Plant Molecular Biology 11:335-344 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

335

Expression of the Escherichia coil glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC6301 causes ammonium tolerance D. A Lightfoot 1, A. J. Baron and J. C. Wootton*

Department of Genetics, University of Leeds, Leeds LS2 9JT, UK (*author for correspondence); 1Present address: Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA Received 8 March 1988; accepted in revised form 10 June 1988

Key words: ammonia assimilation, ammonia toxicity, cyanobacteria, glutamate dehydrogenase, E. coli gdhA gene, Synechococcus PCC6301 Abstract The unicellular cyanobacterium Synechococcus PCC6301 lacks a hybridisable homologue of the strongly conserved gdhA gene of E. coli that encodes NADP-specific glutamate dehydrogenase. This is consistent with the failure to find this enzyme in extracts o f the cyanobacterium. The E. coli gdhA gene was transferred to Synechococcus PCC6301 by transformation with an integrative vector. High levels of glutamate dehydrogenase activity, similar to those found in ammonium grown E. coli cells, were found in these transformants. These transformed cyanobacteria displayed an ammonium tolerant phenotype, consistent with the action of their acquired glutamate dehydrogenase activity as an ammonium detoxification mechanism. Minor differences in colony size and in growth at low light intensity were also observed.

Introduction In all microorganisms and plants that have been suitably studied, the flow of nitrogen from inorganic sources into organic metabolites is funelled through ammonium. All such organisms appear to possess the enzymes glutamine synthetase (E.C. 6.3.1.2) and glutamate synthase (E.C. 1.4.7.1; E.C. 1.4.1.13) which provide an essentially unidirectional, ATPdriven assimilation o f ammonium into the amide group of L-glutamine and the amino group o f Lglutamate [reviews: 18, 32, 36]. The very high affinity o f glutamine synthetase for ammonia, with K m values in the range 10 to 150/~M in microorganisms and plants [17, 32], enables organisms to utilise nitrogen sources that generate only very low intracellular ammonium concentrations, for example soil nitrate or gaseous dinitrogen.

Many prokaryotic and eukaryotic microorganisms also possess an assimilatory NADP-specific glutamate dehydrogenase (E.C. 1.4.1.4) which provides an alternative ATP-independent route for the incorporation o f ammonium nitrogen into the amino group of L-glutamate [reviews: 32, 36]. This enzyme shows approximately 5- to 20-fold weaker ammonia affinity than glutamine synthetase, with K m values around 0.3 to 1.2 mM for the NADP-specific glutamate dehydrogenases of prokaryotes, fungi and green algae for which adequate data are available [35]. NADP-specific glutamate dehydrogenase could give a more energy-efficient pathway for assimilation of moderate concentrations of ammonium than the ATP-driven glutamine synthetase/glutamate synthase cycle. The role of glutamate dehydrogenase in the assimilation of very low ammonium concentrations is limited by the reversibility

336 of its reaction in addition to its affinity [36]. There is strong evidence supporting the predominance of the glutamine synthetase/glutamate synthase pathway for ammonium assimilation in higher plants and some photosynthetic microorganisms [18]. Such organisms have low intracellular ammonium pools. Artificially increasing these pools causes deleterious effects [4] including uncoupling of photosystem II [10]. Many photosynthetic organisms, and also some heterotrophic bacteria, apparently lack NADP-specific glutamate dehydrogenase, consistent with an evolutionary history under nitrogen scavenging conditions of growth [34, 36] or under constraints of symbiotic nitrogen fixation [3]. Cyanobacteria carry out photosynthesis and associated assimilation by processes closely analogous to those of higher plants. The cyanobacterium Synechococcus PCC6301, a strain convenient for genetic manipulation, has been reported to lack glutamate dehydrogenase completely [7, 21], whereas some other cyanobacteria possess NADP-specific glutamate dehydrogenases [21, 31] some apparently functioning in ammonium assimilation [16]. We show here that PCC6301 lacks DNA sequences that might encode a protein of the highly conserved NADP-

specific glutamate dehydrogenase family. We report the construction of stable transformants of PCC6301 carrying the gdhA gene of E. coli which express levels o f NADP-specific glutamate dehydrogenase activity comparable to those found under normal conditions in E. coli. Such engineered strains show an increased tolerance o f high ammonium concentrations and minor differences in growth at low light intensities.

Materials and methods

Bacterial strains and plasmids Strains of Synechococcus PCC6301 and E. coli are listed in Table 1. Plasmids are described in Table 1 and Fig. 1.

