JOURNAL OF BACTERIOLOGY, October 1973, p. 67-73Copyright 0 1973 American Society for Microbiology

Vol. 116, No. 1Printed in U.S.A.

Protein Synthesis During the Differentiation ofSporangia in the Water Mold Achlya

WILLIAM E. TIMBERLAKE, LARRY McDOWELL, JOHN CHENEY, AND DAVID H. GRIFFIN

Department of Botany, State University ofNew York, College ofEnvironmental Science and Forestry, Syracuse,New York 13210

Received for publication 25 May 1973

During the synchronous differentiation of sporangia in the absence of addednutrients, the water mold Achlya bisexualis (Coker and Couch) activelysynthesized protein. Inhibition of protein synthesis at any time during thesporulation process completely inhibited further differentiation. Large changesin the rate of radioactive amino acid uptake resulted in changes in the specificactivity of the cellular amino acid pool. The rate of protein synthesis was

calculated from the amino acid pool specific activity and the incorporation ofisotope into protein. During the 1st h after induction of the sporulation process,

the rate of protein synthesis increased to two times the initial value. The aminoacid precursors for this synthesis were supplied by the degradation of preexistingprotein. Proteolytic enzyme activity assayed in vitro increased in proportion tothe in vivo rates of protein synthesis and degradation. Differentiation was

accompanied by a slight decline in dry weight of the mycelium as well as by a

decrease in the protein content, whereas the relative size of the amino acid poolsremained constant.

The control of the induction of the differen-tiation of sporangia and subsequent formationand release of spores has been well delineatedfor the aquatic o6mycete Achlya bisexualis (3).Furthermore, the morphological changes associ-ated with differentiation have been examinedand correlated with certain biochemical events(4). The high degree of synchrony associatedwith the differentiation of whole cultures ofAchlya, the ability to grow vegetative culturesunder defined conditions, and the ease withwhich both growing and differentiating culturesare handled experimentally make Achlya an

ideal organism for the study of physiologicaland biochemical events associated with theproduction of asexual reproductive structures.The fact that induction of the sporulation

process in Achlya involves the removal of thesupply of organic nutrients (3) shows that thecontinued synthesis of macromolecules mustdepend on the utilization of cellular storesformed during vegetative growth. Although theincorporation of radioactively labeled aminoacids into protein during the differentiation ofreproductive structures by fungi under starva-tion conditions has been shown (7, 9), little isknown about the actual rate of synthesis or theorigin of the cellular precursor pools under theseconditions. The objective of this paper is to

show that Achlya actively synthesizes proteinduring differentiation at a rate considerablygreater than that observed at the time ofinitiation of the sporulation sequence and thatthe source for the amino acids required for thissynthesis is the degradation and reutilization ofpreexisting protein.

MATERIALS AND METHODSCulturing and induction of sporulation. Achlya

bisexualis (65-1) was grown as described by Griffinand Breuker (4) in a medium containing 5.00 g ofD-glucose, 1.25 g of peptone, and 1.25 g of yeastextract per liter of glass-distilled water (PYG). Me-dium (80 ml) in 250-ml Erlenmeyer flasks was inocu-lated with 2 x 106 spores and incubated at 30 C on arotary shaker at 150 rpm for 16 h. Under theseconditions, the fungus grows as a uniform suspensionwithout forming pellets. To induce synchronous spor-ulation, these were harvested and washed with 200 mlof 0.5 mM CaCl, on a sintered glass filter withsuction, taking care not to draw air through themycelium. These mycelia were then transferred into 1liter of 0.5 mM CaCl, in a 2-liter Erlenmeyer flask andincubated at 30 C on a rotary shaker at 60 rpm. Underthese conditions, the differentiation of sporangia andspores was 20 to 30% faster than previously reported(4). Cultures were examined microscopically justprior to induction and at 1-h intervals during differen-tiation for bacterial contamination and normal mor-phological development of reproductive structures.

67

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

TIMBERLAKE ET AL.

