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Arch Microbiol (1987) 148:8 - 13

9 Springer-Verlag1987

Fluoroacetamide resistance mutations in Chlamydomonas reinhardtii R. C. Hodson i and P. M. Gresshoff 2

1 School of Life and Health Sciences, University of Delaware, Newark, DE 19716, USA 2 Botany Department, Australian National University, Canberra, Australia

Abstract. Acetamide, a nitrogen and carbon source for

Chlamydomonas reinhardtii, is hydrolyzed by acetamidase to ammonium and acetate. It also induces urea pathway activities. Fluoroacetamide (F-acetamide) is toxic to wildtype through conversion to F-citrate, a respiratory inhibitor. Resistant mutants were selected on plates of F-acetamide plus urea. When tested on acetamide plates two mutant classes were obtained, acm + (utilized acetamide as sole N source) and acre-. All acre + isolates had acetamidase activity and were obligate phototrophs (i. e. "dark-diers"). A c m isolates had either normal urea assimilation (ure +) or lacked all urea pathway activities, namely transport, urea carboxylase and allophanate hydrolase (ure-). Inheritance patterns for both types indicated single nuclear gene mutations. The a c m - ure + type presumably resulted from a defective acetamidase gene, and the acre- ure- strains might be regulatory gene mutants. Temperature conditional F-acetamide tolerant mutants were also obtained. Acetamidase extracted from one such strain was more thermolabile than the wildtype enzyme, indicating a mutation in the coding region. The hypothesis that acetamidase is involved in urea assimilation was not supported by the genetic and biochemical evidence. Key words: Chlamydomonas reinhardtii - Fluoroacetamide - Fluoroacetate - Acetamide - Mutation Transport - Urea

It was shown in previous studies (Gresshoff 1981 a) that the unicellular green alga Chlamydomonas reinhardtii utilizes acetamide and formamide as sole nitrogen source. The first step in assimilation is hydrolysis by an enzyme subject to induction by acetamide, urea and thiourea, and repression by ammonium. Genetic and biochemical evidence supports the claim that both acetamide and formamide utilization functions are fulfilled by one enzyme, acetamidase, with one active site. This finding is similar to the situation observed with Myeobaeterium (Draper 1967) and Pseudomonas (Kelly and Clark 1962) but contrasts with that of Aspergillus where multiple amidases with differing control systems were found (Hynes and Pateman 1970; Hynes 1975).

Abbreviations. F-aeetarnide, fluoroacetamide; F-acetate, fluoroacetate; TAP, tris-acetate-phosphate medium; CDB, Chlamydomonas dilution buffer; TCA, trichloroacetic acid; AH, allophanate hydrolase; UC, urea carboxylase; PAR, photosynthetically active radiation; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea Offprint requests to: R. C. Hodson

The noninducing acetamide analogue F-acetamide kills cells grown under the appropriate conditions (Gresshoff 1981 a). These conditions involve the absence of ammonium, presence of urea, and a low (or zero) acetate concentration. The toxicity of F-acetamide is based on its conversion by acetamide to F-acetate which enters the tricarboxylic acid cycle, where, converted to fluorocitrate, it inhibits aconitase and slows respiration (Gresshoff 1981 a). Sensitivity to Facetate depends on the amount and form of exogenously supplied carbon source. Thus to produce a certain degree of growth inhibition, mixotrophic conditions with acetate or pyruvate as carbon sources required a higher F-acetate concentration than phototrophic conditions employing carbon dioxide. Analysis of regulatory systems is substantially aided by an interaction of biochemical, physiological and genetic approaches. The possibility of utilizing analogue resistance to isolate mutants affected in the control or function of a particular enzyme has been previously realized in a great diversity of organisms ranging from Escherichia coli to Arabidopsis thaliana. F-acetamide resistant mutants of Aspergillus nidulans allowed Hynes and Pateman (1970) to probe gene regulation in a heterotrophic eukaryote. Very little work of this type has been done with a photosynthetic eukaryote. In this paper we describe physiological, biochemical and some genetic properties of Chlamydomonas reinhardtii mutants selected for resistance to F-acetamide. With Chlamydomonas a study of acetamide assimilation must also consider urea metabolism. Urea is hydrolyzed solely by the sequential action of two enzymes, urea carboxylase (UC) which produces allophanate, and allophanate hydrolase (AH) which produces two molecules each of ammonium and carbon dioxide (Hodson et al. 1975). Inducers of acetamidase, urea a n d acetamide, also induced UC and AH (Hodson and Gresshoff 1979). This indicated that the same regulatory gene or region controls structural genes for both pathways, or that one of the urea pathway enzymes also has acetamidase activity (Gresshoff 1981 b). Therefore, urea assimilation in F-acetamide resistant mutants was also examined. Two major categories of non-conditional F-acetamide resistant mutants were obtained: (i) those unable to hydrolyse the amide group of acetamide, and (ii) those defective in assimilation of the carbon chain. In the former group were two types, ones lacking all known activities required for the liberation of ammonium from acetamide and urea, and others defective only in acetamide utilization. A brief account of some of this work has been published (Hodson and Gresshoff 1979).

