Regulation of nitrogen fixation by different nitrogen sources in the filamentous non-heterocystous cyanobacterium Microcoleus sp.

FEMS Microbiology Letters 153 (1997) 11^15

Regulation of nitrogen ¢xation by di¡erent nitrogen sources in the ¢lamentous non-heterocystous cyanobacterium Microcoleus sp. Grazçyna E. Sroga* Department of Biochemistry, Uppsala Biomedical Center, Box 576, S-75123 Uppsala, Sweden

Received 4 April 1997 ; accepted 9 April 1997

Abstract

The pattern of N2 fixation, the synthesis and activity of nitrogenase under different nitrogen sources was studied in the filamentous, non-heterocystous cyanobacterium Microcoleus sp. grown under defined culture conditions. Cells grown under a 10 h light/14 h dark (10L/14D) cycle with N2 as an inorganic nitrogen source showed highest nitrogenase activity (acetylene reduction) at the end of the light phase and then a decrease after entering the dark phase. Nitrogenase synthesis was neither suppressed after 7 days of growth with 2 mM NaNO3 or 0.2 mM (NH4 )2 SO4 or 0.3 mM urea nor with 20 mM NaNO3 or 3 mM (NH4 )2 SO4 or 4 mM urea under the 10L/14D cycle. Western immunoblots tested with polyclonal antisera against the Feprotein revealed the following: (1) the Fe-protein was synthesized in cells grown with N2 as well as in cells grown with NaNO3 or (NH4 )2 SO4 under the 10L/14D cycle; (2) the Fe-protein was found in cells grown with urea under the 10L/14D cycle, but not in the darkness; (3) only one protein band, corresponding to the Fe-protein, was found in cells harvested during the light phase of the 10L/14D cycle under the tested conditions. No nitrogenase activity was observed when chloramphenicol was added to the cultures 4 h before the onset of the light period. This observation suggest de novo synthesis of nitrogenase in Microcoleus sp. Keywords :

Cyanobacterium;

Microcoleus

sp.; Nitrogen ¢xation; Nitrogenase

1. Introduction

A number of prokaryotic organisms are able to ob`tain their cellular nitrogen from dinitrogen (N2 ) present in the earth's atmosphere. A fundamental property of nitrogenase is its extreme sensitivity to inactivation by O2 . Puri¢ed nitrogenase, regardless of source, is rapidly and permanently inactivated by exposure to O2 . Although N2 ¢xation and O2 * Corresponding author. Department of Biology, Materials Research Center, r. 302, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180-3590, USA. Tel.: +1 (518) 276-6375; Fax: +1 (518) 276-2344.

appear to be incompatible, in practice O2 and N2 ¢xation coexist. Various diazotrophs have reconciled those two incompatible processes in di¡erent ways. For example, certain cyanobacteria form heterocysts [2] that lack the oxygen-evolving photosystem II and are the sites of nitrogen ¢xation [2]. Many non-heterocystous cyanobacteria synthesize nitrogenase only under microaerobic or completely anaerobic conditions [9]. Currently two ¢lamentous non-heterocystous cyanobacteria, Trichodesmium thiebautii [11] and Microcoleus chthonoplastes [7], are known to ¢x N2 under fully aerobic conditions in light. The results of various studies show that utilizable

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G.E. Sroga / FEMS Microbiology Letters 153 (1997) 11^15

12

nitrogen sources are usually assimilated in preference

for the nitrogenase activity assay and immunoblot-

to N2 , and in the presence of such compounds nitro-

ting for the Fe-protein assay were used in order to

genase activity is absent or greatly reduced. N2 ¢x-

examine the regulation of nitrogenase in vivo. The

ation is regulated at the levels of nitrogenase activity

results suggest that the N2 -¢xing system of

Microco-

and nitrogenase synthesis. Activity, for example, is

leus

dependent upon the provision of ATP and reductant.

darkness with urea ; (2) all three tested combined ni-

Metabolism can also in£uence the rate of N2 ¢xation

trogen sources were present in the medium. Since

through the supply of carbon skeletons for assimila-

there was no shift in the electrophoretic mobility of

generated by N2 ¢xation. In most

the Fe-protein under the tested conditions, it is be-

NH‡ 4

tion of the

diazotrophs,

assimilated

lieved that a post-translational modi¢cation of the

through the glutamine synthetase-glutamate synthase

Fe-protein is not involved in regulation of its activity.

pathway.

newly

NH‡ 4

¢xed

nitrogen

is

sp. is fully shut down when : (1) cells grew in

is a toxic metabolite ; it can inhibit

the supply of ATP and reductant to nitrogenase [1,4].

