__ _,__ ...
F~---·-·-------...,~..._-,...._~....---•·--~,_,-,_
l
-~.-...'"""""._....,.._.,
..
~-..-..------·--..~~·-·,......--.~-..--·--......,._,..._......_.....,_....,..-.-,..,.-,___
i
;
California State University, Northridge
LIPID STUDIES IN NEUROSPORA ORASS.l
II
A thesis submitted in partial satisfaction o~ the
requirements for the degree of Master of Science in
Biology
b;y
Roger Hugh ,...-Rich
September, 197::5
l
.. . . ., ,_
L---~-----
______. _______ ....... -··--··-·-···- ---··· ·-·---· ·---- ................. -- ......... ···--··-··-..... _...........................................................
II.
I
I
~
'
l
I
l
The thesis of' Roger Hugh
F~ch
is approved:
Committee Chairma.n
Cal1f'onia state Un1vers1 ty, Northridge
September, 1973
11
.
'
l·
AOKNOWLEDGMDTS
I would 11ke to express my appreciation to Dr.
· Donald Biancl!:d under whose· helpful guidance this work
was done.
I am also grateful to the other members of my
thesis committee, Dr. Joyce Maxwell and Dr. Kenneth
. Jones, for tb.eir assistance in preparing this thesis.
I
I
I
I-----------·------------..·-----------~·_. ______
iii
I
r
·-------··--··--·-----·-
TABLE OF CONTENTS
I
Page
ACKNOWLEDGMEN'fS •••••••••••.••••••••••••••••••••••• 111
TABLE OF CONTENTS•••••••••••••••••••••••••••••••• 1v
T.ABLES •••••••••••••••••••••••••••••••••••••
$
• • •-• •
v
FIGURES •••••••••••••••••••••••••••••••••••••••••• V11
ABSTRACT ••••••••••••••••••••••••••••••••••••••••• V111
INTRODUCTIOB ......................................
1
MATERIALS AND METHODS ••••.••.••••••••••••••••• •..
$
RESULTS ••••••••••••••••••••••••••••••••• • •••••••• ._18
DISCUSSION ••••••••••••••••••••••••••••••••••••••• 58
REFERENCES. • •. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
I
I
I
73
I
.
.
·j
'-------------··----·--------------------·-·--··--·--"·•'-·-----------------iv
I
TABLES
Table
Page
1.
Standard lipid solutions • • • • • • • • •.• • • • • • • • • • • •
13
2.
Gas-liquid chromatograph settings ••••••••••••
15
3 ..
Mean dry weight/50ml of medium for
Lindegren+ grown in 0 and M media ••••••••••••
20
4.
Mean dry weight/50ml of medium for
28A and 7A grown in 0 medium •••••••••••••••••
23
5.
mg extractable lipid/50ml of medium for
Lindegren+ grown on C and
. M media ••••••••••••
'Z7
6.
Change in 1ipid/dry weight for Lindegren•
grown on C and M media •••••••••••••••••••••••
30
Lipids extracted from four day old
cultures of 28A and 7A •••••••••••••••••••••••
33
8.
Rf values and colors of known lipids •••••••••
35
9.
Typical colors and R.p values obtained
from charred chromatOgrams of the lipids
of Lindegren+, 28A and 7A ••••••••••••••s•••••
31
Change in m.g tr1glycer1de/50ml of medium
for Lindegren+ grown on 0 and M media ••••••••
40
Change in triglyceride/dry weight tor
Lindegren-+ grown on C and M media • • • • • • • • • • •.•
43
::10.
11.
12.
13~
Triglyceride/total extractable lipid
for Lindegren+ grown on 0 and M media • • • • • • • •
Efficiency of hydrolysis and methylation
of pure tristearin to stearic acid
methyl ester •••••••••••••••••••••••••••••••••
46
48
14.
Retent~on times of pure fatty acid
methyl esters ••••••••••••••••••••••••••••••~•
15.
Retention times of fatty aoi.d methyl esters
or Lindegren+ grown on 0 and M media •••••••••
52
16 ..
Retention times of fatty acid methyl esters
of 28A and 7A ···········~··········~··••••••
53
II
··-·"·-·····------------·--··-·--··---·----·------·-······---·-·----·---- -------·-·-----..-----
I
I
.
v
---------...,-1
. . :t
••
.
•
~-.
l
·. . .
.
• ..:
.~
..
.......
I
~·
I
I
17.
18.
19.
Fatty acid composition of the
triglycerides of four day old cultures
of Lindegren+ grown on 0 and M media •••••••••
56
Fatty acid composition of the
triglycerides of four day old cultures
of 28A and 7A grown on 0 medium •••••••••••••
57
Instantaneous rate constants of
Lindegren+ grown on 0 and M media ••••••••••••
I·
Vi
~------·---·-·--·-----·
l
FIGURES
i
i
I
Change in dry weight/50ml of medium
for Lindegren+ grown on C and M
media ••••••••••••••••••••••••••••••••••••••••••
22
Change in dry wei ght/50ml of medium .for
28A and 7A •••••••••••••••••••••••••••••••••••••
24
Change in mg extractable lipid/50ml
of medium for cultures of Lindegren•
grown on C and M media •••••••••••••••••••••••••
29
Change in lipid/dry weight for Lindegren+
grown on C and M media •••••••••••••••••••••••••
32
5.
Change in tr1glycer1de/50ml of medium
for Lindegren+ grown on C and M media ••••••••••
42
6.
Change in triglyceride/dry weight for
Lindegren+ grown on C and M media ••••••••••••••
45
Gas chromatogram of methylated fatt7
acids of the triglyeeridea,)of
Lindegren+ grown on C medium •••••••••••••••••••
51
1.
2.
3.
4.
1.
I
\·
I!
II
I
l
I
II
I
I
.
-
.
I
-
I
'----------------··--·-----------------·--·-----~----·--------------_j
v11
I
---'-------------------"'-----·---------~-~---,
I
I
A:BSTRJ.O'.r
LIPID STUDIES IN NEUROSPORA CRASS.&
II
by
Roger Hugh Rich
Master of Science in Biology
Il
September, 1973
I
j
Three strains of Neurospora
c~a~s~
were used to
correlate lipid metabolism With formation of conidia.
Lindegren+, a Wild type strain forms conidia when grown
I
I
~
l
on C medium but forms no conidia or aerial hyphae
When grown on M medium.
I
During the per.iod o-f' maximum
conidia formation o:f' Lindegren+, there
~s
a s1.gni:f'icant
- increase in the ratios of total lipid/dry weight
and triglyceride/dry weight.
When conidia are suppressed
· and growth 1s strictly vegetative, no such inlcrease ia
observed during this period.
!
I
In this case, ·the ratios
_ o:f' total lipid/dry weight and triglyceride/drjj· 1-reight
show a slow but steady
in~rease
after three days.
At
· four days of growth, these ratios are 26% and 39% 1ess
than those of cul. tures grolt"n on C medium and remain lees
-I
;until the fifth and sixth dare.
l
1
l
Wild type strain 28A forms conidia wben grown on
t___________________________
il
I
I
·
----------------.-----·----------~----
Viii
I
... ,___ -··-·------. -~-- ---------- _____. , ____ . ,___________ _,_~1
- - - - - - - - - - - - - --~--~·---
C medium.
S~rain
~--- ~~·-·..........,,.....,.-.~~-~--&""'l
7A, a mutant derived from 28A forma
essentially no conidia.
I
The ratios of lipid/dry weight
and triglycertde/dry weight are 20% higher in strain 28J.
than 7 .A after four days of growth.
The degree of unsaturation of the triglyceride
- fatty acids :is correlated ·with the extension of aerial .
- hyphae, a primary step in the formation ot conidia.
' Fifty six per cent of the fatty acids derived from
cultures of Lindegren+ grown on C medium are
unsaturated wh-ereas, 38% of the fatty acids derived from
· Lindegren+ grown on M medium are unsaturated.
Strains
I
, 28A and 7 A wb1ch both form aerial hyphae have a
!
r~gher
:f'rac·tion of unsaturated fatty acids than
cultures of Lindegren+ grown on M medium.
f
I
·--~----·---•·--~~
-v~-----.-·-----~--
~--·-··-·--·---·····----------J
1
l
I
I
INTRODUCTION
I
. The morphological and biochemical nature of the
I
I growth
cycle of.Wild type and mutant strains of
!I Neurospora crassa has been studied by Tur1an and Bianchi
i
j (1972), Weiss (1965) and Lein (1953), along with others.
I
: Three major phases in the asexual growth cycle of
I crassa can be described as a
I The first of these is marked
!•
result of these studiea.
by the extension of
a
j germination tube from an asexual spore. The second is
i
/ the vegetative phase which is identified by the extension
I
[ of hyphal tips and the formation of hyphal branches behind
1
the elongating tips. This process of elongation and
I branching occurs repeatedly and results in a thickly
l
I intertwined mat or mycelium.
! growth occurs below or at the
I
Initially, this phase of
!
i'
jliqu1d medium.
I1
liquid-air interface in· a
Under favorable condi tiona, narrow
hyphae penetrate the liquid-air interface and elong&te
·I~·into ·the
air above the medium.
I
I
!
The formation of these
I
! aerial hyphae initiates the third phase of growth, a
I phase marked
I
by the differentiation of conidia at the
tips of the aerial hyphae (Cochrane, 1958).
Mature
l conidia form as a string of beaded units loosely
attached to one another •.