Analytical techniques. Dry weight, protein con-

centration, and amino acid pool size were measured as

a function of time after the induction of sporulation.For the measurement of dry weight, 100-ml samples ofthe CaCl, suspension were harvested on preweighedWhatman no. 1 filter papers, washed with 0.5 mMCaCl,, and dried at 60 C. Amino acid and proteinmeasurements were performed by harvesting 50-mlsamples as above and placing the washed myce-lium in 2 ml of ice-cold 1.5 N formic acid. Preliminaryexperiments showed that this technique was as effec-tive as cold 10% (wt/vol) trichloroacetic acid or 1 Nperchloric acid in precipitating proteins within themycelium. After 1 h each sample was filtered througha glass fiber filter (Reeve-Angel 934 AH) and washedwith 1 ml of cold 1.5 N formic acid. The filtrate was

collected and evaporated to dryness at 60 C. Theresidue retained by the filter was placed in 5 ml of 10%(wt/vol) trichloroacetic acid and extracted in a boilingwater bath for 15 min. The hot acid-insoluble mate-rial was collected on a glass fiber filter and washedwith 30 ml of cold trichloroacetic acid and 20 ml ofice-cold ethanol. The residue was then extracted with2 ml of 1 N NH4OH at 80 C. Insoluble material was

removed by filtration through a glass fiber filter, andthe filtrate was dried at 60 C.

Both the cold acid-soluble and hot acid-insolubleextracts were dissolved in 0.2 ml of glass-distilledwater and assayed for Folin phenol reactive materialby a modification of the procedure of Lowry et al. (4,6). The cold acid-soluble extract was also assayed forninhydrin reactive material by using the method ofRosen (8). Standard samples of bovine serum albuminand casein hydrolysate were measured by the same

procedures and used to estimate the amount ofacid-insoluble protein and acid-soluble amino acids.At all stages of differentiation, the estimates of aminoacid pool size by the methods of Lowry and of Rosenagreed within 10%.

Total nucleic acid and protein content of themycelium before induction and in the spores pro-

duced by an equal amount of mycelium was deter-mined by a modification of the procedure of Smillieand Krotkov (10) as described by Griffin and Breuker(4). For these determinations, 10 mg of exponentiallygrowing hyphae were collected by filtration, washed,and killed in cold 10% trichloroacetic acid. Another10-mg sample was collected and induced as above.After essentially all the sporangia had discharged, themycelium was removed by filtration on Mirra clothand washed extensively with 0.5 mM CaCl,. Thespores were collected from the filtrate by centrifuga-tion for 10 min at 5,000 x g and were placed intrichloroacetic acid as before. The samples were thenprocessed in parallel. Protein was quantitated by themethod of Lowry et al. (6) and nucleic acid byabsorbancy at 260 nm.

Measurement of the rates of amino acid uptake,incorporation, and protein synthesis. To measure

the rate of uptake of amino acids, 0.1 MCi of 3H-aminoacid mixture per ml (Schwarz-Mann, yeast profile) was

added to the CaCl, suspension at intervals during thesporulation sequence. Samples (40 ml) were collectedon glass fiber filters, washed with 50 ml of 0.5 mM

CaCl,, and dried at 60 C. These were weighed, andradioactivity was determined in 5 ml of a toluene-based scintillation fluid containing 3.2 g of 2,5-diphenyloxazole (PPO) and 0.3 g of dimethyl 1,4-bis-2-(5-phenyloxazoly benzene (POPOP) per literand 0.05 ml of NCS solubilizer (Amersham-Searle) ona Packard Tri-Carb 3375 scintillation counter.

For determination of incorporation and proteinsynthetic rate, cultures were pulse labeled with 3H-amino acids as above, and amino acids and proteinwere prepared as previously described. Amino acidcontent was determined as described under the para-graph heading "analytical techniques," samples ofeach preparation were dried in scintillation vials, andradioactivity was determined in the toluene-PPO-POPOP-NCS scintillation fluid. The data were usedto calculate the specific activity of the amino acidpools.Measurement of protein degradation, turnover,