Media and growth conditions Growth and maintenance of the E. coli strains has been described elsewhere [13]. Strains of Synechococcus PCC6301 were maintained as described

Table 1. Relevant characteristics of strains and plasmids used (a) Strains

E. coli X5119 E. coli CLR207 recA Synchococcus PCC6301 Synchococcus PCC6301/pDN1 Synchococcus PCC6301/pDGI Nostoc MAC PCC8009 (b) Plamsids

pDN 1 pBG1 pDG1

Source or reference

Relevant characters*

[19] [13] N.G.Carr, Warwick. This work This work N.G.Carr, Warwick

gdhA + recA, gdhA. Wild type Ap. Ap, Cm, Gdh ÷ Wild type

Nature of insert(s)

Vector

Source or reference

Relevant characters

Synechococcus chromosomal DNA E. coli chromosomal DNA pBGI and part of pDNa insert

pBR322

[ 11 ]

Ap

pACYC184

[13]

Cm, Gdh ÷

pBR322

This work

Ap, Cm, Gdh +

* : Gdh ÷ - Assayable glutamate dehydrogenase activity. Ap - Resistance to the antibiotic ampicillin. Cm - Resistance to the antibiotic chloramphenicol.

337 previously [11] with the following modifications: BGll 0 media [24] were supplemented with either ammonium chloride or sodium nitrate as indicated in the text. The antibiotics ampicillin and chloramphenicol were included at 2/~g/ml where indicated. Light intensities for growth as measured at the surface of cultures were 10 W/m 2 (high), 3 W/m 2 (medium) or 0.5 W/m 2 (low). Microtitre dish wells for growth tests contained BGll 0 medium solidified with 1% Difco agar and supplemented with different levels of nitrogen source. Microtitre plates and liquid batch cultures were inoculated with cells washed in BGll 0 medium. The growth o f liquid cultures was measured spectrophotometrically by absorbance (OD) in the range 6 0 0 - 6 6 0 nm [28, 30]. Growth on plates was measured by the diameter of colonies. Growth on microtitre plates was followed by inspection and densitometry.

Construction of a probe for the conserved region of E. coli gdhA The plasmid pGEM4Z10 was constructed in vitro from the recombinant M13 bacteriophage (mp7Z10) which contains nucleotides 376 to 657 of the gdhA sequence (numbered according to [15]) and plasmid vector pGEM4 (Promega Biotech Ltd). A mixture of EcoRI-linearized pGEM4 and the 310 bp EcoRI fragment from mp7-Z10 ds DNA was subjected to ligation and transformation into E. coli CLR207 recA. The 310 bp fragment contains 29 bp o f the M13mp7 polycloning site and was isolated from an agarose gel by electroelution after EcoRI digestion and electrophoresis. Plasmids with inserts were characterised by restriction mapping. Probes were made by nick translation o f the 310 bp fragment reisolated from pGEM4Z10.

Construction of transforming plasmids Transformation protocols Synechococcus PCC6301 was transformed as previously described [11]. E. coli transformation was performed by the method of [5].

Plasmid pDG1 was constructed in vitro from pDN1, which contains an insert of Synechococcus PCC6301 DNA cloned in pBR322 [11], and pBG1 which contains the E. coli gdhA gene [13] as shown in Fig. 1.

Isolation and analysis of DNA Plasmid DNA was isolated from E. coli following the method o f [8]. Total genomic DNA was extracted from cyanobacterial strains by the method of [14]. DNA was further purified by fractionation on caesium chloride density gradients. Analysis o f DNA samples was performed by restriction endonuclease digestion, agarose gel electrophoresis, ethidium bromide staining and Southern blotting and hybridisation [12]. 32p-labelled probes were constructed from isolated DNA fragments and supercoiled plasmid DNA by nicktranslation. Hybridisation and washes were underconditions of either low stringency (55 °C, 6 x SSC) or high stringency (hybridisation at 65 °C in 6 × SSC followed by washes at 65 °C in 0.1 × SSC), where SSC is 0.15 M sodium chloride, 0.015 M tri-sodium citrate.

Preparation of cell free extracts and assays of glutamate dehydrogenase activity Cells of either E. coli or Synechococcus PCC6301 were harvested, washed and lysed by sonication following the methods of [19]. Extracts were clarified by either low speed centrifugation (10000 x g, 5 min) or high speed centrifugation (100000 x g, 1 h) and collection of the supernatant. Spectrophotometric assays for glutamate dehydrogenase activity and activity staining following polyacrylamide gel electrophoresis have been described [19].