L_ _ _ _ _.
.I
I
~-.
Conidiat1on is essentially an aerobic process and
I below an
I form
oxygen tension of 20mm of mercury; no conidia ·
(Kobr
I
I
l
~
!!•,
1965).
Initially, growth in liquid
maintaining a reductive type of metabolism through
.
il glycolytic pathways.
.
However, accompanying the formation
I of aerial hyphae and conidia, there is a shift from
~-
1 reductive to oxidative metabolism.
j
f
During this time,
there are al.so changes 1n the ratio of oxidized and
! reduced
I
pyridine nucleotides (Bar-Lew, 1973).
Turian
I (1965) showed that in static liquid cultures, the develop-
! ment
1
I
of conidia 1 s dependent on the presence of nitrate
.
1
l as the sole source of nitrogen.
When ammonium replaces
1
I·
l
l
i
I
~-
,
no aerial
l
I hyphae and consequently no conidia form and growth
1
! nitrate
as the sole source of nitrogen,
I
'
I
lis exclusively vegetative •
.I
Lipid metabolism of fungi has been the interest of
I
I researchers
!
I (Lein,
for several decades (Prill
1953), (Krzeminski, 1960).
~
!£., 1935),
Until recently,
I
l
!knowledge 1n this area has been limited
I
!I qualitative identification of the
ma~or
mos~ly
to the
lipid classes.
The lipids which have been found to be important to
{metabolism of the fungi are the tr1glycer1des, free
I!
lI
lI
l
!tatty acids, phospholipids, sterols and the caroteno1ds.
IFew studies have been made which correlate quantitative
I
L
I
-------~---.J
I
changes in lipid metabolism w1 th the major developmental
!I stages
.
of the fungi.
Bianchi and Turian (1967) have
shown that the lipids in
1
!•
crassa do undergo changes
at the time of differentiation of macroconid1e. The
j biosynthesis of ce1lular material such as lipid requires
reduced pyridine nucleotides.
1
I
! morphological
Brody and Nyc {1970) used
mutants W1 th reduced levels of reduced
i
I pyridine
nucleotides to correlate the saturated and
!
: unsaturated fatty acid content of the tr1glycerides and
Ii phospholipids
li nucleotides.
Conidia are consid9red to be asexual spores and
1
I
l represent
I
I
1
I
.I
w1 th the levels of reduced pyridine
a res1 stant stage in the life cycle. ·The
longevity of these spores depends in part on their
! storage
reserves which include a lipid fractlon.
Ilikely,
then., that developmental changes Wh1eh occur
I
It is
I during conidiogenesis are reflected in changes in the
I1 lipid
I
traction.
In this study, the role of lipid metabolism and 1ts
I relationship to the differentiation of conidia 1n the
!asexual
growth cyc1e of -N. crassa is studied.
I
...
I
.
i threefo1d approach to the problem is used.
l
i
A
first, a
.
i study of the qualitative changes in the lipi,d classes
l
! which accompany con1d1ation is made.
Secondr. an an.a;t,ysis
l•
\of the changes in the kinetics of growth, 11p1d
l
---·---·-----·---
I
I
I
I
4
accumulatioa., and triglyceride accumulation and their
relationshjp to con1d1at1on is undertaken.
Third, the
fatty acid composition of the triglyceride& is
~·
examined ta <determine if changes in these components
1
are correla~d with the stages of conidial ditterentiati~
I
I
I
I
'
I
I
'
L
---·------------I
·-·--·-·.J
5
------------·--------------------------------------·
MATERIALS AND METHODS
I.
l
Growth.
Three strains of Neurospora crassa were used in
this study.
Lindegren+ was obtained from Dr. G.
Turian, University of Geneva, Switzerland.
Wild type
28A and mutant 7A derived from 28A were obtained from
Dr. R. Siegel, Department of Biology, University of
California at Los Angeles.
Stock cultures were
maintained on. tryptone-soybean-yeast extract slants at
25°0.
Ster11e de-ionized water was added to four day
old cultures and the tubes agitated on a Vortex mixer
to release conidia and hyphal fragments.
All
inoculations of culture media were made by first suspending a known number of conidia and hyphal fragments
in sterile
WD.t~r
and pipeting equivalent"volumes o!
the suspension into each culture flask.
trations of the
~lspensions
The concen-
were determined using a
Model B Coulter Counter With the following settings:
Lower Threshold=lO
Upper threshold=lOO
l/ampl1ficat1on=i
I
1/aperture current=i
l.
Orifice s1ze=100p.
·-·~-------..-..-,
. - -__.,.. . ,______. . - . . ,. . . ._,___________. ---------.-vll
6
·---------------------·-·-----·---------,
l
I
I
The final concentration of the suspension in each
experiment was brought to approximately 1.2x10 6
part1cles/m1.
;
Each culture flask was inoculated
with approximately 1.8x1o 5 particles/50ml of culture·
medium.
Two basic culture media were used for growth.
The
M medium (Turian, 1964) contained:
KH Po
2 4
0.5gm
MgS041H 20
0.25gm
NaCl
o.05gm
CaC122H20
o.osgm
NH4Citrate
1.13gm
Biotin
l.Oml
·sucrose
De-ionized H2 o
I
I
!'
I
I
l
I
1 • 0X 101,.~.-·, :
5.0x10 2m1
i
I
l
{
A solution of trace elements (0.5ml) composed of the
following compounds (Ryan,l950) was added.
II .
I
f
Ne. 2B40iOH 20
o.o44mg
CuSO4?H 2 o
0.19lmg
,
Fe 2 (so4 )j6H 20
o.455gg
MnCl24R2 0
o.o36mg
(NR 4 ) 6Mo 7o2Ji4H 2o
O.. Ol8mg
, This medium supports vegetative gro;.rth..
I
I
I
I
wit~
6N KOH.
A.t 25 0 under
L---·---~--·-------------------~----·-·---·
i!
Il
4.403mg
The pH of the solution was adjusted to 6.2
I
7
---------------------,
I
static conditions, the developing mycelium remains
submerged in the medium and no aerial hyphae·or conidia
form.
The C medium (Turian, 19§4) contained
l.Ogm
10l03
KH Po
2
One-half
o.ssm
4
MgS047H 20
0.25gm
NaCl
0.05gm
CaC122H 20
0~05gm
K 01 trate
1.62gm
Biotin
l.Oml
Sucroae
1.0x101 gm
De-ionized H 0
2
5.0x10 2ml.
m11~111ter
of the trace element solution
used in the M medium was added and the pH of the
solution was adjusted to 6.3 With 6N KOH.
At 25°0,
this medium· :produces aerial hyphae and con1d1a which
develop above the medium !rom submerged vegetative
hyphae.
Growth was sccompl1shed in either 3 liter Fernbach
flasks or in 125ml Erlenmeyer flasks.
To achieve
satisfactory conidiation under static conditions, an
appropriat.e r:atio of airspace to .liquid volume 1 s
necessary.
!
I
in the
~his
Erlen~eyer
is achieved by using 50ml of C medium
flasks.
Five hundred milliliters of
M medium were added to the Fernbach flasks.
L ______;_____________. --·--
l
__l
8
A large number of flasks were inoculated with equal
volumes or the suspension of hyphal particles and
conidia under sterile conditions.
The cultures grown
on the C and M media were harvested by aspiration
in a Buchner funnel on Whatman 11 filter paper at
selected
fn~ervals
beginning ou the second day.
Each
mat obtained. was washed with one 11 ter of de-ionized
water·.
I
I
!
immediately frozen and kept at -10°0 until used.
l
II.
Extraclion of total lipids.
Just prior to lipid extraction, the frozen squares
of
l~euro ~Eora
were put into round bottom flasks
I
I
1
Ii
!
I
I·
I
I
The, mats were cut into small squares and
I
which in turn were plugged into a Vertis Freeze Dryer
and the contents taken to dryness •.The Vertis Freeze
Dryer was oonnected to a Duo-Seal Vacuum Pump and a
trap of acetone and dry ice was used.
A dry weight
after lyoph11.1zation was obtained for each sample.
Extrac,t.ion of the llpids from the lyophilized mats
was carried out using a modification of the procedure
of Radin (1951). Redistilled solvents were used
throughout as was Wha.tma.n. lll f'11 ter paper washed in
chloroform~
~he
procedure is outlined below:
1. Each lyophilized mat was homogenized in a
solutlon of chloroform-methanol (2:1 V/V) in a
Servall Homogenizer ..
l
I
Homoge:n1zat1on w·as
c---·---·-·-•••-··-~•-•-·•·------~-----·•-•·---- . --·---·----·--..-.....1
9
was achieved in three minutes at room
lI
tempe~~~ure.
Seventeen milliliters of the 2:1 solation was
used for up to lgm dry weight.
I
2. The homogenate was filtered and co1lected in
I
a screw top test tube.
;
3. The filtrate containing the lipid
washed w1 th
o. 2
volume~,
(approximately 3ml).
~raction
was
of de-ionized water
The wash mixture was
centrifuged for five minutes in the co1d after
which the bottom layer containing the 11pid
fraction was drawn off and
collectedt~a
50ml
beaker.
4. The solvent was removed with a stream of 1·2
and the lipid residue taken to dryness.
Benzene (5ml) was added and the solvent again
removed under a stream of N2 •
Successive
weighings of the lipid residue were m.ade during
the removal of N2 until two successive we1gh1ngs
differed by 0.2mg or less.
