells prepared as described by
Myrback (1957) were used, the walls disappeared
from the cells in the presence of 2-mercaptoethanol.
The most significant feature of the action of the
myxobacterial culture was that, although in the
absence of 2-mercaptoethanol microscopic examination showed the walls still to be present, they had
lost more than half of their anthrone-positive constituents. Much of this loss was glucose, but small
amounts of polysaccharides containing mannose
and glucose were also released. In the presence of
thiol compounds much more polysaccharide was
brought into solution, mannose being the predominant constituent, and the small final residue when
hydrolysed yielded glucose, with only traces of
mannose.
The best working hypothesis to explain these
results is that two structural systems exist, either of
which when intact will preserve the integrity of the
yeast cell wall and (more or less) its microscopic
appearance. One is the glucan, whose structure
depends throughout on glycosidic linkages; the
other is composed of mannan-protein complexes
1965
associated through disulphide linkages, as suggested
by Nickerson & Falcone (1956). The first can be
dismantled by the myxobacterial enzymes and the
second by thiol compounds or by autoclaving. Only
when both systems are broken does the cell wall
'dissolve'. The 2-mercaptoethanol effect is probably
a chemical one, since a period at 750, which might
be expected to inactivate enzymes (see Table 1),
does not prevent its action. This hypothesis does not
explain the resistance of living yeast cells, which do
not lose their walls when incubated with the myxobacterial enzyme and 2-mercaptoethanol, nor give
rise to protoplasts. It is conceivable that a third
membrane or network exists and that the susceptibility of living cells to certain other microbial
enzymes and snail digestive juice is due to the
presence of enzymes that can attack this further
structural system. The presence of a chitinase in
Cytophaga johnsonii would seem to argue against
this third structure's being of a chitinous nature.
A reasonable extension of the hypothesis would
be to suggest that the action of either 2-mercaptoethanol or of the myxobacterial enzymes alone
should loosen the wall structure and make it more
permeable to large molecules, such as invertase
(mol.wt. about 120000; Andersen, 1960). All the
invertase held in ethyl acetate-treated cells (Burger,
Bacon & Bacon, 1961) was liberated in 4hr. at 30°
by the action of the myxobacterial culture in the
presence of 2-mercaptoethanol at both pH 7-5 and
5 0. In its absence liberation was slower but considerable (about 50% after 4hr.). The thiol alone
had little action at pH 5.0, but liberated practically
all the invertase in 24hr. at pH 7*5.
Andersen, B. (1960). Acta chem. scand. 14, 1849.
Burger, M., Bacon, E. E. & Bacon, J. S. D. (1961). Biochem.
J. 78, 504.
Davies, R. & Elvin, P. A. (1964). Biochem. J. 93, 8P.
Fairbairn, N. J. (1953). Chem. & Ind. p. 86.
Holden, M. & Tracey, M. V. (1950). Biochem. J. 47, 407.
Horikoshi, K. & Sakaguchi, K. I. (1958). J. gen. appl.
.Microbiol., Tokyo, 4, 1.
Masschelein, C. A. (1959a). Rev. Ferment. 14, 59.
Masschelein, C. A. (1959b). Rev. Ferment. 14, 87.
Mendoza, C. G. & Villanueva, J. R. (1962). Nature, Lond.,
195, 1326.
Myers, F. L. & Northcote, D. H. (1958). J. exp. Biol. 35,639.
Myrbiick, K. (1957). Arch. Biochem. Biophys. 69, 138.
Nickerson, W. J. (1963). Bact. Rev. 27, 305.
Nickerson, W. J. & Falcone, G. (1956). Science, 124, 722.
Northcote, D. H. & Home, R. W. (1952). Biochem. J. 51,
232.
Phaff, H. J., Tanaka, H. & Higgins, L. W. (1961). Bact.
Proc. 61st Meet., p. 62.
Salton, M. R. J. (1955). J. gen. Microbiol. 12, 25.
Stanier, R. Y. (1947). J. Bad. 53, 297.
Webley, D. M., Duff, R. B., Bacon, J. S. D. & Farmer, V. C.
(1965). J. Soil Sci. (in the Press).