Results and discussion

Screening of cyanobacterial DNA for gdhA genes gdhA

genes

encoding

assimilatory

NADP-

338 B

,,

l

S

SB

t#

H I

H I

SB I IpBG1

BS

I I

SB

BS

111

I1

I

pDG1

I

pDN1

Fig. 1. Construction of the gdhA plasmid pDG1. A mixture of BamHl-linearised pBG1 and BamHI digested pDNI was subjected to ligation and transformation into E. coli CLR207recA. Ap Cm colonies were selected and screened for the Gdh ÷ phenotype by growth tests on a minimal salts medium containing the necessary supplements except L-glutamate. The restriction map of the plasmid from one such transformant, pDG1, is shown. This plasmid contains the Synechococcus PCC6301 DNA insert of pDNI, except for the 1.3 kbp BamHI fragment which has been replaced by the complete pBG1 DNA in the orientation shown. B: BamHI; H: Hpal; S: Sail. Horizontal arrow: location and direction of transcription of the E. coli gdhA gene. Vector sequences are indicated by open boxes.

d e p e n d e n t g l u t a m a t e d e h y d r o g e n a s e s are u n u s u a l l y s t r o n g l y c o n s e r v e d in a large p a r t o f the c o d i n g sequence [13, 19, 36]. E n t e r o b a c t e r i a l a n d e u k a r y o t i c fungal c o d i n g sequences share a p p r o x i m a t e l y 80% a m i n o acid a n d 7 5 % n u c l e o t i d e sequence i d e n t i t y over the region p r o b e d in the e x p e r i m e n t r e p o r t e d here. In e x p e r i m e n t s using S o u t h e r n blots, p r o b e s c o n t a i n i n g n u c l e o t i d e s 376 to 657 o f E. coli gdhA ( n u m b e r i n g o f [15]) show clear cross h y b r i d i s a t i o n at low stringency to the h o m o l o g o u s genes in the g e n o m i c D N A o f a wide range o f b a c t e r i a a n d fungi [13, 19, 36]. C y n o b a c t e r i a are c o n s i d e r a b l y m o r e closely related to E. coli t h a n are e u k a r y o t i c fungi, b o t h in e v o l u t i o n a r y distance a n d in c o d o n preference. Therefore, if genes e n c o d i n g m e m b e r s o f the conserved NADP-specific glutamate dehydrogenase f a m i l y are present in c y a n o b a c t e r i a , they s h o u l d be d e t e c t a b l e by h y b r i d i s a t i o n at low stringency using the insert o f p G E M 4 Z 1 0 as a probe. N i c k - t r a n s l a t e d p G E M 4 Z 1 0 insert was used to p r o b e S o u t h e r n b l o t s o f g e n o m i c D N A f r o m Synechococcus PCC6301 a n d f r o m N o s t o c M A C

Fig. 2. Southern blot analysis of gdhA and glnA gene homologues in cyanobacterial genomic DNA. Total DNA from Synechococcus PCC6301 was restricted with HindlII. BarnHI and EcoRl (lanes labelled A, B and C respectively). Total DNA from Nostoc MAC PCC8009 was restricted with EcoRl (lanes labelled D). After agarose gel electrophoresis these digests, Southern blot analysis was carried out with the following probes: Panel h the 1.3 kbp EcoRI containing the C-terminal 314 residues of glnA from Anabaena PCC7120 [33]; panel 2: the 310 bp insert cleaved from pGEM4ZI0 (see the Methods section) including the region highly conserved between known gdhA homologues in bacteria and eukaryotic fungi [13]. Hybridisation and washes were under conditions of low stringency. Size markers are in kbp.

PCC8009. T h e latter is a facultative h e t e r o t r o p h [2] typical o f m a n y f i l a m e n t o u s c y a n o b a c t e r i a t h a t produce N A D P - d e p e n d e n t g l u t a m a t e d e h y d r o g e n a s e activity [7, 16, 20, 21]. T h e p r o b e h y b r i d i s e d to several f r a g m e n t s o f N o s t o c M A C P C C 8 0 0 9 D N A at low stringency (Fig. 2, lane 2D), b u t no b a n d s above the n o n - s p e c i f i c b a c k g r o u n d were o b s e r v e d with Synechococcus PCC6301 D N A (Fig. 2, lanes 2A, 2B, 2C). S i m i l a r results to t h o s e shown in Fig. 2 lanes 2A, 2B, 2C a n d 2D were o b t a i n e d with o t h e r p r o b e s p r e p a r e d f r o m n o n - o v e r l a p p i n g sections o f the E. coli gdhA gene ( d a t a n o t shown). These results s u p p o r t the c o n c l u s i o n t h a t Synechococcus PCC6301 lacks a d e c t a b l e gdhA gene h o m o l o g u e , whereas N o s t o c M A C P C C 8 0 0 9 in contrast c o n t a i n s at least one a n d p r o b a b l y two gdhA h o m o l o g u e s . M u l t i p l e genes have been r e p o r t e d p r e v i o u s l y in f i l a m e n t o u s c y a n o b a c t e r i a , for example n i f H a n d p s b A genes [6, 20]. A l t h o u g h lack o f cross h y b r i d i s a t i o n can never p r o v i d e definitive p r o o f o f the a b s e n c e o f a gene, the