I
5. The lipid residue was then red1sso1ved in
j
5ml of chloroform and stored in a screw top
test tube w1 th a teflon cap liner under
refrigeration until further use.
III.
~solat1o~-~d
identification of the general
lipid clJ!;!_Ses.
I!
I
,
Isolation
a~d
identification of the general lipid
L--------~---~-··----·----------·--··-1
10
classes was carried out by thin layer chromatographf
( TLC).
Silica Gel G was used as the adsorbant.
TLO.
plates were poured to a thickness of 0.65mm and were
activated by heating at 105°0 for a minimum of one
day before use.
The lipid extract in chloroform was reduced to
2 to 3ml by evaporation under a stream of N •
2
The
concentrated extract was applied in a narrow band
1.5cm fro·m the bottom of the plate.
Four centimeters
at the right edge were reserved for the appl1cQtion
of a standard solution of triolein at one spot on the
origin and a mixture of known lipids obtained from
commercial sources at another spot. When the plate was
fully developed, the triolein a-pot was used as a
1
reference to indicate the height to which the
'
triglycerid.es had risen.
I:
The spots obtained from the
lipid mixture were used for later identification of
the class'es of lipid pre sent in the extracts.
All TliO p1ates were developed in a tank saturated
with a solvent system of petroleum ether, diethyl
ether and acetic acid in the proportions of 90:10:1
(V/V/V).
The solvent was allowed to rise to about
13cm above the level at which the sample was applied.
After separation of the extract, the right edge of
the plate containing the standard tl"1olein was sprayed
ll
·--------------·--·-----· ---------:-'
'
with a
0.2~
solution of 2,7-dichlorofluorescein in
ethanol and then heated with a stream of warm air to
visualize the triolein.
The plate was then exam1ne.d
0
charred tor 15 minutes at 105 0 to reveal clearlJ
each lipid fraction.
This latter technique was used
to verify that a clean separation of the lipid fraction
I
l
I
I·
. had been made.
Triglycerides were washed from the Silica Gel G
· adsorbant first With a 15ml portion of distilled
'chloroform and then by two successive 5ml portions of
chloroform.
The washings were combined and retained
; as the triglyceride
frac~ion.
The triglyceride
I
:fraction was taken to dryness under a stream of
r.r ..
. J:\2
We1gh1ngs were made during the evaporation
under N2 until two successive weighings differed
I
l
Thin layer plates With known lipids were also
prepared using the specific lipids listed in Table 1.
These TLO plates were developed in the same solvent
system and treated in the same way as the plates
containing the lipid extracts.
The
if
value and
color of each lipid was determined after charring.
These data were used to aid in the identification of
the lipid classes of the extracts.
IV. Hydrolysis and methylation of the tr1glycer1des.
The procedure followed for the hydrolysis and
methylation of the triglyceride fraction o! the lipid
extract was
(1969).
es~entially
that of Morrison and Smith
Tne procedure is outlined below.
1. The triglyceride_ obtained previously from
each TLC plate was evaporated to,dryness under a
stream of N2 •
2. BF -methanol, redistilled benzene and
3
redistilled methanol were added in the
proportions lml:lml:2ml.
This solution was then
put into a screw top test tube with a cap lined
with teflon tape and heated in boiling water
I
f'or 30 minutes.
3. The solution was cooled to room temperature.
I
L.
Pentane (8ml, chromatography quality) and 4ml
of de-ionized water were added and the solution
J
13
~
1
l
·
fable 1,
Standard lipid solutions used for thin layer chromatograph,.
Lipid Class
Speci:t'ic Lipid
:;,
free fatty acid
palmitic acid
myristic acid
stearic acid
phospholipid
phosphatidyl ethanolamine
sterol
cholesterol
stigmasterol
mono glyceride
mono olein
diglycer1de
diolein
triglyceride
triolein
;
trim;rristin
fatty acid
methyl ester
palmitic acid
myristic acid
-
sterol ester
stearic acid
cholesterol n-octanoate
_.1
. 14
shaken.
4. Tb.e solution was transferred to centrifuge
tube.s and centrifuged in the cold !or five
m1nut.ea.
5. The top layer containing the methylated
fatt~
acids in pentane was drawn off.
To determine the efficiency of the hydrolysismethylation procedure, four pure tristearin solutions
were taken through steps 2 through 5 of the preceeding
procedure.
!he stearic acid methyl ester obtained from
hydrolysis; and methylation of the tristearin was
analyzed on a Beckman GC-72-5 Gas Chromatograph using
a Thermal Conductivity Detector and a Beckman Model
1005 Recorder.
A six foot stainless column {O.D.=l/8
inch) packed with 20% diethyl glycol succinate as the
partition liquid and Chromasorb WHP {mesh size=S0/100)
as the sup.port material was used in this study as
I
well as in the folloWing studies.
Helium was used as
the carrier gas.
temper~ture
The gas !low and
settings o.f 'the chromatograph are listed in Table 2.
II
Also analyzed on the chromatograph were two standard
Ij
commercial sources.
'
data obtained from the standard stearic acid methyl
stearic act.d methyl ester solutions obtained from
Comparisons of the chromatographic
l_e_s_t_e_r_s_o_l_u_.t_i_.on,e and the stearic
~c-1d-~-eth~~-es-te:_
15
Table 2.
Gas-liquid chromatograph settings.
Column temperature
180°0
Detector temperature
260°0
Inlet temperature
210°0
Line temperatur,e
230°0
Current
200ma
Air flow rate
300cc/minute
He
flow rate
lOOce/minut•
He flow rate through Rotometer (left)
20cc/m1nute
He flow rate through Rotometer (right)
20cc/m1nute
I
----------
.-.~--~----~----~J
16
,-----~-!
obtained by hydrolysis and methylation of tristearin
were made to determine the per cent conversion of
tristearin to stearic acid methyl ester.
V. Gas chromatograuhic analysis of the hydrolyzed and
methylated triglyceride fraction.
Each hydrolyzed and methylated trig1yceride
fraction obtained from the lipid extracts of
!•
crassa,
strains Lindegren +, 28A and 7 A was evaporated to
dryness under a stream of N2 and redissolved in 0.2111
or 0.5ml of pentane. A known amount of the methylated
fatty· acid sol.ution obtained from the extract was · · ···
injected into the gas chromatograph.
Retention times
of the individual peaks were measured from the point
of injection of the sample to the center of the peak.
The area under each peak was determined by cutting it_
from the recording paper and weighing the paper.
Weights w·ere converted to areas by tbe use of a
I
standard C':urve of weight versus area.
Retention times of known solutions of methylated
fatt~
acids obtained from commercial sources were also
measured on the gas chromatogr.g,.ph.
For :identification,
these retention times were compared to, the retention
l
I
I
times of the unknown peaks obtained from 'the
hyd_rolyzed and methylated. triglycerides' of the lipid ·
l
I
e~tracts.
In one case, a mixture of myristic acid
L--~-·~----·-·---·--·"'-------:...·---·----·----~ ---·--~---------'"""'
17
methyl ester, palmitic acid methyl ester and oleic
-lI
acid methyl ester was injected along with the unkno~
fatty acid methyl esters.
A pattern of known peaks
ll.
consequently overlayed the pattern of unknown peaks. ·
The position of the unknown peaks relative to the known
peaks served as a further means of identification of
the unknown peaks.
I
I·
I
I
L_.- - -..
-------~--------·______· ·-·-··---_]
18
RESULTS
Prior to analysing the lipids of Neurospora crassa,
a series of' experiments were performed to determine
the kinetics of growth on the two types of support
media and to correlate the time of conidial differentiation With the growth phases.
Each experiment
consisted o:f inoculating C and M media w1 th a
suspension of conidia and hyphal fragments taken from
four day o1d stock cultures.
Cultures were incubated
without shaking at 25°0 and harvested on the second,
Table 3
third, fourth, fifth and sixth days of growth.
and Figure 1 contrast the dry weights for cultures
grown in C and M media. over this time period.
data Show
~hat
The
for both cultures, growth increases
steadily through the sixth day.
The M medium supports
an overal1 higher rate of growth than the C medium
a.nd appraa.ches the stationary phase by the sixth
day.
The 0 culture maintains a similar but slower
rate of growth..
The rates of growth on the C and M
media. are g"'.t"eatest between the second and third days.
Assuming logarithmic growth over this interval, the
instantaneous growth rate constants can be deterThe constants k 1 and
k 2 for cultures grown on C and M media are 1.62
mtned (Stanier
I
t
!!:!:. .!!·'
•---··-·--,.----..--.-'
1970).
19
and 1.74 r&spectively.
Between the third an4 fifth
days of gro·wrth the two cultures appear to shift into
a second loga:ri thmic phase of growth.
The instan-
taneous growrth rate constants ki and k2 for the
.
'
cultures grow.n on C and M media between the third and
b
fifth days are .399 and .327 respectively.
The dry weights of !• crassa, strains 28A and
7A were measured from the second to the sixth days of
static grow't:h in C medium at 25°0. Harvesting began
on the secod day.
data.
Table 4 and Figure 2 show these
The rates of growth of 28A and 7A are higher
than for L1m.degren+ grown in either C or M media.
Strain 28A reached 209.5mg/50ml of culture medium
by the sixth day and was still increasing its weight.
1.
By the fifth day, 7A reached 147.7mg/50ml of medium
and by the sixth day was in a declining phase.
