Effect of iron deficiency on ferredoxin levels in Anabaena variabilis PCC 6309

Arch Microbiol (1990) 153 : 528 - 530

Archwes of

Hicrnbiolngy

9 Sprlnger-Verlag1990

Effect of iron deficiency on ferredoxin levels in Anabaena variabilis PCC 6309 M. B. Pardo, C. G6mez-Moreno, and M. L. Peleato Departamento de Bioquimica y Blologia Molecular y Celular, Facultad de Ciencias, Unlversidad de Zaragoza, E-50009 Zaragoza, Spain Received August 10, 1989/Accepted December 21, 1989

Abstract. Cultures of the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 6309 were grown under several iron concentrations, and changes in growth rates and chlorophyll and phycocyanin concentrations were determined. The total amount of ferredoxin present in the cells was found to be dependent on the concentration of iron in the media, as was the pattern distribution of the two different forms of the iron-sulfur protein described in this organism. Flavodoxin was not found to be present in these cells even when they were grown in the absence of added iron, indicating that this flavoprotein does not replace ferredoxin in this particular strain. Key words: Anabaena - Ferredoxin - Flavodoxin Iron deficiency

Iron is involved in many fundamental biological processes. It is required for the growth of all organisms, since it is involved in the biosynthesis of essential iron-sulfur proteins and enzymes such as those participating in oxidation-reduction reactions. The iron level in the growth medium of photosynthetic organisms alters the tylakoidal composition of the light-absorbing pigments, i.e. chlorophyll a and phycocyanine (Guikema and Sherman 1984), by changing the capacity to absorb the light energy (Sandmann and B6ger 1983). Ferredoxin is one of the important iron-containing components of biological electron transport chains. It is involved in cyanobacteria on photosynthesis as well as in the assimilation of different forms of nitrogen compounds (Rogers 1987; Manzano et al. 1976). Two different molecular species of ferredoxins have been documented in cells of some species of cyanobacteria as well as in higher plants and photosynthetic bacteria (Ho and Krogman 1982). They show somewhat similar molecular Offprint requests to: M. L. Peleato

size (from 96 to 99 amino acid residues) but different physico-chemical properties that enable them to be separated. In general, ferredoxin I is the most abundant, although it is admitted that, in some organisms, ferredoxin I could be more similar to ferredoxin II from other organisms (Rogers 1987). No clues have been obtained, however, concerning their physiological role, but some higher nitrogenase activity was found when one species of ferredoxin was used (Wada et al. 1981). This finding is supported by the high differences in redox potentials that have been found in those ferredoxins (Rogers 1987). Most cyanobacteria studied induce the synthesis of a non-iron electron-transfer protein, i. e. flavodoxin, under iron-deficient conditions. This FMN-containing protein has been shown to replace ferredoxin. We have carried out studies on the effect of iron deficiency in the culture medium of Anabaena variabilis and have found that, unlike to most other cyanobacteria, flavodoxin is not synthesized in that particular strain. We have also studied the pattern of distribution of both forms of ferredoxin that are produced as a consequence of limiting iron in the culture medium.

Materials and methods Organism

The cyanobacterium Anabaena variabilis PCC 6309, was obtained from the University of G6ttingen (FRG) collection (strain 1403.4b).

Culture media and conditions A. vanabtlis cells were grown in batch cultures at 28~C. in BGll medium, supplemented with potassium nitrate, as described by Rippka et al. (1979), m 10 liter bottles purged with a 5% COz in air gas mixture. Illumination was provided by a bank of fluorescent tubes with an intensity of 3000 lx at the surface of the flasks. The iron impurities present in the medium were ehmmated by passing all the solutions containing components of the culture medium

529 Table 1. Effect of several iron concentrations on Anabaena variabilis PCC 6309 ferredoxins content. The cells were harvested in exponential growth phase, and 20 g of fresh weight were used

1.6

Fe (mg/l)

Fe in the medium (mg/1)

nmol/g dry weight Ferredoxin I

Ferredoxln II

Chlorophyll a

Phycocyamne

1 0.1 0.05 0.03 0.015

106 37 14 9 3

45 23 10 7 6

26 17 i1 8 3.5

209.1 126.1 79.5 64.8 29.9

Growth rate (doublings/day)

g

-i-YU-

E = 1.2 o o p. uJ O Z 0.8

0.l 0.05 0.03 0.015

1.16 1.09 0.85 0.80

0.000

0.60

O~

\

4

\

2

__.---,--e

,<

,n nO 0.4

0

(t)

a3 <

1

0.0

0

40

80

120

160

TIME (bourn) Fig. 1. Anabaena variabilis growth curves In cultures with different concentrations of iron added to the iron-free medium: [], 1 mg/l: I , 0.I rag/l; ~ , 0.05 rag/l; ik, 0.03 rag/l; e , 0.015 rag/l; I1., no iron m the culture medium. The inset shows the effect of iron on the A. variabdts growth rates

through a chelating resin, except those containing other metals that could be retained. Different amounts of iron were added to the medium in the form if ferric ammonium citrate. 1 rag/1 of iron is equivalent to 17.8 gM.