339 conclusion that Synechococcus PCC6301 lacks an equivalent of gdhA was reinforced by a parallel probing of the same digests with a glnA sequence encoding part of glutamine synthetase. This probe contained a fragment of glnA DNA from the filamentous cyanobacterium Anabaena PCC7120 that encodes the C-terminal region of the polypeptide. Comparisons of glnA sequences [9, 25, 33] shows that this region is strongly conserved (62% amino acid and 60% nucleotide identity between the Anabaena and Salmonella typhimurium sequences), although less so than the segments of different gdhA genes corresponding to the pGEM4Z10 probe. Under conditions of low stringency the glnA probe hybridised strongly to fragments o f Nostoc MAC PCC8009 DNA (Fig. 2, lane 1D) and more weakly but significantly to Synechococcus PCC6301 DNA (Fig. 2, lanes 1A, 1B, 1C; in lane 1C a small band of less than 1 kbp was visible but is not shown in the photograph). This result corresponds to the expected evolutionary relationships of the cyanobacteria: Anabaena is more closely related to Nostoc than to Synechococcus [24, 30]. This strengthens the conclusion that the absence of cross hybridisation o f the E. coli gdhA probe to Synechococcus PCC6301 DNA in three different restriction digests is a significant negative result. The enterobacterial gdhA gene is expected to have approximately equal phylogenetic distances from different cyanobacteria. The apparent lack of a gdhA gene in Synechococcus PCC6301 is consistent with the reported failure to observe glutamate dehydrogenase activity in this strain [7, 21, 22].

Introduction of the E. coli gdhA gene into Synechococcus PCC6301 Antibiotic resistant strains of Synechococcus PCC6301 were selected, after incubation with the DNA of plasmids pBG1, pDN1 and pDG1 (Table 1, Fig. 1), by three different antibiotic selection regimes: ampicillin (Ap), chloramphenicol (Cm) and ampicillin plus chloramphenicol (ApCm) (Table 2). Plasmid pBG1 produced no antibiotic resistant colonies. Plasmid pDN1, an integrative transformation

Table2. Transformation of Synechococcus PCC6301 with plasmid DNA Antibiotic selection regimes

Ampicillin Chloramphenicol Ampicillin and Chloraphenicol

Transformants per/~g of plasmid pDG1

pDNI

pBG1

71 11 10

162 0 0

0 0 0

vector for this cyanobacterium [11], produced Ap (PCC6301/pDN1), but not Cm, colonies. The composite plasmid pDG1 produced seven times as many Ap as Cm or A p C m (PCC6301/pDG1) colonies, which indicates that ampicillin is more efficient than chloramphenicol for the selection o f transformants of Synechococcus PCC6301. Incubation with other internally deleted derivatives of pDN1 routinely gave efficiencies of less than 0.1%0 of pDN1 transformation [11]. Since incubation with pDG1 gave a transformation efficiency 43% of that for pDN1, the large insertion o f foreign DNA appears to overcome the deleterious effects of internal delections on transformation. The presence of E. coli gdhA sequences in the genome of ApCm (PCC6301/pDG1) transformants was demonstrated by hybridisation of Southern blots at high stringency. Digests of total DNA samples extracted from a PCC6301/pDG1 strain and from an untransformed control were probed with gdhA probe pGEM4Z10 and with complete pDG1 (Fig. 3). The 1.3 kbp fragment detected in HpaI digests of this DNA is diagnostic of an essentially complete coding sequence of E. coli gdhA [13, 15, 19]. The pDG1 probe identified two additional large fragments of approximately 27 and 15 kbp (Fig. 3, lane B). These add up to the size of flanking fragments expected if the donor pDGI plasmid integrated into the Synechococcus PCC6301 chromosome by a single homologous crossover event. The 20 kbp fragment, identified in the untransformed Synechococcus PCC6301 DNA by the pDG1 probe (Fig. 3, lane C) and in previous work by probes containing pDN1 sequences [11], is the only region of the recipient chromosome homologous to the pDN1 in-

340 Expression o f E. coli g d h A in S y n e c h o c o c c u s PCC6301

Fig. 3. Southern blot analysis of E. coli gdhA sequences in cyanobacterial DNA after transformation of Synechococcus PCC6301 to Ap Cm with pDG1. Total DNA from Synechococcus PCC6301/pDG1 was digested with HpaI (lanes A and B). After agarose gel electrophoresis of these digests Southern blot analysis was carried out with the following probes: Lane A: the insert of highly conserved gdhA DNA from pGEM4Z10, as in the experiment shown in Fig. 2; lane B: the complete plasmid pDG1. Lane C shows total DNA from untransformed Synechococcus PCC6301 digested with HpaI and hybridized with complete pDG1. Hybridizations and washes were carried out at high stringency. Size markers are in kbp. The three bands observed in lane B (27, 15 and 1.3 kbp) add up in size to the 43 kbp expected if pDG1 has integrated by a single homologous crossover into the 20 kb region that is seen to hybridize in lane C. The 1.3 kbp hpal fragment is diagnostic of the coding sequence of E. coli gdhA [13].