and proteolytic enzymes. Protein was labeled ingrowing cultures by the addition of 2 ,Ci of "C-aminoacid mixture per 80 ml of medium (Schwarz-Mann,yeast profile) at 13 h after inoculation, and incubationat 30 C continued for 2 h. The culture was thenharvested aseptically on sintered glass with suction,washed with 100 ml of unlabeled PYG, and resus-pended in 80 ml of fresh unlabeled medium. After onemore hour of incubation, the culture was harvestedand induced. The net loss of protein, the change in thespecific radioactivity of prelabeled protein, and thespecific rate of protein synthesis were used to calcu-late the in vivo rate of protein degradation andfraction of amino acids degraded from preexistingprotein which were utilized for new synthesis.An estimate of the proteolytic enzyme content of

the differentiating cultures was obtained by homoge-nizing a 200-ml sample harvested as above in 10 ml of10 mM tris(hydroxymethyl)aminomethane (Tris) and1 mM CaCl,, pH 7.5 at 4 C, for 1 min at top speed inan Omnimixer (Sorvall, Inc., Norwalk, Conn.) withglass beads. Cell walls were removed by centrifuga-tion, and protein concentration was determined byabsorbancy at 260 and 280 nm and adjusted to 0.7mg/ml. In vitro protease activity was determined bythe hydrolysis and concomitant increase in absorb-ancy at 247 nm of p-tosyl-L-arginine methyl ester, bythe hydrolysis of N-benzoyl-L-tyrosine ethyl ester andincrease in absorbancy at 256 nm, as described byHummel (5), as well as by the degradation of bovineserum albumin. In this procedure, 5 mg of bovineserum albumin was incubated with 100 Ag of proteinfrom the cell extract in 1 ml of 10 mM Tris, 1 mMCaCl2, pH 7.9 at 25 C for 24 h at 25 C. The reactionwas stopped by adding 1 ml of cold 10% trichloroa-cetic acid. The protein precipitate was removed bycentrifugation, and the absorbancy of the supernatantfluid at 280 nm was measured against a zero timecontrol.

Effect of cycloheximide on sporulation. Cy-cloheximide was added to induced cultures at 1-hintervals starting at 0 h postinduction to a finalconcentration of 2.5 gM, the minimal concentrationwhich completely inhibits vegetative growth andprotein synthesis (W. E. Timberlake, unpublished

68 J. BACTERIOL.

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ACHLYA PROTEIN SYNTHESIS

data). At 5.25 h, the cultures were examined micro-scopically for normal development.

Reagents. Para-tosyl-L-arginine methyl ester hy-drochloride and N-benzoyl-L-tyrosine ethyl ester wereobtained from Schwarz-Mann; Trizma base and PPOwere from Sigma; Folin phenol reagent was fromFisher; dimethyl POPOP was from Amersham-Searle; and cycloheximide was from Calbiochem. Thecomposition and specific radioactivity of the 'H and4C L-amino acid mixtures are listed in Table 1. Allother chemicals were reagent grade or better.

RESULTSInhibition of the differentiation of spor-

angia and spores by cycloheximide. The addi-tion of 2.5 uM cycloheximide, an inhibitor ofprotein synthesis in eukaryotes (2, 13), to sporu-lating cultures arrested the developmental proc-ess at the stage during which the antibiotic wasadministered (Table 2). Complete inhibition ofdevelopment was observed even in the 5-htreatment when sporangia were actively dis-charging spores. The addition of the drugstopped the release of spores by undischarged,but fully cleaved, papillate sporangia. Controlcultures incubated in the absence of cyclohexi-mide differentiated sporangia and releasedspores as described previously (4).Changes in dry weight, protein content,

and amino acid pools during differentiation.Because the induction of sporulation includesthe removal of all exogenous organic nutrients,the developmental process must be maintained

TABLE 1. Composition and specific radioactivity of3Hand 14C L-amino acid mixtures

3H 14C

Amino acid ACi/mCi of specific specificmixture activity activity(Ci/mmole) (mCi/mmole)

Alanine 70 3.5 156Arginine 60 2.78 312Aspartic 100 1.0 210

acidGlutamic 120 15 265

acidGlycine 50 20 104Histidine 20 3.5 312Isoleucine 60 39 312Leucine 100 58 312Lysine 80 30 312Phenylala- 70 7.0 455

nineProline 45 27 258Serine 50 1.2 158Threonine 55 0.82 208Tyrosine 45 52 460Valine 75 6.0 260

