Current 6enetics

Current Genetics (1984) 8:635-640

© Springer-Veriag 1984

Genetic analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardii Emilio Fernfindez 1 and Ren6 F. Matagne

Genetics of Microorganisms,Department of Botany, Universityof Liege, Sart-Tilman,B-4000 Liege, Belgium

Summary. Six mutants (305, 301, 203, 307, 104 and 102) of Chlamydomonas reinhardii, all defective in nitrate reductase (NR) activity, have been genetically analyzed. All except 102 carry single Mendelian mutations. Mutant 305, defective in diaphorase activity and mutant 301, defective in terminal enzyme activity, did not give rise to wild-type recombinants when crossed to each other or with the nit-1 mutant isolated from strain 137c (which is actually a double mutant nit-1 nit-2). Nit-1 was shown to lack both diaphorase and terminal activities. Whether the mutated sites in 305 and 301 are located in a unique cistron (nit-l) or in two adjacent cistrons (nit-la and nit-lb) coding for a diaphorase subunit and a terminal subunit of NR is discussed in the light of previous biochemical findings. The 203 mutation affecting a regulatory gene did not recombine with nit-2, the other mutated locus present in strain 137c. Mutants 307, 104 and 102, all lacking molybdenum cofactor for both NR and xanthine dehydrogenase, where shown to be affected in different loci. The genes mutated in 307 and 104 have been designated nit-3 and nit-4, respectively. The 102 strain is mutated in two nonlinked loci, nit-5 and nit.6, with both mutations required to confer the mutant phenotype. One of these cryptic mutations is present in the "wild" strain 21gr.

Abbreviations: NR, nitrate reductase; MNNG, N-methyl-N'-nitroN-nitrosoguanidine; MoCo, molybdenum-containing cofactor; PD, parental ditype; NPD, non-parental ditype; TT, tetratype; WT, wild type; BVH,reduced benzyl viologen 1 Permanent address: Departamento de Bioqutmica, Facultad de Ciencias, Universitadde C6rdoba. C6rdoba, Spain Offprint requests to: R. F. Matagne

The results indicate that at least six or seven loci are involved in the production of an active NR enzyme: one (nit-l) or two (nit-la and nit-lb) cistrons to produce the NR apoproteins responsible for the partial activities diaphorase and terminal, one locus (nit-2) for the regulation of NR synthesis, and four loci (nit-3, nit-4, nit-5 and nit-6) to produce the molybdenum cofactor. The loci nit-la and nit-2 seem to correspond to the nit-A and nit-B loci described by Nichols and Syrett (J Gen Microbiol 108:71-77, 1978). Key words: Chlamydomonas reinhardii - Nitrate reductase

Introduction

Eukaryotic NAD(P)H-NR 1 (EC 1.6.6.1-3) catalyzes the two electron transfer step of nitrate reduction. The enzyme is highly regulated both at the level of synthesis and activity (Hewitt and Norton 1980; Guerrero et al. 1981; Marzluf 1981). NR displays two partial activities which can be separately assayed, diaphorase or NAD(P)Hcytochrome c reductase and terminal-NR or reduced benzyl viologen-NR. FAD, cytochrome bss 7 and molybdenum are the prosthetic groups of NR driving electrons from NAD(P)H to nitrate (Hewitt and Notton 1980; Guerrero et al. 1981). Molybdenum acts in the form of a labile and low molecular weight cofactor (MoCo) shared by all known molybdenum-containing enzymes, excepting dinitrogenase; this cofactor is essential for both the active site of nitrate reduction and the assembly of subunits into the enzyme complex (Johnson 1980). The structure of the MoCo, a pterin analog, is now becoming elucidated

636

E. Fernandez and R. F. Matagne: C reinhardff nitrate reductase mutants

Table 1. Nitrate reductase activities of WT (21gr and 6145c) and NR mutant strains of Chlarnydomonas reinhardii a Strain

Diaphorase activity related to NR

Terminal activity

Presence of MoCo

Overall-NR activity

WT (+) and (-) 305 (-) 301 (-) 203 (+) 307 (-), 104 (+) and 102 (+)

+ + +

+ + -

+ + + + -

+ -

Nichols and Syrett's mutation sites with the same phenotypeb

nitA nitB nitC

a Fernandez and C~trdenas 1982a, 1982b b Nichols and Syrett 1978; Nichols et al. 1978