sert. A s f u r t h e r evidence s u p p o r t i n g a n i n t e g r a t e d state o f the pDG1 D N A , p l a s m i d sequences were detected in high m o l e c u l a r weight ( 5 0 - 1 0 0 k b p ) u n c u t t o t a l D N A f r o m P C C 6 3 0 1 / p D G 1 t r a n s f o r m a n t s by p r o b e s c o n s t r u c t e d f r o m p B R 3 2 2 ( d a t a n o t shown). T h e s e d a t a s u p p o r t the c o n c l u s i o n t h a t pDG1 integrated into the recipient c h r o m o s o m e by the single h o m o l o g o u s crossover m e c h a n i s m previously prop o s e d for m a n y o t h e r t r a n s f o r m a n t s o f S y n e c h o c o c cus PCC6301 [ll]. T h u s pDN1 provides an effective integrative vector for this c y a n o b a c t e r i a l strain.

Biochemical detection of glutamate dehydrogenase activity in t r a n s f o r m a n t s of Synechococcus PCC6301 required the f r a c t i o n a t i o n o f cell-free extracts b e c a u s e rapid, g l u t a m a t e - i n d e p e n d e n t reduct i o n o f N A D P was o b s e r v e d in s p e c t r o p h o t o m e t r i c assays using b o t h c r u d e extracts a n d s u p e r n a t a n t s . F r a c t i o n a t i o n was achieved by n o n - d e n a t u r i n g polya c r y l a m i d e gel electrophoresis (Fig. 4). T h e glutam a t e d e h y d r o g e n a s e activity detected in strain P C C 6 3 0 1 / p D G 1 (Fig. 4, lane B) m i g r a t e d the s a m e distance as the e n z y m e in extracts o f E. coli (Fig. 4, lane C). N o activity was d e t e c t e d in extracts f r o m u n t r a n s f o r m e d strains o f PCC6301 (Fig. 4, lane A), i n d i c a t i n g the a b s e n c e o f a n e n d o g e n o u s N A D P specific g l u t a m a t e d e h y d r o g e n a s e activity. A b a n d o f m a t e r i a l f r o m S y n e c h o c o c c u s b u t n o t E. coli extracts stained in the absence o f g l u t a m a t e (Fig. 4, lanes A a n d B: b a n d m i g r a t i n g faster t h a n the glutam a t e d e h y d r o g e n a s e activity). This activity m a y be r e s p o n s i b l e for the p r o b l e m o f n o n - s p e c i f i c N A D P r e d u c t i o n in s p e c t r o p h o t o m e t r i c assays. A p p r o x i m a t e l y e q u a l q u a n t i t i e s o f g l u t a m a t e deh y d r o g e n a s e a c t i v i t y were extracted f r o m equal v o l u m e s o f pellets o f E. coli a n d S y n e c h o c o c c u s PCC6301 cells. T h e E. coli activity c o r r e s p o n d e d to a p p r o x i m a t e l y 0.05 ttmoles o f substrate converted

Fig. 4. Analysis of E. coli glutamate dehydrogenase activity in Synechococcus PCC6301. Extracts were electrophoresed on a 5°70 non-denaturing polyacrylamide gel, which was then stained for NADP-dependent glutamate dehydrogenase activity. Lane A: untransformed Synechococcus PCC6301; lane B: Synechococcus PCC6301 transformed with pDG1; lance C: E. coli strain X5119. GDH: L-glutamate-dependent stain. ?, L-glutamateindependent stain.

341 / m i n / m g protein in the sonicated extract, from spectrophotometric assays made as previously described [19]. This is the normal level found in a m m o n i a induced E. coli strains [36]. Therefore expression of the E. coli gdhA gene and assembly o f the enzyme into active hexamers must occur efficiently in the cyanobacterium. Efficient heterologous expression of E. coli gdhA has now been achieved in the diverse bacteria Methylophilus methylotrophus [34], Rhizobium phaseoli [3], Synechococcus PCC6301 (this work) and Klebsiella aerogenes (M. J. McPherson and J. C. W., unpublished). No engineering of promoter sequences was required in any of these cases. It is likely that promiscuous expression of the E. coli gdhA gene is achieved by transcription from its own promoter, which is o f the conventional prokaryotic type [36], as also demonstrated for the high level expression of the Klebsiella aerogenes gdhA gene in E. coli [19]. In pDG1, the E. coligdhA gene is more than 5 kbp downstream of cyanobacterial sequences, separated by the D N A derived from the pACYC184 vector. It is therefore very improbable that the expression of this gene in Synechococcus PCC6301 is directed by PCC6301 sequences, although the involvement of pACYC184 sequences cannot be ruled out in this case.