TABLE 2. Inhibition of sporulation by cycloheximidea

LOI ~

C 0 1-2 hr 3hr 4hr 4.5hr ShrNONE A A

* A

2 A A

3 A A

4 A~ ~~~~A

S A A

a Cycloheximide was added to differentiating cul-tures at the times shown to a final concentrationwhich completely inhibited protein synthesis. At 5.25h after induction, the cultures were scored for round-ing of hyphal tips, refractility, septum and papillaformation, and release of spores. X denotes the stagesobserved at 5.25 h for each treatment. The time atwhich each of these stages normally occurs is shown.

by the utilization of endogenous materialsformed during growth. That cycloheximide in-hibited differentiation at all stages indicatesthat protein synthesis, a process utilizing energyand amino acids, was required. We were there-fore interested in whether there were changes inthe dry weight, protein content, or amino acidpools during the differentiation process.There was a small decline in the dry weight of

the fungus as well as a decrease in proteincontent of the mycelium (Fig. 1). On the otherhand, the acid-extractable amino acid contentof the differentiating mycelium remained con-stant throughout the developmental sequence.There were no detectable amino acids in the 0.5mM CaCl2 wash when it was assayed by thetechniques described in the Materials andMethods section, which showed that there islittle or no loss of intracellular amino acids dueto the procedure used.To estimate the contribution of vegetative

protein and nucleic acid to spore formation, thetotal cellular content of these components wasmeasured in the mycelium before induction andin the spores as described in the Materials andMethods section. The results of this experimentare shown in Table 3 and demonstrate that theamount of these components found in the spores

STAGES OBSERVED AT 5.25 HRAND TIME NORMALLY FORMED

69VOL. 116, 1973

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

TIMBERLAKE ET AL.

2

E

1 2 3

HOURS AFTER INDUCTION

200

S.

100I

#:k

FIG. 1. Dry weight, protein and amino acid contentof the mycelium during differentiation. Samples foreach determination were collected and processed asdescribed under "analytical techniques" in the Mate-rials and Methods section. The observed amino acidconcentrations have been multiplied by 10 and pre-sented on the same scale as protein content.

is a large fraction of that in the vegetativemycelium from which the spores were formed.These data agree with our observations thatunder the conditions described virtually all ofthe hyphal tips are converted to sporangia, anda large fraction of the cytoplasm in the hyphaemigrates into the differentiating tips.Rate of uptake and incorporation of radio-

active amino acids. Because our data indicatedthat protein synthesis was required for thenormal development of sporangia and spores,that protein was degraded during the process,and that amino acid pools were not depleted, wedecided to measure the in vivo rates of proteinsynthesis, degradation, and turnover. It hasbeen shown that the rate of uptake of some

compounds by water molds changes duringsporulation and that these changes may influ-ence the observed rates of radioactive precursorincorporation into macromolecules (7). Anysuch changes in amino acid transport would ofcourse complicate the measurement of in vivorates of protein synthesis by the incorporation ofradioactively labeled amino acids into protein.

Figure 2 shows the results of experimentsmeasuring the rates of uptake and incorporationof 9H-amino acids during differentiation, as

measured over a 30-min time course at eachpoint indicated. After induction there was a

rapid and large increase in the rate of uptakeand incorporation into protein of the isotope.The maximum rate of uptake occurred at about2 h postinduction, whereas the maximum rateof incorporation was at about 3.5 h. Afterreaching the maximum values, both uptake and

incorporation rates declined, but never returnedto the rates observed at zero time. The extremechange in the rate of amino acid uptake indi-cates that the changes in incorporation intoprotein may not accurately reflect changes inthe synthetic rate, but rather differences in thespecific radioactivity of the amino acid pools.Rates of protein synthesis. To determine

the actual in vivo rate of protein synthesisduring differentiation, the specific radioactivityof the amino acid pools was estimated inparallel with the incorporation of 3H-aminoacids into protein, as described in the Materialsand Methods section, and was used to calculatesynthetic rates as micrograms of protein synthe-sized per minute per mg (dry weight) (Fig. 3).At zero time the rate of protein synthesis was

TABLE 3. Nucleic acids and protein in vegetativemycelium and spores

Substance Vegetative SporeSa %myceliuma In sporesb

Nucleic acid 106 68 64Protein 180 83 46

aThe data are shown as gg/mg (dry weight) ofmycelium. For the spores, the values are calculated onthe basis of the dry weight of mycelium used forinduction.