(Johnson and Rajagopalan 1982), but its biosynthetic pathway is completely unknown. NR from Neurospora crassa and Aspergillus nidulans seems to be the assembly product of identical subunits (each bearing diaphorase activity) coded by a single gene, with the MoCo, whose synthesis is dependent upon at least five genes (Garrett and Amy 1978; Cove 1979; Tomsett and Garrett 1980; Marzluf 1981). Those fungal mutants lacking MoCo show diaphorase activity and those affected in the structural gene have no diaphorase activity but may exhibit terminal-NR activity. The phenotypes of Nicotiana tabacum NR-deficient mutants resemble those of N. crassa mutants and biochemical properties of the NR complex from both organisms are also closely related (Mendel and Miiller 1979, 1980). Mutant strains of the green alga C. reinhardii devoid of NR activity have been previously isolated (Sosa et al. 1978; Nichols and Syrett 1978). Three NR-mutants analyzed by Syrett's group (Nichols and Syrett 1978; Nichols et al. 1978) were shown to be mutated in three different Mendelian genes: the nitA mutation determines a loss of diaphorase activity, the nitB mutant is defective in both diaphorase and terminal activities and the nitC mutation makes the mutant strain unable to use hypoxanthine as nitrogen source. Strain 137c carries two mutations, nit-1 and nit-2, most probably located in nitA and nitB loci respectively, and recently mapped to two different linkage groups: nit-1 in group IX and nit-2 in group III (R. D. Smyth, unpublished). Six other NR mutant strains (203, 307, 104, 102, 305 and 301) isolated by Sosa et al. (1978), have been biochemically characterized (Table 1). The occurrence o f a MoCo shared by NR and xanthine dehydrogenase was shown in wild and mutant strains (Fernandez and C~rdenas 1981a, b). The native NR complex was reconstituted by in vitro complementation between terminalNR from mutant 305 and diaphorase subunit donors (e.g. 104 mutant) (Fernandez and C~rdenas 1981a). The

subunit of NR with diaphorase activity was purified and contained FAD and cytochrome bss 7 (Fernandez and Cfirdenas 1983a). The properties of mutant 301 and the in vitro complementation system suggested a heteromultimeric structure for C. reinhardii NR (Fernandez and Cfirdenas 1981a, 1982a). Very recently, a subunit of NR different from the diaphorase subunit has been reported: that subunit has no enzymatic activity unless assembled into the complex in which it bears the MoCo (Franco et al. 1984). In the present work, the genetic analysis of those biochemically well-characterized NR mutants is reported. Four loci have been found to code for MoCo, one for the regulation of the NR induction and one or two for the NR apoproteins.

Materials and methods Strains. The WT strains 21gr (rot +) and 6145c (mr-) were obtained from Prof. R. Sager (Sidney Father Cancer Ctr.) and strain 137c (rot + and mt-) from Prof. R. P. Levine (Harvard University). Mutant strains 305 (mt-), 301 (mr-), 203 (rot+), 307 (rot-), 104 (mt+) and 102 (rot +) were obtained from the corresponding WT by mutagenesis with MNNG (Sosa et al. 1978); they lack overall-NR and exhibit the characteristics described in Table 1. Mutants of opposite m t ( 3 0 5 d , 3 0 1 d . . . . ) were isolated from crosses of original mutants with WT. Media. The strains were grown under continuous light on TAP (Gorman and Levine 1965) or M (Loppes 1966) solidified agar medium (15 g[1 Bacto-agar Difco) containing 0.4 g/1 NH4C1 as sole nitrogen source. To analyse the absence of NR activity, cells were grown on TAP-NOg-medium deprived of NH4C1 and supplemented with 0.4 g/l KNO3. Mutant strains were routinely maintained on TAP medium. Genetic analysis. Crosses, maturation of zygotes and tetrad analysis were carried out according to the methods of Levine and Ebersold (1960). Most often genetic analysis was made by the random spore plating method: after germination of about 50

E. Fern~mdezand R. F. Matagne: C. reinhardii nitrate reductase mutants Table 2. Crosses between NR-deficient mutants from C. reinhardii Cross

nit+ 203 x 305 203 x 301 203 x 307 203 d x 104 203 d x 102 307 x 301 d 307 x 102 307 x 104 104 x 301 104 x 305 104d x 102 102 x 301 102 x 305 301 d x 305 137c x 21gr 137c x 305 137c x 301 137c x 203 137c x 307 137c x 104 137c x 102

Ratio

Progeny

36 11 40 64 170 51 28 31 29 24 176 55 42 0 42 0 0 0 20 48 86

nit99 49 140 176 130 189 28 89 91 96 184 108 85 240 138 180 180 180 160 312 71

nit+ n i t 1 1 1 1 1.3 1 1 1 1 1 1 1 1 0 1 0 0 0 1 1 1.2

2.8 4.5 3.5 2.8 1 3.7 1 2.9 3.1 4 1 2 2 1 3.3 1 1 1 8 6.5 1

mature zygotes in the light (1,000 lux) for 18 h, the spores were allowed to swim in a drop of distilled water and plated on TAP medium. After 5 days, colonies were transferred on TAP medium and after subsequent growth, replica-plated on TAP and TAP-NO~- agar plates. The percentage of WT recombinants was calculated from the number of colonies growing on both media.