Ammonium tolerance of PCC6301/pDG1 strains A p colonies of several different PCC6301/pDG1 isolates grew to a two fold greater diameter on BGll 0 agar plates than PCC6301/pDN1 or other transformants with unrelated integrative plasmid vectors [11]. The greater diameter might reflect increased growth rate a n d / o r greater mobility across the agar surface. To distinguish these possibilities growth rates of strains were compared side by side and in response to nitrogen source concentration on solid media. No significant differences in growth were detected during growth with nitrate as the nitrogen source over a wide range o f conditions (data not shown). Therefore the larger colonies might reflect increased cell mobility or be induced by the presence of ampicillin in the medium. Growth tests on solid media in wells of microtitre

Table 3. C o m p a r i s o n o f g r o w t h o f Synechococcus P C C 6 3 0 1 and transformants on a range of ammonium

concentrations.

M i c r o t i t r e d i s h wells, i n o c u l a t e d w i t h 5/~1 p e r well o f a s u s p e n s i o n o f cells f r o m a o n e h u n d r e d f o l d d i l u t i o n o f a fully g r o w n l i q u i d culture were incubated at 30°C with 3 W/m 2 illumination. At least f o u r r e p l i c a t e s o f e a c h c u l t u r e w e r e tested. Ammonium

(mM)

6301

6301/pDN1

6301/pDGI

6

+++

+++

+++

12

+++

+++

+++

18

+++

+++

+++

+++

+++

24

++

30

+

36

+

++ +

+++ +++

42

-

+

+++

48

-

-

+++

+ + Normal growth. + + Weak growth. + B a r e l y visible g r o w t h . -

Growth not detectable.

dishes containing systematic differences of ammonium concentrations in BGll 0 agar media showed that strain PCC6301/pDG1 had a substantially increased tolerance of high a m m o n i u m concentrations compared with PCC6301/pDN1 and untransformed PCC6301 (Table 3). Tolerance to a m m o n i u m concentrations greater than 25 to 30 m M may be due to characteristics conferred by pBG1, probably glutamate dehydrogenase activity. Several parallel liquid batch cultures of untransformed Synechococcus PCC6301, PCC6301/pDN1 and PCC6301/pDG1 were grown at non-deleterious concentrations of a m m o n i u m or nitrate as nitrogen source at three different light intensities. Culture growth was measured and the mean data for strains of the three types collated. Indices of early and late phases of culture growth, exponential and arithmetic growth rates respectively, and final densities of cultures are compared in Table 4. Considering the variation o f 5-10°70 between replicate cultures, there was no significant difference between the strains compared in any of these growth conditions except in the maximal densities observed in growth at low light intensity (Table 4). PCC6301/pDG1 consistently reached approximately 60°7o greater density than the untransformed con-

342 Table 4. Growth of replicate liquid batch cultures of Synechococcus PCC6301 and transformants with pDN1 and pDG1 at three light intensities. Mean values for strains PCC6301 (n = 2), PCC6301/pDN1 (n = 2) and PCC6301/pDG1 (n = 4) are given : MD; mean maximum OD of cultures; EGR; mean doubling times (h) during exponential growth at low OD (0.1 to 0.2); AGR ; mean rate of increase constant (OD/100 h) during arithmetic growth at moderate to high OD. (N.D. = Not Done). (a) Growth on nitrate Light PCC6301 intensity

PCC6301/ pDN 1

PCC6301/pDG1

MD EGR AGR MD EGR AGR MD EGR AGR High 3.25 18 Medium 0.55 22 Low 0.25* 60

2.5 3.00 19 2.5 2.9 19 0.85 0.65 22 0.85 0.55 19 0.1 N.D. 0.40* 55

(b) Growth on ammonium Light PCC6301 PCC6301/ intensity pDN 1

2.0 0.9 0.17

PCC6301/pDG1

MD EGR AGR MD EGR AGR MD EGR AGR High 0.50 32 Medium 0.62 42 Low 0.22* 44

0.37 0.55 45 0.48 0.45 45 0.55 0.65 40 0.55 0.57 35 0.05 N.D. 0.35* 44

0.48 0.55 0.07

* These values are significantly different between strains.

trol. The reason for this difference is u n k n o w n but it is possible that, u n d e r energy-limited growth at low light intensity, glutamate dehydrogenase might confer an advantage to cells otherwise dependent on the ATP-requiring glutamine synthetase/glutamate synthase pathway for a m m o n i a assimilation. A n energy advantage o f this type for cells containing glutamate dehydrogenase activity has previously been suggested for Methylophilus methylotrophus [34] and E. coli [36].