The
instantane:ous growth rate constants k
~nd k
4 for
3
28A and 7 A b·e·tween the secon4 and third days of growth
are 2.37 an:d. 2.16 respectively.
Conid1at1on began about the third day of growth
for 28A and continued profusely through the sixth day
of growth.
Strain 7A began to form small amounts of
i
conidia around the fifth day and the total formation
I
of conidia was estimated to be less than 5% of that o!
I
28A..
l
Lindegran + began to form aerial hyphae and conidia
L _____
- - - - ·-·---J
~--·-·-··----------------.--------·------·-···-------·-··-·-
..
--·--·-------·-··· ....- .......................... _________
,
_______ _. _______________________________ __________ __
.....
..
.
I
I
!
ll
i
I
Table 3.
r
I Mean
I
·,
ot cultures ot NeurosJ!_ora crassa,
L1ndegren+ srown on c and Mmedia at as•o. The data represent the results ot
four dupl1cate experiments.
dry weights alld firl;alu.l~rd d$v1a,t1on.s
mg dry we1ght/50ml
of M culture
medium
2 Days
3 Days
4 Days
5 Days
-
48.30+5.60
-
71.33,!5.71
92.85.;!7 .67
-'
22.99~.07
8.47+. 705
!>,'
6 Days
-
I'
102.0+8.55
mg d17 we1ght,f50ml
of C culture
medium
2.99.,!.86
15.05+2.62
'
33.52.;!4.83
-
41.80+3.35
'
i
I
I!
!_________________________________,_____________
~
21
Figure 1.
Change in mg dry we1ght/50ml of medium for cultures
of Neurospora orassa, Lindegren+ grown on C and
0
M culture med1aiat 25
o.
I
I
l
L..-~-----. . . .-
I
. . .-,,..~. ---~-----------· _______. __;
22
M
100
I
.,I
50
I
'
0
·.,a
i
lI .
s
::s
.....
'tj
II
Q)
El
CD
! ~::s
I ~
l!
r-1
0 ..10
-
I
ft-4
::s
I
I
l
0
i
l 0
II lC\
..........
....,
5
~
.....
Q)
!
~
I ta
~
~
s
2
J
\.
3
4
Da.rs
5
6
,-- _____ __
..
.................................. ..
·-----·--·-·---~---~
I
!
I Mean
II
Table 4.
l
I
di7 we1ght/50ml of medium and av:era;g:&.... deviations for cultures of
l~m!X:SG9,tl! ,CF!II!' 28A and 7 A. grown on 0 medium at 25• c.
l
.
--
I
.
'
!!
!
!
2 Days
:3 Days
4 Days
5 Days
6 Days
,I
I
!
weight/50ml
of C culture medium
wild type strain 28A
I
mg dry
mg dry we1ght/50ml
of M culture medium
mutant
strain 7A
----·
-
60. 9.,:t. 80 '
-
51.5+.80
5.71+.41
3.25+.04
-
140.0.±3.50
176.5.±3.25
-
110.7 ·.±13. 3 147 .7+.75
-
209.5+3.60
-
125.2+1.1
I
I
t
I
i
.I
t
J
l
L.___ . __ _,._,_., ___
. ---·-----·-·-------··--·---..-·--·------------···-
1\J
\JI
24
----;,:--,
1 - - - - - - ·-.------------------~------
.-....-.. !
200
I
-----I
100
I
70
I
I
Figure 2.
Mean dry we1ght/50ml ot
medium for cultures ot
Neurospora crassa, 28A and
grown on o medium at 25•0.
j
2
I
I
7AI
1
!
I
5
6
I
___I
25
by the second day when grown on 0 medium.
The
great bulk of the growth on M medium took place below
the liquid-air interface or at the surface.
By the sixth
day, small amounts of hyphae had grown up along the
walls of the culture vessel and occasional small
patches of conidia were observed in some of the
cultures.
_ _,__j
26
Total Lipid Content
Table 5 and Figure 3 show the rate pt change ot
total lipid for cultures of
grown in C and M media.
!· crassa, Lindegren+
The rate constants k
5
which describe the changes in lipid during the
and t
I
logarithmic phases of increase between two and three
i
days are 1.05 and 1.46 respectively.
:~
logar1 thmic: phase is approximated by the increase
I
between the fourth and sixth days.
I
k5
I
I
l
and
k6 for
A second
The rate constants
this interva1 are 0.18 and 0.22.
Figure 4 and Table 6 show the change in lipid/dry
weight for the cultures of Lindegren+ grown on C and
I . M media.
I
6
In both cultures, the lipid/dry weight is
greatest on the second day of growth although this
ratio for the cultures grown on M medium is only
I
I
0.65 of the ratio for the cultures grown on C medium.
The lipid to dry weight ratio of the cultures grown
on C medius remains significantly greater than
that of the cultures grown. on M medium until the
fifth and sixth days.
Table 7 shows the lipid/dry w·eight for strains
28A and 7A after four days of growth on C medium.
These values are .. 066 and .054 re·spectively.
to dry
weigh~
The lipid
ratio of 7A is 0.81 times that ot 28A.
These lipid levels are 1.67 and 1.35 times higher than
the corresponding levels of Lindegren+ grown in 0 medium.
Table 5.
mg extractable lipid/50ml of medium and sta.n.dard deviations for oul turee
of ~e~rospor~ ~rassf, L1ndegren+ grown on 0 and M media at 25•0. The data
represemt the results of tour duplicate experiments.
4 Days
2 Days
3 Days
mg 11p1d/50ml
of M culture
medium
.33,!.. 084
1.43,!.171
2.20_±.382
.I mg 11pid/50ml
of C culture
I medium
.178.±.01
.513+.005
-
.882+.16
l
-
-
5 Days
6 Days
-
3.42.,±0.0
2.86+.164
-
1.09+.141
-
1.28+.164
t:·
I
I
I
II
1'\)
-4
28
r
Figure 3.
!
'
Change in mg extractable 11p1d/50ml of medium for
!
cultures of Neurospora crassa, Lindegren +
I
grown on C and M media at 25°0.
I
I
I
I
L_
29
'T""
2
5
Days
--C----
·--
__ _______
--
....... ·---- ··-·-- . ·----·--.... ........._, ..____ , ..............____ ............,,__...................... ... ..,
........
- - ............................ _____________,_,_.__________
_,
Table 6.
-l
Change in lipid/dry we1gltt/50ml ot culture medium and standard deviations tor cultures
of
~~uros~ora £rass~,
Lindegren+ grown on
c and M media at 25QO. The data
i represent the results of four duplicate experiments.
!
!
i
II
I
2 Days
3 Days
i4 Days
5 Days
we1ght/50ml
I 11p1d/dry
of M culture medium
• 0 39..:t• 002
we1ght/50ml
II 11p1d/dr.y
of 0 culture medium
.059+.0041 • 0 350.!• 0088 .0395+.0042 .0329 +.0032
i
-
.0287,!.001
6 Days
.0309+.0013
-
.0314,.:t.0022 .0317.;t.001C .0334+.0026
-
-
'
I
!
I
l
I
I
1_ _ _ .., _... _. _ _ _ _
. - - - - - - - ·- - - - - - - · - ' - - - - - - - - - - -..--..· - - - - · - - · - - - - - - - - - - - - -
~
31
.Figure .\.
Change in 11p1d/dry we1ght/50ml of medium tor
cu1tures of Neurospora crassa, L1ndegren+
grown on 0 and M media at 25°0.
·---·-·------------------'
}2
c
.06
a
:::s
I.,..
.'t:f
.05
4l
a
4l
k
:::s
,...
~
:::s .04
0
.01
i
2
I
3
Days. 4
5
6
-------------------
I
Table 7.
Beurospo~! ~t!!~q,
The lipids extracted !rom tour day old cultures ot
2SA and 7! grown on
a med~um
strains
at 25°0.
,,,.
: C>\
i.
C)""' ca...a.n
!
mg d
1t.tht/50mll
of c medium
11p1d/d
of C medium
1~ht/50ml
28!
127+5.36
-
0. 0 660.!• 0102
7!.
122+13.9
-
0. 05 35 +. 00425
triJZ:l
ide/d
Of C medium
0.0153,!.00141 .
-
-
0.0127+.0028 .
0.81
7A/28J.
1ght/50m1
0.83
. !) \.
-.,,,
t·;;
,·
_,-···
{o~_,,.
ttJ
\
:?
'-·~-
._j
;;
\JI
VI
34
Separation and Identification of Lipid
~The
Rr
Fracti~na
values and colors of the known lipid
fractions after charring, used for the identif'i68.ttiJjc.
of the unknowns,are given in Table 8.
Table 9 is a representation of typical TLC data
obtained for cultures of Lindegren+ grown on C andhM
media over six days and for 28A and ?A grown for tour
days on C medium.
Seven distinct lipid fractions
were obtained from Lindegren+ cultures grown on M
medium for each day of the six day interval.
Six
distinct lipid fractions were obtained from the
cultures of Lindegren+ grown on c medium for each day
of the six day intervale
The cultures grown on X
medium consistently showed an additional polar
fraction between the phospholipid fraction and the sterol
fraction~
The 28A and 7A strains each demonstrated
seven distinct lipid fractions.
Both 28A and 7&
differed from Lindegren+ in that an additional
fraction appea=ed between the sterols and the free
fatty acids•
35
Table 8.
