Molecular characterization of the gene encoding glutamine synthetase in the cyanobacterium Calothrix sp. PCC 7601

Vol. 189, No. 3, 1992 December 30, 1992

MOLECULAR

BlO&lEMlCAL

CHARACTERIZATION

AND BIOPHYSICAL RESEARCH COMMUNICAJONS Pages 1296-l 302

OF THE GENE ENCODING GLUTAMINE

SYNTHETASE IN THE CYANOBACTERIUM

Calorhrix sp. PCC 7601

Khalil Elmorjani, Sylviane Liotenberg, Jean Houmard, and Nicole Tandeau de Marsac*

Physiologie Microbienne, Departement de Biochimie et GCnCtiqueMoleculaire, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France

Received

October

28,

1992

SUMMARY: In order to study the regulation of the synthesis of glutamine synthetase in response to changes in environmental parameters (light and nitrogen sources), we have cloned and sequenced the glnA gene from the filamentous cyanobacterium Calothrix PCC 7601. This gene consists of 472 codons and encodes a polypeptide of Mr 52,290 highly homologous to that from Anabaena PCC 7120, but more distant from those identified from other procaryotes. The relative abundance of the two glnA transcripts (1.6 and 1.8 kb) is equivalent in cells grown under either red or green light, but the 1.6-kb species predominates in nitrate-grown cells and the 1.8-kb species in ammonia-grown cells. The very high identity (74%) observed between the 374-bp long nucleotide sequence upstream from the Calothrix and Anabaena glnA genes suggests the existence of similar regulatory signals for the control of glnA expression in both cyanobacteria. 0 1992Academic Pre**, Inc.

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme in nitrogen metabolism for all organisms, but so far little is known about the regulation of the activity and synthesis of this enzyme in cyanobacteria. GS has been purified to homogeneity from cyanobacteria belonging to various genera, Synechococcus (l), Synechocystis (2), Anabaena (3,4,5,6), Nostoc (4), Phormidium (7,8) and Calothrix (2). In each case, the enzyme consists of twelve identical subunits of approximately 50 kDa. The nucleotide sequence of the corresponding glnA gene, has been reported for Anabaena PCC 7120 (9). Different amino acids, divalent cations, nucleotides and thiols have been shown to modify GS activity (10) and, more recently, the loss of GS activity observed in the presence of ammonium has been attributed to the noncovalent binding of a phosphorylated compound to the enzyme in Synechocystis PCC 6803 (2,ll). To date neither a control of GS synthesis via a transcriptional regulation involving a two-component system, nor a modulation of GS activity by adenylylation/deadenylylation of the enzyme has been found in cyanobacteria (3,12,13). This was evidence for the absence of an enterobacterial-type nitrogen regulated (Ntr) system and thus of PII, protein known to play a central role in the modulation of both activity and synthesis of GS in enteric bacteria (14). However, it is now established that cyanobacteria do possess a PI1 protein highly related to its counterpart in enteric bacteria, but, in * To whom correspondenceshould be addressed.Fax number: (1) 40 61 30 42. Elecmonic mail: [email protected] 0006-291X/92

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cyanobacteria, the covalent modification of the PI1 protein depends on both the nitrogen status and the photosynthetic activity of the cells (15). As part of a study aiming at elucidating the signal transduction pathways that allow cells to sense changes in environmental parameters (light intensity, spectral light quality and nitrogen sources) and the regulatory processes that maintain a proper carbon/nitrogen balance in the cells, we have characterized the glnA gene from Calothrix PCC 7601. This filamentous nitrogen-fixing cyanobacterium has been extensively studied for its ability to adapt its light-harvesting antenna complexes to the incident wavelength and to respond to environmental

stresses by differentiating

nucleotide sequence of the Calothrix

hormogonia (16). We present here the complete

glnA gene, a comparison of the deduced amino acid

sequence with that known from other procaryotes and measurements of the expression of the gene in cells under different growth conditions. MATERIALS

AND METHODS

Culture Conditions and Isolation of DNA and RNA - For cultures of Calofhrix sp. strain PCC 7601 (= Fremyellu diplosiphon UTEX 481) grown under red and green illuminations, Na2C03 concentration of the BG-11 medium was increased 10 times (17). When the source of nitrogen was varied (17.65 mM NaN03 or 8 mM NH&l), Na2C03 concentration was doubled and the medium was supplemented with 10 mM NaHC03. All cultures were bubbled with 1% CO;! in air. Total DNA and total RNA purifications have been described previously (18). Smalland large-scale plasmid extractions from cultures of Escherichiu coli, DNA fragment isolation, and agarose gel electrophoresis were performed as described by Maniatis er al. (19). Genomic Library Construction and Hybridization - The partial DNA library was constructed by ligation of Hind111 DNA fragments of approximately 4.3 kilobases (kb) into the Hind111 site of plasmid pTZl8R. Standard methods were used for in situ colony hybridization (19). DNA-DNA and DNA-RNA hybridizations were performed on nylon membranes (HybondN, Amhersham) as described elsewhere (20). The DNA probes used were a 455 bp EcoRI fragment from plasmid pAn503 carrying the glnA gene from Anabuena PCC 7 120 (9; Gift from R. Haselkom, USA) or 400 bp fragments internal to the Calothrix PCC 7601 glnA gene. DNA Sequence Analysis - Overlapping clones, from the recombinant plasmid carrying the glnA gene, were obtained by using the Cyclone System from IBI adapted for single-stranded DNAs of the pTZ18R clones (21). To complete the sequence, specific synthetic oligonucleotides, kindly provided by G. Rapoport (Unite de Biochimie Microbienne, Institut Pasteur), were used as primers. DNA sequence analysis was performed by the chain-termination method of Sanger et al. (22) on single-stranded DNA templates according to the protocol from Amersham. Computer analysis of the DNA sequence was carried out using the programs developed by the Unitt d’Informatique Scientifique of the Institut Pasteur, Paris. RESULTS