Generally

NH‡ 4

is

released

by

diazotrophs

2. Materials and methods

when assimilation is inhibited [1,4]. Nitrogenase synthesis is regulated by the availability of ¢xed nitrogen.

NH‡ 4

repress synthesis of nitrogenase activity. However,

NH‡ 4

2.1. Organism and growth conditions

and other forms of combined nitrogen

is not itself a repressor molecule. Repression

‡ NH‡ 4 requires NH4 as3 3 similation. On the other hand, NO3 and NO2 , which of nitrogenase synthesis by

also inhibit N2 ¢xation, do not always act through their conversion to

NH‡ 4

[1,4].

Axenic cultures of

Microcoleus

sp. were estab-

lished from a ¢eld sample of non-heterocystous cyaè sk, Poland). nobacteria obtained from J. Barcz (Gdan A single ¢lament transfer technique was applied. The organism

was

grown

in

an

Arti¢cial

Sea

Water

(ASW) medium [10] with 7 mM CaCl2 . For N2 -¢xing

Nitrogenase is composed of two proteins, a Fe-

cultures, a nitrogen source was omitted from the

protein and a Mo-Fe protein, that are highly con-

medium. The cultures were grown in 150 ml Erlen-

Trichodesmi-

meyer £asks containing 50^75 ml of the growth me-

served among N2 -¢xing organisms. In

um

cells that actively ¢x N2 , the Fe-protein exists in

dium on a rotary shaker at 150 rpm. N2 -¢xing cul-

two di¡erent molecular sizes, with a greater content

tures were kept under a 10 h light/14 h dark (10L/

of the smaller form. Only the larger form was found

14D) cycle or in darkness at 28³C. Non-¢xing cul-

Trichodesmium cells that did not ¢x nitrogen and 3 ‡ were grown on NO3 or NH4 [5]. In two ¢lamentous non-heterocystous cyanobacteria, Oscillatoria limosa [13] and Trichodesmium thiebautii [16], the Fe-protein

tures were grown in the presence of the following

exists in two di¡erent apparent molecular sizes. The

used in the experiments presented here.

in

high-molecular-mass kDa

in

size,

(HMM)

while

the

form

is

about

nitrogen sources : 2 mM or 20 mM NaNO3 , or 0.2 mM or 3 mM (NH4 )2 SO4 , or 0.3 mM or 4 mM urea. A 10 mM concentration of chloramphenicol was

40.5

lower-molecular-mass

2.2. Nitrogenase activity

(LMM) form is about 39.5 kDa. It was suggested that the modi¢ed HMM form resulted from the con-

Nitrogenase activity was estimated indirectly by

version of the LMM form in response to O2 expo-

the standard acetylene reduction technique [14]. To

sure. The only ¢lamentous non-heterocystous cyano-

20 ml of the culture in a 40 ml test bottle, 1 ml

bacterium which does not modify the Fe-protein is

commercial acetylene was added. The bottles were

Plectonema boryanum

can

incubated for 1 h and sampling was done every 15

¢x N2 only anaerobically or microaerobically at the

min. Samples were analyzed by gas chromatography.