Materials and methods

Media. Cells were cultured mixotrophically in TAP (trisacetate-phosphate) medium as described by Gresshoff (1976) or in the medium of Orth et al. (1966) as described by Hodson et al. (1975). TAP is buffered with tris, and the Orth et al. medium is buffered with phosphate. The use of two basal media simply reflects convention in two different laboratories and is insignificant to the results obtained. NMinus medium (e. g. N-TAP) lacked NH4C1, and minimal medium was TAP with acetate replaced by chloride. All supplements were filter sterilized and added after autoclaving and cooling the basal medium. For solid media unrefined agar (Moorehead and Co., Inc., Van Nuys, CA, USA) was added to 1.5%. Chlamydomonas dilution buffer (CDB) is nitrogen and acetate free TAP medium containing 0.1 M mannitol (pH 6.8). Strains and mutant selection. The "wild-type" strain of Chlamydomonas reinhardtii used to generate mutants (AN228) is a clone of 137c mating-type plus (Gresshoff 1981 a). Its origin traces back to the Levine laboratory and it therefore lacks nitrate reductase. Wild-type strains CC124 and CC-125 used in crosses were obtained from the Chlamydomonas Genetics Stock Center at Duke University. Mutant strains are designated AN reflecting their geographic origin. Genetic nomenclature complies with current practice for the nuclear genome in Chlamydomonas (Harris 1984). For mutant selection, logarithmically growing cells were washed and suspended in CDB. Agar plates containing Nminus medium supplemented with 5 mM urea and 150 ~tM F-acetamide were inoculated with 1 - 1.5 x I 0 6 viable cells, allowed to dry, and incubated under continuous light for 7 days. At this stage dark green colonies of various sizes were picked and streaked on plates of basal medium. Subsequently single colony isolates were grown up on basal medium and retested on the selection medium to confirm mutant status. The initial phenotype designation was fam ' (F-acetamide resistance). Mutant storage was always on nonseletive (TAP) medium. Culture conditions. Stock cultures were kept on TAP agar slants at 20~ and in dim light. Inoculum for agar plates was produced in 24-well culture dishes. Cells for enzyme and transport assays were grown in liquid culture at 25~ on a rotary shaker under cool-white fluorescent lamps producing a PAR flux of 70 gmol photons m-2 s-1. The cells were harvested at mid-exponential phase (A = 100) as determined by absorption measurements with a Klett-Summerson colorimeter fitted with a 540 nm filter. An absorption value of 100 corresponds to approximately 2.4 x 1 0 6 cells ml-1. Growth tests on agar plates. Cultures were passed through N-minus medium (0.1 ml culture into 1 ml medium) for 2 to 3 days to deplete nitrogen reserves. Subsequently a drop of culture was placed on plates of N-minus medium supplemented with the tested nitrogen source. A second drop was spread on the same plate to produce isolated colonies. Incubation was at 25 ~C with a PAR flux of 40 gmol photons m-2 s- 1 of cool-white fluorescent light. Plates were observed at 3 and 7 days. Enzyme assays. Acetamidase was determined with a modification of the static induction method decribed by