Analytical methods Chlorophyll a was determined spectrophotometrically as described by Parsons and Strickland (1973). Phycocyanins were estimated in the crude extract, after chlorophyll removal, by the absorption spectra as described by Glazer (1976). Ferredoxin was purified as described by Buchanan and Arnon (1971), from acetone dried cells and the ratios A 4 2 o / A 2 7 5 = 0.49 and A 3 3 o / A 2 7 5 = 0.65 were taken as the criterion for purity. Ferredoxin was quantified from the visible absorbtion spectrum using an extinction coefficient of 9.7 raM- lcm- 1 at 422 nm. Iron was determined by atomic absorption using a Pye-Umcam model SP.9 spectrophotometer after digestion of the sample.

Results and discussion T h e g r o w t h rate o f Anabaena variabilis cells was determ i n e d in cultures c o n t a i n i n g 1, 0.1, 0.05, 0.03 a n d 0.015 mg/1 o f iron, as well as in one with n o i r o n added. T h e i r o n - f r e e m e d i u m c o n t a i n e d less t h a n 0.001 mg/1 as d e t e r m i n e d by m o l e c u l a r or a t o m i c a b s o r p t i o n m e a s u r e -

100 200 Time (hours)

300

Fig. 2. Iron content in cells of Anabaena variabilis transferred from complete to iron-free medium. Ceils that were grown in complete medium were transfered to an iron-free medium after washing them twice with the medium not containing the metal. Aliquots of the culture were withdrawn at the times indicated and used to determine the iron content in the cells, according to Material and methods

0.5

0.5

o cN

0.4

0.4

uJ

0.3

0.3

0.2

0.2

E

(J

Z < m nO m

:i w

iii

Z 0.1

0.1

0.0 . . . . . . . . . . . . . . . . . . . 5 10 15 20 0

0.0

25

FRACTION NUMBE R

Fig. 3. DEAE-cellulose elutlon profile of an extract of cells grown m a medium containing 0.03 mg/1 of iron. After acetone and ammonium sulfate precipitations of the cell-free extract, the sample was applied to the column equilibrated in 50 mM tris/HC1, pH 8.0 and, after washing, eluted with a linear gradient of NaC1 (0 - 0.5 M). The arrow indicates the salt concentration at which flavodoxin would elute in other Anabaena strains

ment. F i g u r e 1 shows t h a t the g r o w t h o f A. variabilis is d e p e n d e n t o n the i r o n c o n t e n t in the culture m e d i u m . T h e g r o w t h rates, s h o w n in the inset, i n d i c a t e g o o d correl a t i o n w i t h respect to the i r o n c o n t e n t o f the culture m e d i a . Cells g r o w t h in the absence o f a d d e d i r o n s h o w s o m e g r o w t h , p r o b a b l y based o n the i r o n reserves. This m a r k e d effect o f the i r o n c o n c e n t r a t i o n in the m e d i u m o n the g r o w t h o f this strain o f Anabaena suggests that it is n o t able to a d a p t its m e t a b o l i s m to i r o n - d e f i c i e n t