I; Rf values and colors of known lipids determined after
0
1
j charring at 105 C for fifteen minutes •
Class
Phospha.tidyl
ethanolamine
o.oo
Color after
charring W1 th
1~ ~so 4eth ol
Dark brown
Mono olein
Cholesterol
Stigmasterol
0.02
0 .. 11
0.12
Light. tan
Red-gra1
Blue-gray
I Diglyc erlde
Diolein
0.14
Light tan
!I Free
Palmitic acid
Myristic acid
Stearic acid
0.25
.Light tan
Light tan
Light tan
Triolein
Tr1myristin
0.44
0.43
Tan
Tan
Palmitic acid
Myristic acid
Stearic acid
0.76
0.72
0.73
Tan
Tan
I! Phospholipid
I
Ii Monoglyceride
!
Sterol
1
I
fatty acid
I
I
lI
Triglyce~ide
I
I
! Fatty
acid
[ methyl ester
Sterol ester
. Lipid
0.33
0.29
Cholesterol
n-octanoe.te
L~--·-------·---··------
Tan
Red-gray
36
I
I
Table 9 •
Typical colors and Rf values obtained from the charred
chromatograms of the lipid extracts of cultures of
Neurospora crasaa, strains Lindegren+, 28A and 7 A.
I
L_-----------------~~-
r--
----····-·-----·-··------------------------·------·-··--------------·--·---·-······-----··------·---
Lipid
1
! s'tor-o1
seter
fatty acid
methyl ester
R.,. values and
colors of
lipid fractions
of Lindegren+
cultures grown
on M medium
Rf' values and
colors of
lipid fractions
of Lindegren+
cultures grown
on 0 medium
.89 brown-gray .S? gray
-
,,
------···--·---:j
R.,. values and
colors of
lipid fractions
of four day old
cultures of 28A
grown on 0 medium
R.,. values and
colors of
lipid fractions
of four day old
cultures of 7A
grown on 0 medium
.S9 brown-gra1
.89 brown-gray
-
-
trigl7oeride
.44 tan .
.46
tan
.4o tan
.39 tan
free fatty f\C1d
.23 tan
.26
tan
.27
.18 light brown
.22
.17
.12 dark gra;r
.13 blue-gray
.04 light tan
.05 light tan
.oo
.oo
unidentified
unidentified
.12
.oa
unidentified.
.03 light gra1
phospholipid
•.oo
sterol
blue-gray
.15 dark gra;r
tan
.o6
dark
bro~
.oo
tan
tan
light brown
light tan
dark brown
dark brown
dark brown
L-
~
38
Quantitative Analysis of the Triglyceride&
Table 10 and Figure 5 show the changes in total
triglyceride for cultures of Lindegren+ grown on 0
and M media.
The data show that the rate of change
o~
triglyceride content is greater on M medium than on
C medium.
Between two and four days in the 0 medium
there is a rapid increase in the rate of accumulation
of triglyceride
fo~lowed
a stationary phase.
by a sharp leveling off into
On M medium, the culture maintains
a high rate of triglyceride accumulation through·
the sixth day.
Assuming a logarithmic increase
between the second and third days, the rate constants
k?
and k 8 for the change in triglyceride over this time
interval are 0.709 ~d 1.42 for the cultures grown on
0 and M media respectively.
Table '11 and Figure 6 show the chan.ge in
triglyceride/dry weight for cultures of Lindegren+
grown on 0 and M media.
The triglyceride to dry
weight ratio is 1.86 times greater on the 0 medium
than on the M medium in1tial.ly •. /The triglyceride to
dry weight ratio increases at a fairly constant. rate
between the third and sixth days of growth on M
medium whereas on C medium, a sharp increase between
the third and fourth days followed by a decl.ine i.a
39
observed in each experiment.
This increase occurs
over the tlme interval when conid1ation is observed
to be the greatest. The same increase between three
and four days appears when the triglyceride to lipid
ratios are examined (Table 12).
The triglyceride to dry weight ratio was determined
for 28A and 7A after four days of growth in C medium
(Table 7).
.I.- - - - .· ·-· · - - - ·- - - - · · - - .- - - .-·-· · ·- - - ·- - - ·-·-· · · ·- · · · . . _._ _ _ _ _ _ _ _ _ _ _ ... . . . ______,_: .___,__.. ---l
l
Table 10.
t~~glrcer1de/50ml
of medium and standard deviations tor cultures ot
Neurospora crassa, Lindegren+ grown on o·and M media at 25°0.
The data Tepresent the results of four duplicate experiments.
Change in mg
I!
l
3 Days
4 Days
5 Days
6 Days
.0248+.0010
-
.0539 +.0120
-
-
-
2 Days
mg tr1glycer1de/50ml
of M culture medium
.0122+.0030
-
.0144+.0220 .0596+.0174 .1020 +.0142 .1596+.0197 • 2198 +. 0380 '·
mg triglyceride/50ml
ot 0 culture medium
I
·----·----
........
-
-
.0463+.0038 .0542+.0017
.
__________
~.:;.~
tl
Figure 5.
Change in mg tr1glycer1de/50ml of medium tor
cultures of Neurospora orassa, Lindegren+ grown on
C and M media at 25•0.
42
M
.....~
20
~
Q)
Jal
•
k
.a
r-f
:::s
()
io
8
'H
0
El0
~
II\
..........
Cl)
'CI
4
.....
k
CD
()
~
I ~.....
I
k
~
~.
l<f
-a
0
.-1
w
!l
Day-s
J
·:w
r··· -··· -··-··· -··· .·-·--····---····-··
- --·----~~---·----------·- -·
ll
I Change in triglyceride/dry weight/50ml of medium and
I cultures of Neu.,rospor,! crassa, Lindegren+ grown on 0
--l
i
Table 11.
I
\
i'
-··------~-----
standard deviations tor
and M culture media at 25°0.
!he data represent the results ot tour duplicate experiments.
2 Days
3 Days
4 Days
5 Days
6 Days
.00131
+.00040
.00140
+.00005
-
.00172
+.00024
-
.00204
,t.00031
.00230
+.00063
.00145
+.00019
.00127
+.00001
triglyceride/
dry weight/50ml
of M culture
medium
-
-
triglycerid-e/
dry weight/50ml
of 0 culture
medium
.00366
.,:t.00052
.00167
+.00030
.00195
-+.00034
.
-
.
-
-
-
'
I
I!
l
l[.
_______
j
~
j
!"
~igure
I! .
I
6.
Change in triglyceride/dry weight/50ml ot
medium for cultures of Neurospora crassa,
0
Lindegren+ grown on C and M media at 25
c.
45
-.,...
If\
0
H
S4
::s
. ..-f
0
oC
G)
s
'H·
0
i3
0
t!\
''6b
~
~-
••"""':c2t:
00
'co'tJ
.....
~
~1
~
,-t
·~
.....
k
~
2
3
Days
4
5
6
~-------------------------------------------·
Table 12.
Triglyceride/total extractable lipid for cultures
Lindegren+ grown on o and M media
at 25•0.
2 Days
3 Days
4 Days
5 Days
6 Days
.0435
.0415
.0465
.0555
.0640
Triglyceride/lipid
.0630
for cul turee grown '"··.
on 0 medium
.0483
.0610
.0425
.0425
Trlglycer1de/11pid
for cultures grown
on M medium
I
of Neurq&mora crassa,
L
--·
.J=-
0\
47
Fat~7
Acid Composition of the
Triglyce~des
The procedure of Morrison and Smith (1969) used
for the h7drolysis and methylation of the fatty acids
of the triglycerides proved to be effective in this
study as shown by the data in Table 13.
An average
of 87.1% for the conversion of pure tristearin to
stearic ac1d methyl ester was obtained.
The retention times of pure fatty acid metbyl
esters app1ied individually and in combination to the
GLC column are listed in Table 14.
A typical recorder printout shoWing the fatty acid
methyl ester peaks derived from the tr1glycer1des
of a culture of Lindegren+ grown on M medium is
shown in ]>'1.gure 7.
The retention times of the methylated fatty acids
from the triglyceride fractions of cultures of
Lindegren+· grown on C
Table 15
peak.
e~d
M media are listed in
along with the probable identity of each
The same peaks are found in each of the
different
~ultures.
Table 1.6 shows the retention t.ime and probable
identity
o~
each fatty acid methyl ester peak obtained
from 28A and 7 A cul turea grrnnJ. on C medium. Strains
28A and 7 A_b_o_t_h
--di-~fe~
from Lindegren+ in that ne1 ther
J
.,\
r--··-------~--·-·-
,._, _ _
·-----~
.... -·--·----·
l
I.
moles of stearic
acid methyl este~
expeoted if conversion
ot tristearin is 10~
~
actual Jl moles
detected by gasliquid chromatographJ
%conversion
39xlo-3
33.6xlo .. 3
86.2
24xlo-3
24xlo-3
100
32xlo-3
27 .oxlo-3
84.4
16.5xlo-3
77,8
21.2%10_,
::
'fable
I
I
I
•.
f
1~
Ett1c1enoy of hydrolysis and methylation ot pure tristearin to stearic
acid methyl eater.
I
IIL---:---..-
-
··---
__ j
I
ct
49
Table 14.
Retention
~1mes
of known pure fatt7 acid meth7l esters.