Gresshoff (1981 b). Cells were harvested aseptically by centrifugation, suspended to one-half density (1 x 106/ml) in Nminus medium plus 10 mM acetamide, and cultured 24 h. These cells were again harvested by centrifugation, suspended in the same volume of N-minus medium and incubated an additional 2.5 h. Portions of culture in duplicate ( 2 0 - 4 0 ml) were trapped on Gelman type A/E fiberglass filters (47 mm diameter) and rinsed with 100 mM potassium phosphate buffer, pH 7.5. The filters were immersed in 5 ml buffer, frozen and stored in a - 2 0 ~ freezer overnight. The frozen material was thawed to 4~ and held at that temperature for 1 h with occasional agitation to extract enzyme. The extract was used without centrifugation. The reaction mixture consisted of extract ( 0 . 2 0.4 ml) and 100 mM potassium phosphate, pH 7.5, in a total volume of 0.5 ml. The reaction was started by adding formamide to 25 raM, continued for 60 rain at 30 ~C, and terminated by heating 5 rain at 80~ After cooling and adding an equal volume of water, ammonium was measured by a phenol-hypochlorite procedure (Muftic 1964). Absorbancy of controls with heat denatured enzyme were subtracted to yield enzyme activity. Urea carboxylase (UC) and allophanate hydrolase (AH) were assayed according to Hodson et al. (1975). UC induction (3 mM urea for 4 h) and subsequent extraction by sonication were as previously described (Hodson et al. 1975). AH was induced with urea and extracted as for UC, and it was also induced with acetamide for 24 h. Transport assays. The derepressed level of urea transport was determined essentially by the method of Williams and Hodson (1977). Cells harvested by centrifugation were suspended in N-minus medium and incubated 4 h. To 11-ml of suspension in a 125-ml flask, [C14]urea (430 Bq/nmol) was added to a final concentration of 60 gM. Incubation was at 25 ~C with a PAR (photosynthetically active radiation, i.e. 4 0 0 - 700 rim) flux of 170 ~tmol photons m - 2 s- 1 tungsten illumination (GE Gro and Sho). Duplicate 2 ml portions were removed at 0.5, 1, 1.5, 2 and 3 min, filtered on 25 mm diameter Gelman type A/E fiber-glass filters, and rinsed with 20 ml of ice-cold 20 mM urea. Radioactivity retained on the filters was determined by liquid scintillation with correction for counting efficiency. The derepressed level of arginine transport was similarly determined, except that the substrate was 11 laM [2,3-~H]L-arginine (1.3 Bq/pmol). The induced level of urea transport was measured in cell suspension induced with acetamide and conditioned in Nminus medium as for the acetamidase assay. One portion of suspension was made 10 gM in DCMU and treated as for the derepressed urea transport assay except that duplicate samples were taken at a single time of 5 min. Also, filters with retained cells were soaked overnight with I ml 10% TCA in an open liquid scintillation bottle to remove radioactive carbon dioxide. The residual radioactivity was called "cell label". A second portion of suspension was used to determine the amount of urea assimilated to carbon dioxide. The technique was based on the assay for urea carboxylase and allophanate hydrolase (Hodson et al. 1975). Two milliliters of suspension containing DCMU (10 laM) were placed in the outer well of the 25 ml flask and 10% KOH went into the center well. The flask was sealed, radioactive urea was added (to 60 gM), the contents were incubated for 5 rain at 25~ with illumination, and the radioactivity

10 Table 1. Chlamydomonas reinhardtii strains used in this study Strain AN109 AN114 AN207 AN214 AN228 AN248 AN266 AN277 AN278 AN281 AN281R

Mutagen a

Mutant phenotypes b

S S S S None S UV S S S S

fam~fac~ famr fac~ famr acrefamr acre- urefam~facSacre § ure § fam~fac~ famr(ts) famr acmfamr acmfamr acm- urerams facs acre + ure +

Table 2. Fluoroacetate resistant (fac~) mutants fail to grow in dark culture"

Source AN228 AN228 AN228 AN228 [wild-type strain] AN228 AN228 AN228 AN228 AN228 revertantofAN281

a S, spontaneous; UV, ultraviolet radiation b ramr, resistant to F-acetamide; famr(ts), resistant to F-acetamide at elevated temperature; fac~, resistant to F-acetate; acm-, unable to utilize acetamide as sole nitrogen source; ure-, unable to use urea as sole nitrogen source. All wild-type phenotypes in mutants have been omitted

volatilized by sulfuric acid a n d trapped in K O H was determined by liquid scintillation. The result was called the "carbon dioxide fraction". The sum of the cell label a n d carbon dioxide fraction was used to determine urea flux.