Conclusions

We have failed to detect in Synechococcus PCC6301 either a hybridisable h o m o l o g u e o f the E. coli gdhA gene or N A D P - d e p e n d e n t glutamate dehydrogenase activity. O u r observations and those o f [7, 21, 31] support the conclusion that this strain, unlike several other cyanobacteria, lacks a m e m b e r o f this strongly

conserved gene and enzyme family. There is one report o f accumulation o f glutamate in this strain when glutamine synthetase activity is inhibited [16], but it is not necessary to postulate the presence o f glutamate dehydrogenase to explain the data o f [16]. The E. coli gdhA gene is expressed efficiently in transformants o f Synechococcus PCC6301 from an integrated c h r o m o s o m a l copy, producing levels o f enzyme activity similar to those in a m m o n i u m grown E. coli cells. These transformants show a markedly increased a m m o n i u m tolerance c o m p a r e d with the u n t r a n s f o r m e d PCC6301 which is strongly inhibited by levels o f 30 m M a m m o n i u m . M i n o r differences in colony size and in growth at low light intensity were also observed in the transformants expressing glutamate dehydrogenase activity. The causes o f these m i n o r effects are u n k n o w n but they are reminiscent o f effects o f engineered increases in glutamate dehydrogenase activity noted previously in growth o f E. coli [36] and Methylophilus methylotrophus [34]. The increased a m m o n i u m tolerance o f these transformants is likely to reflect a reduction in the intracellular a m m o n i u m pools resulting f r o m an increased capacity for a m m o n i u m assimilation by glutamate dehydrogenase activity. This would avoid several possibly deleterious effects o f high intracellular a m m o n i u m concentrations [4], including the saturation o f p H regulation [29], uncoupling o f photosynthetic electron transport [10] a n d an inhibition o f certain enzymes [1]. It would be interesting to investigate these mechanisms further, and possibly to extend the strategy o f engineering glutamate dehydrogenase activity to higher plants. The productivity o f m a n y higher plant species, when grown on a m m o n i u m salts, is related to their ability to detoxify a m m o n i u m [1, 4]. Metabolic poisoning by increased intracellular a m m o n i a pools has been observed in plant tissues treated with methionine sulphoxamine, an inhibitor o f glutamine synthetase [23, 26]. A similar suggestion has been m a d e to explain inhibitory effects o f a m m o n i u m on Nostoc strains [27]. In view o f our results it would be valuable to study in detail the possible roles for glutamate dehydrogenase activity in a m m o n i u m detoxification in b o t h cyanobacteria and higher plants.

343

Acknowledgements W e t h a n k M . J. M c P h e r s o n a d v i c e , J. M c C h e s n e y

and A. Mountain

for

for technical assistance and

D. A l l e y a n d N . G. C a r r f o r s u p p l y i n g c y a n o b a c t e r i al s t r a i n s a n d D N A Financial GR/C/45836

support

from Nostoc MAC PCC8009. was

from

SERC

grant

t o J. C. W.

References 1. Beevers L, Hageman RH: Nitrate and nitrite reduction. In: Miflin BJ (ed.) The Biochemistry of Plants, Volume 5, pp. 115-168. Academic Press, New York (1980). 2. Bottomly P J, van Balen C: Characterization of heterotrophic growth in the blue-green alga Nostoc sp. strain MAC. J Gen Microbiol 107: 309-318 (1978). 3. Bravo A, Becerril B, Mora J: Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation. J Bact 170:985-988 (1988). 4. Givan CV: Metabolic detoxification of ammonia in higher plants. Phytochem 18:375-382 (1979). 5. Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557-580 (1983). 6. Haselkorn R: Organisation of the genes for nitrogen fixation in photosynthetic bacteria and cyanobacteria. Ann Rev Microbiol 40: 5 2 5 - 547 (1986). 7. Hoare DS, Hoare SL, Moore RB: The photoassimilation of organic compounds by autotrophic blue-green algae. J Gen Microbiol 49: 351-370 (1967). 8. Ish-Horowicz D, Burke JF: Rapid and efficient cosmid vector cloning. Nucl Acids Res 9:2989-2998 (1981). 9. Janson CA, Kayne PS, Almassy R J, Grunstein M, Eisenberg D: Sequence of glutamine synthetase from Salmonella typhimurium and implications for the protein structure. Gene 4 6 : 2 9 7 - 3 0 0 (1986). 10. Krogmann DW, JagendorfAT, Avron M: Uncouplers of spinach chloroplast photosynthetic phosphorylation. Plant Physiol 34:272-277 (1959). 11. Lightfoot DA, Waiters DE, Wootton JC: Transformation of the cyanobacterium Synechococcus PCC6301 using cloned DNA. J Gen Microbiol 134:1509-1514 (1988). 12. Maniatis T, Fritsch E, Sambrook J: Molecular Cloning: A Laboratory Manual, 1st edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). 13. Mattaj IW, McPherson M J, Wootton JC: Localization of a strongly conserved section of coding sequence in glutamate dehydrogenase genes. FEBS Lett 147:21-25 (1982). 14. Mazur BJ, Rice D, Haselkorn R: Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Nat Acad Sci USA 77: 186-190 (1980). 15. McPherson M J, Wootton JC: Complete nucleotide sequence