530 conditions, probably due to its inability to replace ferredoxin by another carrier not containing iron, namely flavodoxin, as occurs with other cyanobacteria. In cells grown in a complete medium and later transferred to an iron-free medium, we observed (Fig. 2) a pronounced loss of the iron content inside the cells, reaching approximately one fifth of the concentration found at iron-saturating conditions. Parallel to the loss of iron in the cells, a decrease in the level of ferredoxin was observed. Two brown proteins were obtained, after passing crude extracts through a DEAE-cellulose column and eluting at salt concentrations that correpond to those described for ferredoxin. Figure 3 shows the typical elution profile for substances absorbing at 420 nm. Both bands, obtained at approximately 0.25 M and 0.4 M NaC1 concentrations, were collected and their absorption spectra indicate that both correspond to plant-type ferredoxins. N o indication of the presence of flavodoxin was found in any of the conditions assayed, even when the cells died. The absence o f flavodoxin in the crude extract of cells of A. variabilis grown in iron deficient conditions was confirmed by the absence of reactivity with antibodies prepared against flavodoxin obtained from Anabaena PCC 7119 by Fillat et al. (1988). Figure 3 shows the position at which flavodoxin elutes when extracts o f Anabaena PCC 7119 grown in iron-deficient conditions are treated. The strain PCC 6309 of Anabaena variabilis appears, then, as genetically unable to induce flavodoxin synthesis in conditions in which other cyanobacteria do. Table 1 presents the different parameters determined in cells grown in media containing 1, 0.1, 0.05, 0.03, 0.015 mg/1 of iron. Ferredoxin II which, according to different authors ( C a m m a c k et al. 1977; Chan and Markley 1983), corresponds to the first eluting band, amounts to aproximately 30% of the total ferredoxin in cells grown in complete medium while it becomes the m o s t a b u n d a n t form at low iron concentrations. Iron deprivation of A. variabilis cells also causes a loss of both chlorophyll and phycocyanin content, as shown in Table 1. This effect of iron deprivation on the chlorophyll content of cyanobacteria, and other photosynthetic organisms, has already been related to the effect of iron on the synthesis of the thylakoid membranes, ( G u i k e m a and Sherman 1983), which includes the synthesis of proteins not containing iron. Synthesis of phycobilins in A. variabilis also depends on the iron content in the culture media (Table 1) as has been reported for Anacystis nidulans by Pakrasi et al. (1969). We have not determined whether the limited content of phycocyanins in iron-deprived cells is due to a specific effect on the synthesis of the pigment or to the lower capacity of the cells to assimilate nitrogen c o m p o u n d s (nitrate in our conditions), which has been reported to

have a strong influence in the phycobiliprotein content in cyanobacteria (Allen and Smith, 1969).

Acknowledgements. This work was supported in part by Grants PR84-0792 from CAICYT, Spain and CM-2/85 from Diputaci6n General de Arag6n.

References Allen MM, Smith AJ (1969) Nitrogen chlorosis in blue-green algae. Arch Microbiol 69:114-120 Buchanan B, Arnon DI (1971) Ferredoxins from photosynthetic bacteria, algae and higher plants. Methods Enzymol 23:413-441 Cammack R, Rao KK, Bargeron CP, Hutson KG, Andrew PW, Rogers LJ (1977) Midpoint redox potentials of plant and algal ferredoxins. Biochem J 168:205-209 Chan TM, Markley JL (1983) Nuclear magnetic resonance studies of two-iron-two-sulfur ferredoxins. 1. Properties of the histidine residues. Biochemistry 22 : 5982 - 5987 Fillat MF, Sandmann G, G6mez-Moreno C (1988) Flavodoxin from the nitrogen-fixing cyanobacterlum Anabaena PCC 7119. Arch Microbiol 150:160-164 Glazer AN (1976) Phycocyanins: structure and function. Photochem Photobiol Rev 1 : 7J - 115 Guikema JA, Sherman LA (1983) Organization and function of chlorophyll in membranes of cyanobacteria during iron starvation. Plant Physiol 73 : 250 - 256 Guikema JA, Sherman LA (1984) Influence of iron deprivation on the membrane composition of Anacystis nidulans. Plant Physiol 74:90-95 Ho KK, Krogman DW (1982) Photosynthesis. In: Carr NG, Whitton BA (eds) The biology of cyanobacteria. Blackwell, London, pp 191-214 Manzano C, Candau P, G6mez-Moreno C, Relimpio AM, Losada M (1976) Ferredoxin dependent photosynthetic reduction of nitrate and nitrite by particules of Anacystis nidulans. Mol Cell Biochem 10:161 -- 169 Pakrasi HB, Goldenberg A, Sherman LA (1969) Membrane development in the cyanobacterium Anacyst~s nidulans during recovery from iron starvation. Plant Physiol 79:290- 295 Parsons HB, Strickland TR (1973) Pigment analysis. In: Stein JR (ed) Handbook of phycological methods: culture methods and growth measurements. Cambridge University Press. Cambridge, pp 360- 367 Rippka R, Deruelles J, Waterbury JB, Herman M, Stanier RY (1979) Generic assignements, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111 : t - 61 Rogers LJ (1987) Ferredoxins, flavodoxins and related proteins: structure, function and evolution. In: Van Baalen P, Fay C (eds) The cyanobacteria. Elsevier, Amsterdam, pp 35-67 Sandmann G, B6ger P (1983) The enzymological function of heavy metals and their role in electron transfer processes of plants. In: Lanchli A, Bileski RK (eds) Inorganic plant nutrition. Encyclopedia of plant physiology vol 12. Springer, Berlin Heidelberg New York, pp 563-596 Wada K, Matsubara H, Chain RK, Arnon DI (1981) A comparative study of the biological activities of two molecular species of chloroplast-type ferredoxins. Plant Cell Physlol 22:275281