Genetic techniques. The procedures for gamete induction, mating plus and minus strains, germinating zygotes, a n d analyzing tetrads were adopted from Levine a n d Ebersold (1960). Zygotes were matured in c o n t i n u o u s light on Nminus m e d i u m with 4% agar (van Winkle-Swift 1977). Reagents. [14C]Urea and [2,3-3H]L-arginine were purchased from New E n g l a n d Nuclear. [14C]Potassium allophanate was synthesized by a chemical method (Whitney a n d Cooper 1972) as described by H o d s o n et al. (1975).

Results M u t a n t selection The frequency of spontaneous fam ~(F-acetamide resistance) m u t a n t s in the wild-type strain was in the range of 1 in 104 to 105 viable plated cells. O n urea plus F-acetamide selection plates two sizes of green m u t a n t colonies arose in a lawn of chlorotic wild-type cells. It was hypothesized that the large colonies were assimilating urea a n d the tiny colonies lacked urea assimilation but grew for a time on the trace of a m m o n i u m present in the agar. Both size classes were picked. After more detailed analysis, several classes of m u t a n t s were found in the selected fam ~ clones. The majority of m u t a n t s (about 90%) were fac s a c m - (F-acetate sensitive and u n a b l e to utilize acetamide as sole nitrogren source), while a b o u t 10% were fac r acre § A small percentage of other m u t a n t types were also found. F o r example, m u t a n t s with an fac ~ a c m - phenotype arose, just as m u t a n t s which were fac s acre + (unexplained faro r phenotype). Strains which were used in experiments are listed in Table 1.

F a d acre + mutants Wild-type cells grown mixotrophically on acetate (16 m M ) had a higher m i n i m u m inhibitory concentration value for

TAP

TAP + TAP + 3 g M F a 60gM Fa

TAP + 100gM Fa

Continuous light AN228 (wt) fac~ AN109 facr ANl14 facr AN248 facr

+ + + + + + + + + +++

+ + + + + + + + + +++

+ + + + + + + + +++

+ + + + + + + +++

Continuous dark AN228 (wt) fac~ AN109 facr AN114 fac~ AN248 facr

+ + +/-

+ + +/---

+ +/-

-

Dark 14 days then light AN228 (wt) facs AN109 fac* ANl14 facr AN248 fac~

+ + + n.d. + + + + + +

+ + + n.d. + + + -

+ + n.d. + + + -

+ n.d. + -

" Fa, F-acetate; n.d., not done; + + +, colony diameter of 1 2 mm after 14 days growth; + +, colony diameter of 0 . 3 - 1 mm; +, just noticeable colony; + / - , colony of (+) category, but extremely pale and hard t.0 detect; - , no apparent growth and presence of only small cell/aggregates on agar surface if viewed by inverted microscopy

F-acetate than cells grown phototropically. For example, AN228 (wild-type) cells plated on 20 g M F-acetate on m i n i m a l m e d i u m were killed, while identical cells plated onto TAP plus 60 gM F-acetate still grew as well as controls. Partial inhibition of wild-type cell growth was noticeable only on TAP m e d i u m supplemented with 100 gM F-acetate or higher. In contrast to this, facr m u t a n t s (such as AN109 a n d A N I I 4 ) were capable of normal growth on minimal m e d i u m supplemented with 1 m M F-acetate. The fac r mutation also allowed these two m u t a n t strains to utilize Facetamide as a nitrogen source, suggesting that the internal F-acetate produced by the utilization of F-acetamide was also non-toxic. Preliminary tests showed that m u t a n t AN114 possessed no general resistance against fluorinated comp o u n d s as both fluorodeoxyuridine and para-fluorophenylalanine were toxic as in wild-type cells. A n interesting observation a b o u t the fac r m u t a n t s came from a series of tests which were designed to investigate the role of photosynthetically derived carbon sources on the Facetate tolerance. Three separate fac ~ m u t a n t s were plated at single colony density onto agar plates containing TAP plus varying concentrations of F-acetate. The plates were incubated in the light and the dark u n d e r otherwise similar conditions. The results are shown in Table 2. I n the light the fac ~phenotype was clear only at F-acetate concentrations in excess of 100 gM. This was presumed to stem from the interaction between the medium-derived acetate and photosynthetic activity. Similarly inoculated plates when placed in the dark gave a very different result. Wild-type cells were n o longer capable of colony formation at 100 IxM F-acetate, but grew well at lower levels. I n contrast all fac ~ m u t a n t s tested showed a n inability to grow on F-acetate in the dark. More importantly this inability was caused by the general failure of the fac r m u t a n t s to grow in the dark, even