Fatty acid methyl
ester
Number of
carbon atoms
and degree of
unsaturation
Retention time
in minutes
~
capric
lauric
myristic
palmitic
palmitoleic
stearic
oleic
linoleic
linolenic
arachidic
10:0
12:0
14:0
16:0
16:1
18:0
18:1
18:2
18:3
20:0
0.71
1.09
1.68
2.75
3.14
4.99
5.85
6.85
9.07.
9.65
50
Figure 7•
Gas chrome..togrmJt of the methylated fatty acids
obtained from the triglycerides of a culture ot
Lindegren+ grown on C medium.
,_,
l
_________ __ ___________ ________
.
._,.,.,..,,
,
1
I
I
Injection of sample
.....
IU
l
\;t
~
Myristic acid (14:0)
....1-:ii
am
I
I
!.
-1>
-·...,.s
\.1'1
f;::
0\
i'"I;::S
.::__
c:::::::::--=-=
c:::_______
OJ
I.
l
Stearic acid ( 18 1 0 l
Oleic acid (18:1)
(J)
I .
I
Unidentified
~
c+
.I
I
=
Palmitic acid (16:0)
II
I
I
Linoleic acid (18:2)
-4
CD
\0
)"
..
Linolenic acid (18:3)
~----------·J
~
52
0.
Table 15.
Retention times and standard deviations of the fatty
acid methyl esters of cultures of Neurospora crassa,
Lindegren+ grown on C an~ M media for four days at
25~ C. The data represent the results of five duplicate ·
experiments.
Cultures grown on M medium
Peak
1
2
3
4
5
6
1
Average
Retention Time
1.63.,!'.11
2.80+.23
3.91,!-.41
4.93_!.53
5.40+.-81
6.77:z.46
9.04.:!:.76
-
-
Cultures gro"WD. on a medium
1
1.56+.44
2
2.67.z.48
3.98,!62
3
4
5.03+.53
5.63,.t.74
5
6
6.75+54
8.71•·.76
i
7
-
I
-
Identity
myristic ac1d
palmitic acid
unidentified
stearic acid
oleic acid
linoleic acid
linolenic
myristic acid
palmitic acid
unidentified
stearic acid
oleic acid
11nol eic acid
linolenic acid
'
I
I
II
!..
I
~_j
5:5
Table 16.
Retention times and standard deviations of the fatt7
acid methyl esters of the triglycerides of
Neurospora crassa, strains 28A and ?A grown on C
medium for four days at 25°0. The data represent the
results of three duplicate experiments.
28A
Peak
Average Retention
Time
1
2
1.56.:,t.l4
3
5.02,±.17
5 .. 76.;!:·.10
6 .. 69_:t.l:l.
4
5
·~.68_!.1"4
Identit7
myristic acid
palmi tic acid
stearic acid
oleic acid
linoleic
7A
1
2
3
1.49.;t.l2
2.52+.00
4.7l_t.23
4
5
6.91. +.17
5 .. 66+.08
-
-
acid
palmi·tic acid
s•cearic n.cid
oleic acid
lin-oleic acid
myris"~io
54
contains the peak identified as linolenic acid or the
unidentified peak With a retention time of approximately
3.90 minutes.
The .fraction of the total which each .fatty acid
represents can be determined by taking the ratio
of the area beneath one fatty acid methyl ester peak
to the total area. under all peaks.
This was done
With the GLC data of the Lindegren+ fatty acids and
the 28A and 7A fatty acids.
Table 17 gives the
average fraction of the total whi.ch each fatty acid
I
methyl ester represents and contrasts the
I
grown in 0 and M media.
cultures
The data show that the
primary difference is in the saturated and
unsaturated o18 fatty acids. The total o18 content
of both differs by 3.8%. However, the total fraction
of unsaturated :fatty acids in the cultures grown on
C medium is .558 or 47.3~ greater the~ theM medium
cultures.
Table .18 gives the average fraction of each fatty
acid to the total fatty acid content of 28A and 7A.
The data shnw that the primary difference between the
two is that 7.! contains a small fraction of linolenic
acid ( .026) Whereas linolenic acid 1s not detectable in
28A..
Strains 28A a.nd 7 A differ from the Lindegren+
L_:.ul ture s 1n that neither has detectable amounts of
I
i
55
linolenic acid (18:3) and that both have a higher
total fraction of c18 fatty acids.
I
··----·-------·-·~---J
------
--------------·
I
I'
Table 17.
Fatty acid compoa1·t.1on
ar~d
standard ,deviations of the tr1_glycer1des of :f'ou.r
day old cultures of Neurp~pora crases., L1ndegren+ grown on 0 and M media
at 25°0. !he data represent the reaulta of five duplicate experiments.
l.i'attr acid
myristic (14:0)
palmitic (16:0)
unidentified
stearic (18:0)
oleic (18:1)
linoleic (18:2)
linolenic (18:3)
I
l_
-
Average fraction
of the total for
cultures grown in
M medium
-
.044+.015
• 281,±.007
.007+.000
• 242_±.027
.026+.015
.299+.022
.127+.006
-
Average fraction
of the total for
cultures grown in
0 medium
-
.• 046+.004
.311+.033
.010.,!.002
.090.,!040
.163+.037
• 332+.033
.063+.027
-
..
.
IJ1
0'\
"---------------·
·---------··~---
Table 18.
Fatty acid composition and standard deviations of the triglycerides of four
day old cultures of Neuros~o~a £ra~sa, strains 28A and 7A grown in 0 medium
at
2s•o.
!he data represent the results ot three duplicate experiments.
Fatty acid
myri a tic (14:0)
palmitic (16:0)
stearic (18 :o}
oleic (18:1)
linoleic (18:2)
linolenic (18:3)
Average fraction
ot the total for
cultures of 28A
.Oll.z.002
.232.z.026
.269.,:t.080
.476,.:t.02l
Trace
not detectable
Average fraction
of the total for
cultures of 7A
.011.,!.003
.256..±.074
.245+.016
,.459 +.026
--
.026+~000
not detectable
I
I
I
L
..
... -· -- I
~
58
.-1
r
I
i
DISOUSSIOI'
The developmental events leading to the formation
of aerial hyphae and conidia can be described as taking
place in an ordered sequence:
asexual
vegetative
extension
conidia
spore
--4•-growth
--.-or aerial__._formation
germination
hyphae
Lindegren~
I
II
l
I
grown on conidia suppressing media (M)
: and conidia :promoti.ng media ( 0) as well as strains of
I
I
Neuro_sl!_or!
whiah form aerial hyphae w1 th conidia (28A.)
• e.nd aerial hyphae w1 th essentially no conidia {71) have
proyidsd in£ormation that affords a comparison between
.!
I
·lipid content·. triglyceride content and stages in the
:above sequence.
I
Growth of Lindegren+ on M medium
!
'has allowed f'or the examination of lipid and triglyceride
i
content at the stage of vegetative growth only.
!
The
1
I
growth of 7A on C medium has enabled the lipid and
1
!
I
triglyceride content at the stage of extension of
aerial hy-phae to be examined.
The groll"'th of L1ndegren+
:ceride content at the stages of aerial hyphae extension
I
Il-··--·------·---~--------"·"·-·--··----··-·-·---·
I
1
I
:and 28A on 0 medium has allowed the lipid and trigly-
·and conidia form.ation to be exa.miued.
I
l
i
I
I
___.,_J.
59
II· Identification
or
Lipid 01asses '
The lipid extracts of the cultures of Lindegren+
grown on 0 and M media and the cultures of 28A. and 7A.
!grown on C medium were all found to contain phospho-
!!lipids,
I
·~sterol
stero1s, free fatty acids, triglycerides and
,by Bianchi and
i
This complement of lipids has been found·
esters.
~urian
(1967) and Mull;
!! !!• (1944).
jin addition to these components, Lindegren+ grown on M
!medium had an additional polar fraction Which
icpromatographed between the phospholipid fraction and
lthe sterol fraction.
Since this fraction was absent
t
!from the cultures ot Lindegren+ grown on 0 medium even
I1on
the second da7 when most of the groWing mycelium was
!submerged and growth wes principally anaerobic, it
I11s
probable that its
form~tion
was not a function of
!aerobic or anaerobic respl.ration..
Rather, 1 ts formation
lin the cultures grown ser.tl.-e.erobicall:r on M medium
I1could
have been
a reflection of the differences in
.
jcul ture media i.e., a:mmonium ion :ra:ther than nitrate ion.
Both 28A and 7A had a 11p1d fraction which was
intermedia.te in pols.r1 ty between
1
lrree fa tty acids
lthe
wh:1.~h
th~
sterols and the
was not pre sent in either ot
Lin~e~en~ cUltures,
The Rf value indicates the.t
1-t----~
60
r
!
is probably a diglyceride.
The presence of this
fraction cou1d not be correlated specifically With the
. ;
i
formation of' aerial hyphae or conidia. since it was absent
from the Lindegren+ cultures grown on 0 medium Which do
Strains 28A and 7A both
form aerial hyphae and conidia.
have much higher levels o! lipid/dry weight and triglyceride/dry weight ratios than L1ndegren+ grown on
C or M medium. The triglyceride/dry weight ratio of
28A in particular is 6.5 times greater than that of
The detection ot
Lindegren+ grown on 0 medium.
diglycerides in these cultures may
. level of triglyceride formation.
b~
due to the high
Diglycerides are an
intermediate in the formation of tr1glycer1des from
: oe -:Phosphatidic acid (Wh1 te !!