of the Escherichia coli gdhA gene. Nucl Acids Res 11: 5257-5266 (1983). 16. Meeks JC, Wolk CP, Lockau W, Schilling N, Shaffer W, Chen WS: Pathways of assimilation of [13N]N2 and [13N]NH4 + by cyanobacteria with and without heterocysts. J Bact 134:125-130 (1976). 17. Meister A: Catalytic mechanism of glutamine synthetase: overview of glutamine metabolism. In: Mora J, Palacios R (eds) Glutamine: Metabolism, Enzymes and Regulation, pp. 1-40. Academic Press, New York (1980). 18. Miflin BJ, Lea P J: Ammonia assimilation. In: Miflin BJ (ed.) The Biochemistry of Plants, Volume 5, pp. 169-202. Academic Press, New York (1980). 19. Mountain A, McPherson M J, Baron A J, Wootton JC: The Klebsiella aerogenes glutamate dehydrogenase gene: cloning, high level expression and hybrid enzyme formation in Escherichia coli. Mol Gen Genet 199:141-145 0985). 20. Mulligan B, Schultes N, Chen L, Bogorad L: Nucleotide sequence of a multi-copy gene for the B-protein of photosystem II of a cyanobacterium. Proc Nat Acad Sci USA 81: 2693-2697 (1984). 21. NeilsonnAH, Doudoroff M:Ammoniaassimilationinbluegreen algae. Archiv Microbiol 8 9 : 1 5 - 2 2 (1973). 22. Pearce J, Leach CK, Carr NG: The incomplete tricarboxylic acid cycle in Anabaena variabilis. J Gen Microbiol 55: 371-378 (1969). 23. Platt SG, Anthon GE: Ammonia accumulation and inhibition of photosynthesis in methionine sulphoxamine treated spinach. Plant Physiol 67:509-513 0981). 24. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY: Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1-61 (1979). 25. Rocha M, Vazquez M, Garciarrubio A, Covarrubias AA. Nucletide sequences of the glnA-glnL intercistronic region of Escherichia coli. Gene 3 7 : 9 1 - 9 9 (1985). 26. Sinden SL, Durbin RD: Glutamine synthetase inhibition: possible mode of action of wildfire toxin from Pseudomonas tabaci. Nature 219: 379-380 (1968). 27. Singh HN, Ladha JK, Kumar HD: Genetic control of heterocyst formation in the blue-green algae Nostoc muscorum and Nostoc linkea. Archiv Microbiol 144:155-159 (1987). 28. Singh HN, Sonie KC, Singh HN: Nitrate regulation of heterocyst differentiation and nitrogen fixation in a chlorate resistant mutant of the blue-green alga Nostoc muscorum. Mutation Res 42:447-452 (1977). 29. Smith FA, Raven JA: Intracellular pH and its regulation. Ann Rev Plant Physiol 30:289-311 (1979). 30. Stanier RY, Adelberg EA, Ingraham JL: General Microbiology, 4th edn. McMillan Press, London (1977). 31. Stewart WDP: Some aspects of structure and function in N 2fixing cyanobacteria. Ann Rev Microbiol 3 4 : 4 9 7 - 5 3 6 (1980). 32. Stewart GR, Mann AF, Fentem PA: Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase and glutamate synthase. In: Miflin BJ (ed.) The Biochemistry

344 of Plants, Volume 5, pp. 169-202. Academic Press, New York (1980). 33. Tumer NE, Robinson SJ, Haselkorn R: Different promoters for the Anabaena glutamine synthetase gene during growth using molecular or fixed nitrogen. Nature 306:337-343 (1983). 34. Windass JD, Worsey M J, Pioli EM, Pioli D, Barth PT, Atherton KT, Byrom D, Powell K, Senior PJ: Inproved conversion of methanol to single-cell protein by Methylophilus

methylotrophus. Nature 287:396-401 (1981). 35. Wootton JC: Reassessment of ammonium ion affinities of NADP-specific glutamate dehydrogenase: activation of the Neurospora crassa enzyme by ammonium and rubidium ions. Biochem J 209:527-531 (1983). 36. Wootton JC, McPherson M J: Genes of nitrate and ammonium assimilation. Ann Proc Phytochem Soc Europe 23: 89-114 (1984).

Expression of the Escherichia coli glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC6301 causes ammonium tolerance.

The unicellular cyanobacterium Synechococcus PCC6301 lacks a hybridisable homologue of the strongly conserved gdhA gene of E. coli that encodes NADP-s...
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