11 Table 3. Enzyme and transport activities of C. reinhardtii mutants compared to wild-type" Strain

Induced enzyme activity (% of wild-type)

Transport activity (% of wild-type)

Acetamidase

Urea carboxylase

Allophanate hydrolase b

Urea Urea (derepressed) (induced)

Arginine (derepressed)

(A)

(13)

(C)

(D)

(E)

(F)

(G)

0 0 1 1

75 78 93 39

110 130 90 50

230 200 170 140

140 96 110 140

79 75 83 54

79 69 19 41

2 0

0 0

8 9

4 2

5 2

1 0

33 93

100

100

100

100

100

100

100

n.d. c

n.d.

160

n.d.

62

n.d.

a c m - ure + strains

AN207 AN215 AN277 AN278 acre- u r e - strains

AN214 AN281 acm + ure + strains

AN228 (wild type) AN281R

38

" Wild-type specific activities in Ixmoles - 1 0 6 cells-a, min-x: column (A) 19, (B) 0.012, (C) 0.21, (D) 1.5, (E) 0.038, (F) 1.0, (G) 0.058 b Induced 4 h (column C) and 24 h (column D). See Methods for details c n.d., not done

if supplied with acetate (i. e. TAP medium). In other words, these mutants appeared to be "dark-diers". Following this line of reasoning, a transfer experiment was set up in which facr and facs cells were exposed for 14 days to darkness after which the plates were transferred to the light. Wild-type cells (fac s) recovered quickly and showed good colony formation as expected from continuous light control platings. However, recovery of colony forming ability of mutant AN248 (fac~) was severely limited suggesting that growth on Facetate was no more possible. Cells kept on TAP medium without F-acetate, however, completely recovered suggesting that cell growth arrest, rather than cell death, occurred during the 2 weeks dark period. Mutant A N I I 4 , in contrast, was capable of growth recovery on F-acetate containing media after dark exposure. Dark cultured facr strains failed to grow even if maintained for 9 weeks of culture in the dark. Fac s acm-

mutants

N u t r i e n t u t i l i z a t i o n . Six isolates, representing both large and

small colony sizes from the original selection plates, were chosen for further study. Nutritional phenotypes were determined by plate tests with acetamide, urea or urea plus Facetamide as nitrogen source. Three phenotype classes were obtained. Four strains (AN207, 215, 277 and 278) were unable to utilize acetamide but grew normally with urea (acm- ure + phenotypes). Two strains (AN214 and AN281) were unable to utilize either acetamide or urea (acm- urephenotypes). Two strains (wild-type AN228, and a revertant of AN281, AN281R) utilized both acetamide and urea as sole N sources and were sensitive to F-acetamide (acm + ure + phenotypes). E n z y m e a c t i v i t i e s . The specific activities of acetamidase, UC

and AH were measured in extracts obtained from mutant cells and compared to wild-type (Table 3, columns A - D ) . The four acm- ure § strains lacked acetamidase, but UC and A H activities were high enough to indicate no significant

defect. These mutants also lacked acetamidase activity when induced with urea instead of acetamide (data not shown). Likewise they lacked the "constitutive" activity seen when wild-type cells are transferred into N-TAP medium. This suggests that a basic parameter, most probably a regulatory or structural gene, had been affected. The two acre- urestrains lacked acetamidase and UC and showed AH activities less than 10% that of wild-type. Revertant AN281R regained acetamidase and AH activity; UC was not measured. T r a n s p o r t a c t i v i t i e s . Assimilation of exogenous urea involves a high affinity active transport system (Williams and Hodson t977), and so it was of interest to find out if acmure- mutants have defective urea transport. Both mutant strains lacked derepressible and inducible urea transport (Table 3, columns E and F). The acm + ure § revertant AN281R had normal transport, as did the four acre- ure + strains. Arginine transport was also examined and found to be present in all strains (Table 3, column G). A similar study of acetamide transport was not undertaken because it has not been demonstrated to occur in wild-type. I n h e r i t a n c e . Both acm- ure + and acm- ure- mutants had