'Percent in spores is the amount of either fractionremaining in the spores over the amount in thevegetative mycelium X 100.

10--

E.*0

0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~S

otj.

HOURS AFTER INDUCTION

FIG. 2. The rates of uptake and incorporation intoprotein of 3H-amino acids as a function of time afterinduction of the sporulation process. At intervalsduring the differentiation of sporangia, 9H-aminoacids were added to cultures, and samples were

collected and treated as described in the Materialsand Methods section. The initial rates of incorpora-tion and uptake were calculated from the time course

with points at 0, 10, 20, and 30 min after the additionof isotope and are shown as per minute values.

_* *

_~~~~O

70 J. BACTERIOL.

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ACHLYA PROTEIN SYNTHESIS

2

zilz

O 1 2 3 4

HOURS AFTER INDUCTION

FIG. 3. Rate of in vivo protein synthesis duringdifferentiation. The specific radioactivity of theamino acid pools and the incorporation of label intoprotein were determined by collecting and processingsamples as described in the Materials and Methodssection at 0, 10, 20, and 30 min after the addition ofisotope. The time course of specific radioactivity ofthe amino acid pools and incorporation into proteinwere used to calculate the specific rate of proteinsynthesis.

0.72 gg per min per mg (dry weight). By usingthe protein content of the mycelium, the proteindoubling time was determined to be 3.1 h, avalue in close agreement with the dry weightdoubling time of noninduced cultures on PYGmedium (2.8 h). Because it has been shown thatprotein concentration as a function of dryweight is constant in exponentially growingcultures of Achlya (D. H. Griffiln, unpublisheddata), these calculations indicate that themethods employed to determine in vivo rates ofprotein synthesis are valid.During the lst h after induction of the sporu-

lation sequence, the rate of protein synthesisrapidly increased to a value two times greaterthan that observed at zero time. After thisinitial increase, the rate of synthesis remainedconstant throughout the rest of the develop-mental sequence (Fig. 3).Rates of protein degradation and turnover.

The fact that protein synthesis proceeds at arapid rate during sporulation, whereas there is adecline in total protein content of the myceliumand the precursor pools are not depleted, showsthat protein degradation must also occur at arapid rate. Amino acids so released could bereturned to the precursor pools and incorpo-rated into new protein. To determine the rate ofturnover, protein was labeled with 'IC-aminoacids, as described in the Materials andMethods section, during the growth phase of the

organism, and protein radioactivity was meas-ured in the absence of added label duringdifferentiation (Fig. 4). During the sporulationprocess there was a gradual decline in thespecific radioactivity of the protein, which re-sulted in a 24% decrease in specific activity atthe time of spore discharge.By using the protein content of the mycelium,

the rate of protein synthesis, and the "4Cprelabeled protein specific activity, the in vivorate of protein degradation and the fraction ofthe amino acids released, which were utilized inde novo protein synthesis, were calculated. The10-min rate of protein degradation, the fractionof degraded protein reutilized in synthesis, andthe fraction of the cellular protein turned overare shown as a function of time after induction(Table 4). Both the rate of protein degradationand turnover increased rapidly during the 1st hafter induction, and were then maintained at ahigh, constant value.

In another experiment, cultures were labeledduring growth with "4C-amino acids, induced,and pulse labeled with 3H-amino acids for 10min at 0.5-h intervals during sporulation. Afterpulse labeling, the cultures were harvested byfiltration and suspended in fresh CaCl2 with 50gg of casein hydrolysate per ml, and 2.5 uMcycloheximide was added to halt further incor-poration of isotope into protein. Protein radio-activity was measured immediately after the"chase-inhibition" was started and at 15, 30,and 45 min. The ratio of "4C to 3H radioactivity

g 20

0

w

uacW

0 1 2 3 4

HOURS A"ER INDUCTION

FIG. 4. The loss of radioactivity from protein la-beled with "4C-amino acids during the growth of theorganism. Protein was prelabeled during growth as

described in the Materials and Methods section. Atintervals during the differentiation of sporangia,50-mI samples were taken, and the amount of proteinand protein radioactivity was determined. Thesevalues were used to calculate the protein-specificradioactivity shown in the figure.