[8]. However,

P. boryanum

dark phase of the dark/light cycle. Here the e¡ects of three di¡erent nitrogen sources,

‡ NO3 3 , NH4 ,

and urea, as well as chloramphenicol on

Microcoleus sp. are reported. The acetylene reduction method

2.3. Determination of chlorophyll a and protein concentration

the synthesis and activity of nitrogenase of

Cyanobacterial

FEMSLE 7606 22-10-97

suspension

(1

ml)

was

¢ltered

G.E. Sroga / FEMS Microbiology Letters 153 (1997) 11^15

13

the primary antibodies was done overnight. Following four 10 min washes in PST bu¡er with 1% BSA, the membrane was incubated for 2 h with blotting grade a¤nity puri¢ed goat anti-rabbit IgG (H+L) alkaline conjugate (Bio-Rad). 3. Results and discussion

3.1. Rates of acetylene reduction

Aerobic nitrogenase activity associated with Misp. in axenic cultures appeared about 5 days after the transfer of the washed cells to the nitrogen-free medium. The maximum activity (9.0^ 12.8 nmol C2 H4 [Wg chla]31 h31 ) was seen late (after 8^9 h) in the light phase of the 10L/14D cycle (Fig. 1). It was followed by a decline of acetylene reduction when cells entered the dark phase. However, the activity did not decrease abruptly and was still detectable after 4^5 h of darkness. After illumination nitrogenase activity increased after about a 3^4 h lag period. The pattern of N2 ¢xation of Microcoleus sp. di¡ers from that of Trichodesmium sp. NIBB 1067, for which activity decreased through the latter half of the light period and was undetectable prior to the start of the dark period [6]. No acetylene reduction was observed in the samples treated with chloramphenicol, grown with NO33 , NH‡ 4 or urea as a sole nitrogen source. Addition of chloramphenicol stopped the synthesis of nitrogenase, suggesting that under the experimental conditions nitrogenase needed to be newly synthesized each light/dark cycle. crocoleus

Fig. 1. Nitrogenase activity (acetylene reduction) in cultures of Microcoleus sp. grown under a 10L/14D cycle.

through the Millipore Fritted Funnel ¢tted with a glass micro¢ber ¢lter GF/C (diameter 2.4 cm; Whatman). The pellet was rinsed with 10 ml deionized water and extracted twice with 90% (v/v) methanol for 1 h at 4³C, in dim light, followed by centrifugation at 10 000Ug for 10 min at 4³C. The chlorophyll a content was calculated from the absorbance of the methanolic extract at 665 nm, using the following equation: C (Wg ml31 ) = OD665 U13.9. Protein concentrations were determined by the use of the BCA Protein Assay Kit (Pierce, USA). 2.4. Western blot

A sample (0.1 g) of Microcoleus sp. was heated at 100³C for 5 min in a loading bu¡er (200 mM TrisHCl pH 6.8, 2% SDS, 10% glycerol, 2 mM PMSF). Routinely 5 Wl of the crude cell lysate was electrophoresed on a 15% (w/v) SDS-PAGE and then transferred to 0.45 Wm PVDF transfer membrane (Millipore). Non-speci¢c binding sites were blocked with phosphate-bu¡ered saline, 3% (w/v) bovine serum albumin (BSA). An antiserum was obtained from rabbits immunized with the recombinant fusion MBP-357Tt protein. The 375-Tt-DNA fragment of nitrogenase gene from Trichodesmium sp. was cloned by PCR [12]. All other cloning steps and puri¢cation of the recombinant fusion protein were done as described by Zehr et al. [15]. The antiserum was used at 1:1000 dilution. After 5 h blocking, incubation with

3.2. Nitrogenase protein

The Fe-protein of nitrogenase is a highly conserved prokaryotic enzyme. Antisera raised against the Fe-protein from one organism normally crossreact with the corresponding protein of another organism. The antisera raised against the Fe-protein from cyanobacterium Trichodesmium were used in this study. Immunological studies of the Fe-protein in Microcoleus sp. have revealed that the apparent molecular size is about 32 kDa, larger than the same protein from the heterocystous cyanobacterium Anabaena

FEMSLE 7606 22-10-97

G.E. Sroga / FEMS Microbiology Letters 153 (1997) 11^15

14

Fig. 2. SDS-Page pattern (right) and Western immunoblot analysis of the Fe-protein (left) from the crude protein extracts of

Microcoleus

sp. grown with di¡erent nitrogen sources : (1) 2 mM NaNO3 ; (2) 20 mM NaNO3 ; (3) 4 mM urea in darkness ; (4) all three combined nitrogen sources at lower concentrations ; (5) 0.3 mM urea ; (6) 4 mM urea. Controls : (7)

Microcoleus

sp. and (8)

Anabaena

sp. strain PCC

7120 grown under N2 -¢xing conditions ; (M) SDS-PAGE pattern of the protein molecular mass markers. If not indicated otherwise, cells were collected at the end of the light phase.

sp. strain PCC 7120 (about 30 kDa) (Fig. 2, lane 8

starting around that concentration) in the medium,

and Fig. 3, lane 1). Although nitrogenase activity

the Fe-protein was still detected by Western blot.