normal gametogenesis. A representative of the acm- ure + class (AN207 m t + ) and the acm- ure- class (AN281 m t - ) were separately crossed to wild-type strains commonly used in genetic analysis (CC-124 m t - and CC-125 rot+). No unusually high amount of lethality in the zygotic products was observed. Complete tetrads from about 20 zygotes for each cross were tested for acm phenotype. Both crosses gave an a c m - : acm + ratio of 2: 2. In addition, the acre- and urephenotypes of AN281 did not segregate from each other. Ts mutants

It was also possible to use the general selection scheme to isolate temperature sensitive (ts) mutants in the acetamidase system. To do so, wild-type midlogarithmic cultures ( 1 -

12 Table 4. Thermal stability test of acetamidase in cell extracts from wild type and ts-mutant Enzyme activity " Time of exposure (min) 0 Mutant (AN266)

Wild-type (AN228)

1

l0

30

37~ 60~ 98~

8.3 n.d) n.d.

8.1 4.1 0

8.4 0.7 0

7.6 0 n.d

37~ 60~ 98~

12.0 n.d. n.d.

12.6 9.8 1.2

11.7 9.1 0

11.9 7.6 n.d

Crude extract was assayed for acetamidase activity at 37~ as described in Material and methods. Activities are expressed as ~tmoles ammonia produced 9 10 .6 cells-1. Results are averages of three separate readings from one single experiment b n.d., not done a

3 x 1 0 6 cells m l - 1) were mutagenized with ultraviolet radiation prior to plating. Agar plates and general manipulations were in near darkness for the first 2 4 - 36 h prior to normal light incubation to prevent photoreactivation. Selection was at 33~ (restrictive temperature) and 22~ (permissive temperature). Wild-type strain AN228 grew without difficulty at 33 ~C, while 38~ was growth inhibitory. Also, urea utilization and urea induction of acetamidase were normal at 33 ~C. The frequency of resistant clones appearing after 14 days incubation on N-TAP medium supplemented with 5 m M urea and 150 g M F-acetamide was determined. The values obtained were 1 x 10 -1 for selection at 22~ and 1 x 10 -3 for selection at 33~ A n unirradiated control selected at 22~ also yielded fam r mutants at useful frequencies (1.7 x 10-5) so that mutagenesis might not have been required. However, we rationalized the use of mutagenesis as the type of allelic variation, which may lead to temperature sensitivity, may not be frequent a m o n g spontaneous mutants. The frequency of fam r mutants after mutagenesis appeared exceedingly high (i.e. 1%), but this was confirmed and is still unexplained. The fam r colonies (over 150 in total) isolated at 33~ were purified and retested at 22~ and 33~ for their (a) stability of fam r and (b) ability to utilize acetamide as a nitrogen source. The majority (about 85%) o f colonies maintained their mutant phenotype at the permissive temperature. The 15% remaining became sensitive to Facetamide and capable of acetamide utilization to varying degrees. These mutants were termed famr-33-n (where n ranged up to 21). One mutant isolate, famr-33-7 ( = AN266) showed pronounced temperature sensitivity o f the acetamidase as verified by thermal stabilities of crude enzyme extracts. Other ts-mutants did not. The results of one such test are shown in Table 4. The mutant enzyme extract clearly showed decreased thermal stability, although this difference from wild-type enzyme was not expressed at 37~ but clearly at 60~ This suggested that enzymes when in situ have different characteristics to enzyme in extracts. Discussion Two general classes o f mutants resistant to F-acetamide (fam r) were obtained: those that can utilize acetamide as