VOL. 116, 1973 71

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

TIMBERLAKE ET AL.

TABLE 4. Summary of protein degradation andturnover during the differentiation of sporangia

Protein de- ProteinHours after graded per 10 Reutilization turned overinduction min per mg (%)b per 10 mindry weight ()

(,g)a M

0 8.4 85.9 5.31 14.4 91.6 10.22 16.2 92.5 12.33 16.2 92.4 12.84 16.2 92.7 13.9

a The rate of protein degradation is equal to the rateof synthesis plus the rate of loss of protein as shown inFig. 1.

bReutilization was calculated from the actual lossof 14C radioactivity from prelabeled protein and thepredicted loss of radioactivity as determined fromFig. 1 and 4.

c The turnover value is the product of the degrada-tion and the reutilization divided by the proteincontent of the mycelium.

was calculated to ascertain whether there waspreferential degradation of either prelabeled ornewly labeled protein. At all times duringdifferentiation the isotope ratios remained con-stant after the "chase-inhibition" was begun,showing that degradation was general.Measurement of protease activity in vitro.

Protease activity was assayed in three differentways from whole-cell extracts prepared fromdifferentiating cultures at 1-h intervals afterinduction (Table 5). Protease specific activitymeasured under each condition paralleled invivo rates of protein degradation and synthesis,providing further support for the in vivo meas-urements.

DISCUSSIONThe water mold Achlya provides a valuable

system for the study of the biochemical eventsinvolved in the development of asexual repro-ductive structures. The differentiation of spo-rangia of whole culture's can be induced bysimple techniques, proceeds with a high degreeof synchrony, and has distinct changes in mor-phology which can be correlated with biochemi-cal changes. Furthermore, our data indicatethat a large fraction of the mycelium contrib-utes to the formation of spores. This means thatit is unlikely that biochemical events associatedwith the developmental process will be maskedby uninvolved processes. The developmentalsequence has been well documented under avariety of conditions (3, 11).As was shown by Griffin and Breuker (4),

ribonucleic acid (RNA) synthesis is required forthe normal sequence of developmental events inthis organism. We have shown that the ad-ministration of cycloheximide at any time dur-ing differentiation completely inhibits furtherdevelopment. Although it is generally assumedthat cycloheximide is a specific inhibitor ofeukaryotic protein synthesis, Cameron and Le-John (1) have shown that this antibiotic effec-tively inhibits both protein and RNA synthesisin Achlya. Thus, the cycloheximide data donot conclusively show that protein synthesis isrequired for the completion of the develop-mental sequence resulting in mature sporangiaand spores. However, as shown here, proteinsynthesis proceeds at a rapid rate throughoutthe process, indicating that it is an importantfunction during normal development.

Although the whole process of differentiationoccurs in the absence of exogenous nutrients,Achlya has the ability to convert endogenousstores to forms needed for the formation ofreproductive structures. The high rate of pro-tein turnover shown by this study is an excellentexample of the efficiency of reutilization by thisorganism. Protein turnover during the differen-tiation of spores has been demonstrated inbacteria (12) and the slime mold Physarumpolycephalum (9), a primitive eukaryote. Theprotein turnover observed in our experimentswas similar to that observed in bacterial sporeformation in that there was general degradationof proteins rather than the preferential degrada-tion of proteins formed during the growth phaseas observed in Physarum. However, the rate ofturnover during differentiation is extremely

TABLE 5. Protease specific activity duringdifferentiationa

Hours after TAME" BTEEc BSAdinduction

0 0.204 0.75 0.3071 0.461 1.77 0.6212 0.374 1.45 0.6723 0.503 1.93 0.7044 0.433 1.73 0.602

a Protease enzymes were extracted and assayed invitro during differentiation as described in the Mate-rials and Methods section. All data are presented asunits per milligram of protein.

b One unit is 1 Umol of p-tosyl-L-arginine methylester hydrolyzed per minute at 25 C.

c One unit is 1 Mmol of N-benzoyl-L-tyrosine ethylester hydrolyzed per min at 25 C.dOne unit is equal to a 280 nm absorbancy of

trichloroacetic acid-soluble hydrolysis products of0.001 per min per ml at 25 C.