Reversion experiments. Spontaneous reversion frequency was calculated from the ratio: number of colonies growing on TAPNOg- plates vs total number of plated living ceils, counted by appropriate dilutions on TAP plates. UV-induced reversion frequency was calculated as above by plating cells which had been irradiated with a short wavelength UV-lamp Mineralight mod. R52 (Ultra-violet Prod. Inc., San Gabriel, Calif.) to yield approximatively 10% survival.

Enzymatic activities. NADPH-NR (overall) and BVH-NR (terminal) activities were determined as described elsewhere (Barea and C~irdenas1975). The diaphorase subunit of NR was detected by electrophoresis of crude extracts on 7.5 per cent polyacrylamide gels as previously described (Fermtndez and C~irdenas 1982b).

Results The six NR-deficient mutants were first crossed with the wild-type strain of opposite mating-type (21gr m t ÷ or 6145c m t - ) (results not shown). In crosses envolving 203, 307, 104 and 305, nit + and n i t - meiotic products segregated in a ratio very close to 1 : 1, indicating that each of the m u t a n t strains differed from wild-type by a

637

single mutation. In each case, both mating-type a n d y - l ("yellow" in the dark, present in 6145c and derived m u t a n t strains) markers segregated independently of the n i t - character. An unusually high frequency of WT segregants (about 5 nit + for 1 n i t - ) was obtained in the cross 102 x 6145c and could not be explained on the basis of a single Mendelian mutation in 102. The results of crosses between different nit mutants are shown in Table 2. The cross 301d x 305 was the only one where no WT recombinant was obtained, indicating that the mutations were either alMic or very closely linked. As 301 showed a faint residual growth on nitrate, its phenotype could be distinguished from the 305 null-phenotype: among the 240 n i t - colonies issued from the meiotic progeny, 105 showed a residual growth (expected ratio: 1 : 1). In the other crosses (except those involving 102), nit + and n i t - characters segregated in a ratio close to 1 : 3 which is indicative of the absence of linkage between the different mutations. Again, the crosses involving 102 gave rise to WT recombinants at a higher frequency than that expected for crosses between single mutants. The segregation observed in the cross between WT 21gr and the double m u t a n t (nit-1 nit-2) 137c strain was as expected for a cross involving two unlinked mutations (Table 2) if both mutations are required to confer the m u t a n t phenotype. In crosses with 137c, the n i t - allele from 307 or 104 segregated independently of nit.1 and nit.2 whereas the cross 137c x 102 yielded an excess of WT progeny (Table 2). In contrast, no WT recombinant was obtained in crosses 137c x 305, 137c x 301 and 137c x 203; as 301 and 305 are allelic and unlinked to 203 (see above), this indicates that both 301 and 305 mutations are allelic to one of the n i t - mutations present in 137c and that 203 is allelic to the other.

Nit-B and 203 are regulatory mutations conferring a very similar phenotype (Nichols and Syrett 1978; Nichols et al. 1978; Fernfindez and C~irdenas 1982b). As nit-B and nit-2 are allelic or very closely linked, it is likely that the mutations 203 and nit-2 affect the same gene. On the other hand, 305 and 301 are phenotypically different and well.defined structural mutants of NR (Fernfindez and C~rdenas 1982a, 1983b), b u t the biochemical characteristics of the m u t a n t nit-1 are unknown. The single m u t a n t nit-1 was isolated as follows. Clones unable to grow on nitrate were isolated from the cross 137c x 21gr: they are expected to be nit-l, nit-2 or nit-1 nit-2. Gametes from these clones were mixed with gametes of 203 of opposite mating-type and the copulation mixture was spread over TAP-NO~- agar medium. After 6 days incubation in the light, colonies were observed on some plates. These phenotypically wild colonies had to arise from vegetative zygotes dividing mitoti-

E. Fern~mdez and R. F. Matagne: C. reinhardii nitrate reductase mutants

6 38

Table 3. Reversion frequency of structural mutants from C.

reinhardii Strain

Reversion frequency

301 305

nit-1

Spontaneous

UV-induced

4. 10 - 5 < 6 . 10 - 9 6. 10 - 9

10 - 3