~·,
1968):
L- o<. -phospha:tidic acid~hg.:Eh~tas!I.D-1, 2-diglyceride + P1
fatt,- ao,-1 OoJ. +
J
triglyceride·
Syste~e
producing re1atively
l~rge
amounts of
i
; tr1glycer1des could be expected to ha·ve correspondingly
large quantities of triglyceride precursors such as
. diglycerides~
S1Lce the Lindegren+ strain produced
· lower levels of tr1g1ycer1des, the diglyceride level
!
may have been too low to detect.
I
IL
..
·--------·-~-· --·~-·------·
61
--l
II. Kinetic Analysis.
In the individual curves ot dry weight, total lipid
content and triglyceride content (Figures 1,3,5), two
intervals of logarithmic increase can be identified.
The
.first interval occurs between two and three days and is
I
i the customar;y pert.od or logari trJ.DliC growth determined
I!by
Emerson (1950), Cochrane (1958) and
l
IAlexander
(1960).
Marshall~
and
The data show that growth after three
, days is well approxim'\ted by a second logs.ri·tbmic phase.
The idealized model of the growth of bacterial
I
populations is generally divided into three sequential
phases: 1) lag phase with no apparent increase in
population, 2) log phase in which the
in~reaae
1s rapid
and exponential and 3) stationary phase With no net
Iincrease
I(1959)
{Mandelst, 1965).
It has been shown by Zalokar
and Stadler (1952) that growth occurs only at the
hyphal tips in the filamentous fungi and that growth is
not a function o.! cell number as 1.t is w1 th bacterle.,
but a. function of the number o! tips an.d thei:r rates
of growth.
Tb.eratore, variations .from the idealized
kinetic model, can be expected for the fungi.
Indeed,
variations have li>·een observed in this experiment.
In
particular, there appears to be a second logarithmic phase
- - - - - · - - - - ·--·---·--·_____j
62
Ipreceeding the stationary phase between the third and
fifth or sixth da7s for the increase in dry weight,
!lipid content and triglyceride content.
I
The instantaneous rate constant is a measure of
the relative increase of a parameter per unit of time
!during logarithmic increase.
These constants have
!been calculated for the periods of logarithmic increase
ll
!encountered .in this experiment and are summarized in
ITable 19.
A. Dr;r we1gh't
The constants k 1 and k 2 for the logarithmic increases
in the dry weights of cultures of Lindegren+ grown on
lc
and M med1.a (Table 19) are 1.62 and 1.74 respectively,
!
I
i1nd1cat1ng that both cultures are initially growing
I
1
.
at approximately tb:e same rate.
The difference in dry
, weight at the end of' three days of growth (Figure 1}
1
Ii may
I
reflect a sl.1ght1y longer lag phase for growth in
~ C medium.
~ul:tures enter into
Both
l
a second loge.r1 tbm1c
!phase between the 'third and f'if'th days.
constants ki end
t2 for
The rate
this interval of time are .399
and • 327 for the cul:tures grown on C and M media
I
jrespecti,rel;y.
The significance of this difference is
that now the growth rate in the·,_ a medium is 22% greater
than that in the .M m.edium.
This difference in the rates
, indicates th.at the cultures grown on M medium exhaust
lthe1r nutrients at a greater rate then in the 0 med1wa _ _j
63
·-~----,..-
Table 19.
Instantane•s rate constants determined :tor
periods of logari'thmic increase in dry weight,
lipid
accu~latlon
and triglyceride accumulation·
for oul turs of !T:eurosnora eras sa, Lindegren+
grown on C and X media at 25°0.
______
_:,....;.;_~_....;_...J
L
J
65
r-------------------~---:-"'--·--
initially and that once aerial hyphae break.the surface
in the 0 medium, the shift from reductive to oxidative
metabolism enables the cultures grown on .0 mediWI to
increase their rates of growth relative to the cultures
grown on M medium.
Cultures grown on M medium remain
mostly submerged and metabolism is primarily reductive.
It can be concluded that growth which is dependent on
· oxidative metabolism results in a faster rate of
accumulation of cellular materials than
. metabolism.
redu~tive
Indeed, Bianchi and Turian (1972) have
shown that the cell wall dry weight to total dry weight
ratio is greater in cultures grown on 0 medium than on
· M mediu.m.
The rate constants k
and k 4 determined for the
dry weights of strains 28A and 7A in the period
3
between two and three days are 2.37 and 2.15 respective17 •
. Growth of 28A. and 7 A 1 s simil.ar through the first five _
days even though. 7 A. forms aerial hyphae but no conidia.
Both 28A and 7A grow at a !aster rate than Lindegren+ on
C and M media.
The conidiating strain, 28A, and the
non-conidiating strain 7A, both form aerial hyphae
and undergo a much greater rate ot growth than Lindegrea+
I
which grows strictly vegetat1ve1T on M medium.
!his
further supports the contention that the formation of
aerial hyphae and the accompanying shift to
oxidative metabolism allows for an increased rate
l----------·--·-------------·--..----··----·-·
l
I.
I
66
,-------·----·---·
I
: of . growth in fungal cultures.
I
B. Lipid
Table 19 gives the instantaneous rate constants for
• the logarithmic phases With respect to lipid increase for
the cultures of
~indegren+
grown on 0 and M media.
These are 1.05 and 1.46 respectively.
The constants for
the second logari thmio phases, ks and
k6 , '·are
.184 and
.220 for the cultures grown on 0 and M media respectively.
When these constants are compared to the dry weight
constants over similar time intervals, one obser1es that
'the rates of increase in dry weight in cultures grown
on C and M media are greater than the rates of increase
ot lipid.
This shows that the amount of non-lipid
material accumulates at a faster rate than lipid material
regardless of whether or not conidia are forming or if
the culture is
i
grow~ng
strictly vegetatively.
The cultures grown on M medium in this experiment
had higher
rat~s
of lipid accumulation than cultures
grolm on 0 medium.
The carbon-nitrogen ratio (Cochrane,
1958) is an :lmportant factor in the rate or lipid
formation.
Morton and MacMillan
'that n1 t!"ogen concentration in
(1954) showed
ft.~ng1
1s higher
when
ammonium salts are used tha.:n when n1 trates are used in
culture medium.
Since M medium is prepared With
ammonium citrate while 0 medium is prepared with
I
.I
___,_j
67
I
l
~:
-~-
_____________
,
68
production of ethanol.
Reoxidation of this
c2
compound
to acetate during the shift to oxidative metabolism
induced by the extension of aerial hyphae in 0 medium
would provide a marked increase of substrate for lipid .
. s,ynthesis at the time when conidia formation is at a
lmaximum.
The decline in the lipid to dry weight ratio
!noted between four and five days in cultures grown on
IjO
medium could be attributed to the exhaustion of
!ethanol
in the medium.
I
Oulevey and Turian (1968) have
!round that ethanol gradually disappears from cultures
!grown in 0 medium after three days i.e. during the time
I
of maximum conidiation.
l
C. Tri glyceride
The kinetics of triglyceride metabolism appear to
Ijparal1~1: .c1oitel:t
the pattern for total lipid accumulation
lin cultures grown on M medium as shown in Figures 3 and 5.
!The rate constan-t, k8 for the first logarithmic phase of
!triglyceride accumulation in cultures grown on M medium
Ilis
1.42 as compared to 1.46 (k 6 ) for total lipid
1aoeumulat1on over the same interval. After three days,
!the rate of increase of triglyceride is slightly greater
I
lthan that of total lipid.
I
Triglyceride accumulation 1.n cultures of Lindegren+
grown on C medium deviates substantially from total lipid
/accumulation.
fhe initial rate constant,
k.r•
ts .709
'
las
oom:ared_ to 1.05 (k5 J for total lipid accumulation C=·
J
69
lin C medium and implies that triglyceride accumulation
1
lags behind total lipid accumulation. After four days,
1
triglyceride accumulation reaches a stationary phase
I!With
•
no net increase.
This appears to be associated
Jwith the differentiation o:f conid1a 1 :for after :four days
lfew conidia are formed.
The peaks in the triglyceride
Ito dry weight ratio and the triglyceride to lipid
!ratio (Figure 6, Table 12) for cultures grown on 0
I1 medium
[With
.
also seem to reflect events which are correlated
the differentiation of conidia and which do not
·take place in cultures which grow strictly vegetatively.
/It is apparent that after :four days,when tew conidia are
!formed, these events are no longer operative, and the
!ratios consequently decline.
D. Mutant Studies
One of the characteristics of the mutant 7A under
!the conditions of this experiment is that it does not
lrorm conidia at four days of growth, a time when the
I
! parent strain 28A formed large quanti ties of conidia.
I
'strain 28A was found to contain nearly 20% more total
lipid/dry weight and triglyceride/dry weight than did
.7A.
This supports the conclusions drawn from the data
lor Lindegren+ grown on C and M media with respect to
Ilipid and triglyceride accumulation, n""'ely, that
~lipid
Jclosely related to the :formation of conidia.
I
Jl
metabolism and t:riglycertde me·tabolism are
·-""------,-----·-
·
-~--
70
l
III. Gas-liquid Chromatographic Analysis •
•
Gas-liquid chromatography of the .fatty acids from
the triglycerides of both the cultures ofLindegren+
l!grown on C and M media
showed that both were composed
o~
!the same fatty acids (Table 15), but that there was a
I
!significant difference in the fraction of unsaturated
i
I
!fatty acids
!c
{~able
17).