72 J. BACTERIOL.

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ACHLYA PROTEIN SYNTHESIS

high, i.e., almost four turnovers during thedevelopment of sporangia, or about 75% perhour. Spudich and Kornberg (12) reported atumover rate of 18% per hour during the forma-tion of spores of Bacillus subtilis, with a devel-opmental time of about 14 h. Thus, althoughthe rate of tumover in Achlya is 4.2 timesgreater than that of B. subtilis, its develop-mental time is 3.1 times shorter. Because thevalue presented by Spudich and Kornberg isminimal, there is reasonable agreement be-tween the total number of turnovers during thedevelopmental times. The decrease in dryweight during differentiation could be the resultof the oxidation of cellular storage productsused for the production of high-energy com-pounds needed for both protein and RNA syn-thesis.The water mold A. bisexualis synthesized

protein at a high rate during the differentiationof sporangia under starvation conditions. Theactual rate of synthesis doubled soon after theinduction of the sporulation process. The pre-cursors for this synthesis were supplied by rapiddegradation and turnover of preexisting protein.Furthermore, changes in the capacity of thecells to transport radioactive amino acids dur-ing differentiation made it impossible to deter-mine in vivo rates of synthesis or degradation ofprotein without determining the specific activ-ity of the precursor pools. Further studies on thebiochemical events associated with sporulationshould provide valuable information regardingthe control of development in eukaryotes.

ACKNOWLEDGMENTSThis study was supported in part by grants from the

Brown-Hazen Fund of the Research Corporation, Providence,

R.I., and the Research Foundation of the State University ofNew York.We wish to thank Sally Timberlake for her technical

assistance in the preparation of the manuscript.

LITERATURE CITED1. Cameron, L. E., and H. B. LeJohn. 1972. On the

involvement of calcium in amino acid transport andgrowth of the fungus Achlya. J. Biol. Chem.247:4729-4739.

2. deKloet, S. R. 1965. The effect of cycloheximide on thesynthesis of ribonucleic acid in Saccharomyces carts-bergensis. Biochem. J. 99:566-581.

3. Griffin, D. H. 1966. Effect of electrolytes on differentia-tion in Achlya sp. Plant Physiol. 41:1254-1256.

4. Griffin, D. H., and C. Breuker. 1969. Ribonucleic acidsynthesis during the differentiation of sporangia in thewater mold Achlya. J. Bacteriol. 98:689-696.

5. Hummel, B. C. W. 1959. A modified spectrophotometricdetermination of chymotrypsin, trypsin, and thrombin.Can. J. Biochem. Physiol. 37:1393-1399.

6. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

7. Murphy, S. M. N., and J. S. Lovett. 1966. RNA andprotein synthesis during zoospore differentiation insynchronized cultures of Blastocladiella. Develop. Biol.14:68-95.

8. Rosen, H. 1957. A modified ninhydrin colorometric analy-sis for amino acids. Arch. Biochem. Biophys. 67:10-15.

9. Sauer, H. W., K. L. Babcock, and H. P. Rusch. 1970.Changes in nucleic acid and protein synthesis duringstarvation and spherule formation in Physarum poly-cephalum. Wilhelm Roux' Arch. Entwicklungmech.Organismen. 165:110-124.

10. Smillie, R. M., and G. Krotkov. 1960. The estimation ofnucleic acids in algae and higher plants. Can. J. Bot.38:31-49.

11. Sparrow, F. K., Jr. 1960. Aquatic phycomycetes, 2nd ed.University of Michigan Press, Ann Arbor.

12. Spudich, J. A., and A. Kornberg. 1968. Biochemicalstudies of bacterial sporulation and germination. VII.Protein tumover during sporulation of Bacillus subtilis.J. Biol. Chem. 243:4600-4605.

13. Viau, J., and F. F. Davis. 1970. Effect of cycloheximide onthe synthesis and modification of ribosomal RNA inNeurospora crassa. Biochem. Biophys. Acta 209:190-195.

73VOL. 116, 1973

on February 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from