sole nitrogen source (acm +) and those that cannot (acm-). Six a c m - isolates were extensively characterized. They fit into one of two nutritional phenotypes, a c m - ure + or a c m u r e - . Their enzymatic and physiological properties were consistent with the nutrient utilization data. The four a c m ure + strains were unable to assimilate acetamide but grew normally on urea. Cell extracts from these strains lacked acetamidase but contained the two acitivities of urea hydrolysis, urea carboxylase (UC) and allophanate hydrolase (AH). Whole cells had normal derepressed and induced urea transport activities. The two a c m - u r e - strains were unable to grow with both urea and acetamide. Consistent with this, cell extracts lacked acetamidase, U C and AH, and intact cells did not have derepressible or inducible urea transport. F r o m the genetic evidence, back mutation rates and properties o f a revertant we conclude that both acre- ure + and a c m - u r e - phenotype classes result from single mutations. The unmapped mutation in AN207 giving the a c m - ure + phenotype may be in the structural gene for acetamidase or a regulatory gene specific to acetamidase expression. A c m - u r e - strains behave as if they are constitutively repressed. This could result from a mutation in a positive acting regulatory gene which either prevents synthesis o f its product or produces an inactive product, or from a mutation in a nitrogen control gene which codes for a repressor that does not require a m m o n i u m (or a related substance) for activity. Similarities in substrate structure and pathway regulation have led to speculation that acetamide and either urea or aUophanate may be substrates for the same enzyme, i.e. U C or A H may have acetamidase activity (Gresshoff 1981 b). This possibility for U C can be dismissed on the basis of enzyme properties. U C has a biotin cofactor and requires Mg 2+ and ATP, and in crude extracts it is inactivated by freezing (Hodson et al. 1975). Acetamidase has no known cofactor or activator requirements, ATP is not required, it is stable during freeze-thaw cycles, and urea is not a substrate (Gresshoff 1981a). In terms of resistance to freeze-thaw inactivation A H and acetamidase are similar. However, the reaction rate of A H with 1 m M allophanate was not depressed by either acetamide or formamide at 25 m M (Hodson, unpublished work). The evidence presented in this paper also supports the conclusion that acetamidase and U C / A H are distinct catalytic entities. A H and U C are present in mutants lacking acetamidase. Moreover, in those mutants lacking all three enzymes urea transport is also affected which, as we noted above, suggests a defect in a regulatory gene rather than a structural one. Partial characterization of acm + isolates showed the possibility of using tolerance to F-acetate (fac r) as a selection for mutants which are unable to grow in the dark despite a supply of fixed carbon (i. e. acetate). This general phenotype was also noted for mitochondria defect mutants of Chlamydomonas (Wiseman et al. 1977), and our observation thus might suggest that a functional chloroplast can compensate for mutational deficiencies in a mitochondrion as caused by the F-acetate resistance mutation. F-acetate is converted to F-citrate which inhibits aconitase of the citric acid cycle. Mutants altered in aconitase activity may require a functional chloroplast to remain viable or at least capable of growth. The ability to select for temperature sensitive mutants by positive fam r selection allowed the isolation of mutant material which appeared to be altered in the structural gene

13 for acetamidase. Further biochemical analyses, however, will be required to verify the thermal stability tests as illustrated in Table 4. Still unexplained is the fact that the mutant enzyme was stable at 37~ which in vivo (i.e. at 33 ~C) was already selective enough to presumably inactivate the enzyme activity. If conditional mutations affecting acetamidase are confirmed, these markers will allow mapping the acetamidase structural gene. Gametogenesis in Chlamydomonas is suppressed by nitrogen sufficiency. One hypothesis for a mechanism is that it is controlled by a component in the repression of nitrogen assimilation pathways (i. e. nitrogen control). In connection with this, we anticipated that uninducible strains AN214 and AN281 would be non-gametic. However the opposite was found, both strains produced gametes u p o n nitrogen deprivation. Therefore these strains do not appear to provide material for testing the hypothesis. The acm + gene may be suitable as a homologous selectable marker for molecular transformation into a c m Chlamydomonas hosts. Galloway and G o o d e n o u g h (1985) have shown that acm + is expressed in acm + a c m - diploids. Transformation with an acm + marker in Aspergillus has been successful (Kelly and Hynes 1985; Tilburn et al. 1983).

Acknowledgements. Mrs. Tessa Raath is thanked for technical assistance. This material is based upon work supported in part by the US Department of Agriculture under Agreement No. 59-21011-1-721-0.

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Received September 1, 1986/Accepted January 12, 1987