In the cultures grown on
medium, 56% of the total fatty acids of the
I
ltr1glycerides were unsaturated,
w~reas,
jgrown on M medium., 38% were unsaturated.
I
in the cultures
Cultures
grown on M medium appear to have a reduced capability
to carry out the desaturation steps for the series of
reactions:
stearic ac1d(1B:O)
• oiefc a.cid(18:1)
----•-linoleic acid(l8:2)
,_
-linolenic ac1d(l8:3).
71
0
.
runsaturation in the cultures grown on C med1um·consistent with the known biosynthetic pathways of fatty
acid formation.
These pathways have been elucidated
by many workers (Majerus and Vagelos, 1967), (Green
and Allman, 1968), (Erwin and Block, 1964).
Brody
and Nyc (1970) have shown ·that the levels of reduced
pyridine nticl.eotides are directly related to the degree
of unsaturation of the fatty acids of the triglycerides
-
of N. crassa.
....
Bloomfield and
Block
.
(~960)
have shown
that the unsaturated fatty acids arise from the saturated
fatty acids and James (1962) has shown that unsaturation requires molecular oxygen and reduced
pyridine nuc1eotides.
The increased capability of the
cultures :grown on C medium to carry out the unsaturation
steps seems to be directly related to a major developmental stage in the :morphogenesis of conidia, n8JJ1ely,
the extension of aerial hyphae and the accompanying
increase in molecular oxygen.
The GLO data obtained from the methylated fatty
acids of the triglycerides of 28A and 7A provides
I
additional support :f'or the conclusion that the extens1ol1
j
of aerial hyphae leads to a higher degree of unsaturation.
1.
1
Although both cultures grow with an extensive system ot
aerial hyphae and desaturation stops for the most parl
st the production of oleic acid (Table 18), the total
II
..
.
·
II
L _ _ _ ,_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _:.._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _.,
72
uneat~~t;.;:- o;~th ~8J.-.,;d 7~~s
1
nearly
th~l
(.476 and .485) and significantly, these values are
I
higher than for cultures of Lindegren+ gr.own on X
j
medium which form no aerial hyphae.
The use of Lindegren+, 28A and 7A in this study has
brought out the direct correlation of lipid metabolism 1•
general and tr1glycer1pe metabolism in particular With
the deveiopmental events that lead to the formation of
· conidia.
Singularly important to conidiation is the
.extension of aeria.l hyphae from submerged mycelia.
The
: analysis of growth kinetics and lipid accumulation 1A
Lindegren+ has shown that the extension of aerial hyphae
is directly related to the rate ot lipid accumulation,
triglyceride accumul.ation and tatty acid unsaturation.
The peaks in the ratios of lipid/dry weight and
triglyceride/dry weight during maximum conidia formation
of Lindegren+ are particularly significant since these ·
peaks were not observed in the
non-con1d1~ting
cultures.
Growth of 28A and 7A revealed that conidiation itself 1a
correlated with the increase in lipid and triglyceride
content.
Tbe questions of what roles are played by the
increases in unsaturat1on of the triglyceride !atty_ac1ds
and the rate of lipid accumulation in the formation of
conidia remain unanswered and the abject of future
• studies.
_,__________
·-·--·~--~----·--------·--·-·--·--·~·-·---------··-----------J
73
r------.
-------------
----~---
REFERENCES
· 1.
2.
Bar-Lew, s. 1973. Masters Degree Thesis. California
State University, Northridge.
D.E. and G. Turian. 1967a. Lipid content of
conidia of Neurospora crassa. Nature. 214:1344-1345.
Bianchi~
_____ and -----· 1967b. The effect of nitrogen
source and cysteine on the morphology, conidiation
and cell wall fraction of conidial and aconidial
M!urospora crassa. Zeitschr. Allg. Mikrobiol.
7:257-262.
-I
· 4.
: 5 ..
Bloomfield, D.K. and X. Block. 1960. The !ormation
of' /). 9 unsaturated fatty acids. J .Biol. Chem.
235:337-345.
Brody,
s., ·and
Il
l
J.F. Nyc. 1970. Altered fatty acid
d1stibut1on in mutants of Neurospora crassa.
J. Bacterial. 104:781-786.
6.
Cochrane, v.w. 1958. Physiology of Fungi. John
Wiley and Sons, New York.
: 7.
Emerson, s. 1950. The growth phase in Neurospora
craasa corresponding to the log phase 1n unicellular
organisms. J. Bacterial. 60:221•223.
' 8..
Green, D.E. s.nd D. W; Allman. 1968. The biosynthesis
of fatty acids. In D.M. Greenberg (ed.), Metabolic
Pathways, vol. 2. Academic Press, New York.
i ·.
I
.
I
t. . _. . . . . .:~~---~----·------·-~--'''"'~'- •···--~·~··------·~·~·--·-~------ ~--·---------~-A-~---···----- ---~~--~~---. -1
74
~-·-------~
9.
l
James, A.T. 1962. The biosynthesis o:t unsaturated
fatty acids in isolated-plant leaves. Biochem.
Bio:phy's .. Acta. 57:167-169'.
10. Xobr, J.M., Bianchi,D.E., Oulevey, N. and G. Turian.
1965. The e~fect o:t oxygen tension on growth,
conidiation and alcohol production of Neurospora
crassa. Canad. J. Microbiol. 13:805-809.
11. Krzeminski, L.F. 1960. Conversion of some c14
labelled compounds into neutral lipids ot
Neurosnora crassa. J. Am. Oil Chemists Soc.
37:371-373.
12. Lein, J. 1953. Unsaturated fatty acid metabolism
in Neuro~l{Ot!• Arch. Biochem. Biophys. 45:434-442.
I
.I
II
I
· 13. Majerus, P.W. and P.R. Vagelos. 1967. Fatty acid
biosynthesis and the role o:t acyl carrier proteiD..
Adv. Lipid Res. 5:1-33.
· 14. Mandels, G.B. .. 1965. The kinetics of fungal growth.
In G.o. Ainsworth and A.S. ~ussman (eds.), The
Fungi, vol. 2. Academic Press, New York.
15. Marshall, K.o. a.nd M. Alexander. 1960. Growth
charaote:rtstics of fungi and actinomycetes. J.
· Bacteriol. 80:412-416.
16. Mazl1ak, P. 1973. L1p1.d metabolism in plants.
Ann.
Rev~
PJ.aut Physiol. 24:287-310.
· 17. Morrison, W.R~ and L.M .. Smith. 1969. Preparation ot
tatty acid methyl esters and dimethyl acetyls from
i
l1p1ds With boron-!luoride-mothanol. J. L1p1d Res.
L--··--··--------------·-··--·------·----·-·-·-·-·--·---~- ..-.--------·----·-·-----..---..
•
I
Jl
75
~----­
!!
5:600-608.
· 18. Morton, l.G. and A.M. MacMillan. 1954. The·
assimilation of nitrogen from ammonium salta and
nitrate in fungi. J. Exp. Bot. 5:232-252.
· 19. Mull, R.P. and F.F. Nord. 1944. On the mechanism
of enzyme action. Part 24. Fat formation 1n 1usar1a.
Arch. Biochem. 5:283-290.
i
I
Turian •. 1968. Centrale
.metabolique et aspects ultrastruoturaux .de la
conidiatton de Neurosp2r~ crassa. Arch. Mikrob1ol.
20. Ouleve:r, N. and G.
60:35-58.
I
21. Prill, E.A., Wenck, P.R. and W.H. Peterson. 1935•
III. The chemistry of mould tissue. VI. Factors
influencing the amount and nature of the !at
produced by Aspergillus Fischer!. B1ochem. J.
29:21-33.
I
22. Radin, c. 1957. Preparation of lipid extracts.
In s.P. OoloWick and N.O. Kaplan (eds.), Methods
in Enzymology, vol.14. Academic Press, New Yo~k.
!
. 23, Ryan, FsJ. 1950. Methods in Neurospt!!'_E! genetics.
In R.W. Gerard {ed.), Methods in Medical Research,
vol. 3· The Year Book Publishers, Inc. Chicago.
2.l~ ..
Stadler, D.R. 1952. Chemotropism in Rhizopus
Nigrlcans. J. Cell. Oomp~ Phys1ol. 39:459-474.
25o S·tanier, R.I.,
Doudero~f',
M. and E.A. Adelberg. 1970.
The Microbial World, 3rd ed. Prentice Hall, Inc.,
l
I
I
I
I
I
76
,----------·-------------
l
1
26. Turian, G. 1964. ~nthetic conid1ogenous media tor
Neurospora crassa. Nature vol. 202, no. 4938.
21.
l
• 1965. Reserches sur la morphogenese dee
Neurospo:t,!. Arch. Sci., . Geneve. 18:371-381.
28.
and D.E. Bianchi. 1972. Conidiation in ·
Neurospora. Bot. Rev. 38:119-154.
29. Weiss, B. 1965. An electron microscope study
Neur~spor! crassa during development.
J. Gen. Microbial. 38:85-94 •
o~
. 30. White, A., Handler, P. and E.L. Sm1 th. 1968.
Principles ot Biochemistry, 4th ed. McGraw-Bill
Book Co., New York
. 31. Zalokar, M. 1956. Growth and differentiation of
Neurospora hyphae. Amer. J. Bot. 46:602-610.
I
I
I
I
i
I
I
!
-
.
!
L------------·~----.----------------------------···------------------------~