Microcoleus sp. cells

(acetylene reduction) was not detectable when cells

Suppression of the Fe-protein in

grew under various nitrogen sources, Western blots

grown with urea in the darkness (Fig. 2, lane 3), but

of the Fe-protein indicate that the protein : (1) was

in the presence of it during growth under the light/

(Fig. 2, lanes 1 and

dark cycle (Fig. 2, lanes 5 and 6) needs further in-

(Fig. 3, lanes 3 and 4), or urea (Fig. 2,

vestigation. However, the data suggest that regula-

present in cells grown with 2), or

NH‡ 4

NO3 3

Microcoleus

lanes 5 and 6) under the 10L/14D cycle ; (2) was

tion of nitrogenase synthesis in

present in cells grown with {FUNC {NO}} _{3}^{-}

be di¡erent from that of another ¢lamentous non-

sp. may

Trichodesmium, capa-

in darkness (Fig. 3, lane 5) ; (3) was absent in cells

heterocystous cyanobacterium,

grown with urea in constant darkness (Fig. 2, lane 3)

ble of ¢xing N2 aerobically in the light [5]. It seems

or with all three combined nitrogen sources under

that the Fe-protein, and so nitrogenase, is not regu-

the 10L/14D cycle (Fig. 2, lane 4). Prolonged incu-

lated at the transcriptional or post-transcriptional

bation of cells (more than 3 days) with urea, as well

level by urea during growth during the light/dark

in the darkness caused bleaching and

cycle, but its synthesis is shut down by urea in the

as with

NH‡ 4,

subsequent death of cells. In that case,

NH‡ 4

sis in

NO3 3

and

did not suppress nitrogenase Fe-protein synthe-

Microcoleus

sp. during growth under the 10L/

14D cycle. Even at 3 mM (NH4 )2 SO4 concentration (the toxic e¡ect of high

NH‡ 4

levels was observed

constant darkness. Post-translational regulation by reversible modi¢cation of the Fe-protein similar to [16] or

Oscillatoria limosa

Trichodesmium

[13] had not been found

under the tested conditions. However,

Microcoleus

sp. ¢xes N2 aerobically in the light. Since preliminary data revealed nitrogenase activity in

Microcoleus

sp.

in the presence of H2 S (G.E. Sroga, unpublished), perhaps anoxygenic respiration is involved in the protection mechanism against the oxygen degradation or is capable of transient support of the nitrogenase activity. If so, Fig. 3.

Western immunoblot analysis of the Fe-protein from the

crude protein extracts of

Microcoleus

sp. grown with : (3) 0.2

mM (NH4 )2 SO4 ; (4) 3 mM (NH4 )2 SO4 ; (5) 2 mM NaNO3 in darkness ; (6) 20 mM NaNO3 . Controls : (1) PCC 7120 and (2)

Microcoleus

Anabaena

sp. strain

sp. grown under N2 -¢xing condi-

Microcoleus

sp. may be similar

to those cyanobacteria that are able to perform anoxygenic photosynthesis with H2 S as an electron donor and can switch totally or partly from a normal green plant-type oxygenic photosynthesis to a bacte-

tions. If not indicated otherwise, cells were collected at the end

rial-type anoxygenic photosynthesis when ambient

of the light phase.

sul¢de concentration becomes su¤ciently high [3].

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G.E. Sroga / FEMS Microbiology Letters 153 (1997) 11^15

Acknowledgments

15

non-heterocystous cyanobacterium. FEMS Microbiol. Lett. 5, 163^167.

The work was supported by an MBP Grant.

[8] Rai, A.N., Borthakur, M. and Bergman, B. (1992) Nitrogenase derepression, its regulation and metabolic changes associated with diazotrophy in the non-heterocystous cyanobac-

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