AND

DISCUSSION

A partial Hind111 gene library from Calorhrix PCC 7601 was screened, using the 455 bp EcoRI DNA fragment internal to the glnA gene from Anabaena PCC 7120 as a probe, and a recombinant plasmid pPM123 containing a 4.3 kb HindIII DNA insert was isolated. To sequence the g1n.A gene in the insert, subclones were generated by the Cyclone System (see Materials and Methods). For sequencing the complementary strand, the 4.3 kb Hind111 fragment from pPM123 was cloned in the reverse orientation in pTZl8R and its size reduced to 3.0 kb after PstI digestion and subsequent intramolecular religation. Among the different subclones, only those containing the complete glnA gene complemented the E. coli glutamine auxotroph strain, ET8051 (23). A physical map of the genomic region carrying the Calothrix glnA gene is presented in figure 1A. The complete nucleotide sequence of the noncoding strand is shown in figure 1B. The glnA gene 1297

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A

H2

ti3

X

Rl H2

H2

Ii2

HP

HP

Pl

B 1 81 161 241 321 401 481 561 641 721 801 881 961 1041 1121 1201 1281 1361 1441 1521 1601 1681

20 40 60 80 TGTTACTGCATCGCGCATTCCGATTTCTCCCAATCATTACTATCTATGGTTTGATATT~TTTGGCTGCTTCATGCACCA GTCATTTTTTGTGCCAATTACAATI\TATAT-TCGGCAC-CAAGTACCCGCTATTTMGGATTCTATGCC-GTTGA CCCCTCTGAGATGGTATGTTCGATTTTTGTACAGGTATGAGGC-GGTTMTA-CTGT-CCGGAGAATCTGT~ CAAAGACTACRAlVVLTTCTTARTGTCATATCATATCCTTAGGATATTCCAGGTGTGTCAC~TTGGTTCATCTCAGCAGGGTCAG CGTGGTTTAARGATTCTAGTTTGAGTTTATTTARTTGGCA ATTGATTCAAGACCAAAAAATTCAGATGATTGATCTGAACGTGT ACTACAACCAAATCGATGAAAGTTCATTCATTCACT~TGGCGTACCTTTCGACGGTTCCAGTATTAGGGGTTGGAAAGGCATC GAAGAATCAGACATGACAATGGTTTTAGATCC-CACTGCCTGGATC~CCCATTCATG-GAGCC~CTCTMGTAT AATTTGTAGCATT-GICCTCGCACAGGCGAATGAGACT ATTTAGTTTCTACTGGCCTTGGTGACACAGCTTTCTTTGGCCCAG~GCTGAGTTCTTTATCTTT~TGATGCCCGTTTT GACC-CTGCCIVLCTCAGGTTATTACTAGRCTC CCTCGCTTACAAACCACGCTTCAAAGAAGGTTACTTACTTCCCAGTTGCGCCCACTGATACTTTCCAGGATATGCGTACAGTGCTGTTGACGATGGCAGCTTGCGGCGTACCCATTGAIUTG GGTTTCCGCTTTGGTAAGCTCATCGMGCTGCTGACTGGTTGATGACTTACAAGTATGTATGTCATCAAG~CGTTGCCAAG~ ATATGGTAG~CCGTGACCTTTATGCCA-CC~TTTTTGGTGATMTGGTTCGGGGATGCACTGTCACCAGTCTATTT GGAAGGATGGTAI\ACCTCTATTTGGAGGTGATAAGTATGCTGGTTTGAGTWLCATGGCACTGTATTACATCGGTGGTATC CTCAIIVLCACGCACCAGCATTGTTGGGCATCACAAACCCCCACCACC~CTCTTAC~GCGCTTAGTACCTGGTTATG~GC ACCTGTTAACTTGGCTTACTCCC~GGTTCTGCGCTA AACGTTTAGAGTTCCGTTGCCCAGATGCTACCTCTAACCCCTTACTTGGCATTTGCTGCTATGCTTTGTGCTGGTATTGAT GGTATCAI\GAAT-TCCATCCTGAACCCTTAGACGT TccTTCTACTCCTGGTTCTTTGGAACTGGCATTAWULGCACTGG-C~TCACGCCTTCTTGACTG-GCGGTGTAT TCACAGAI\GACTTTATCCAAAACTGGATCGATC~GTAC~GCTAGTTMCG~GTT~GCAGCTAC~TTACGTCCTCATCCC

1921

AATCTTGAGCTTTATGCCTGCA

FIG. 1. (A) Physical map of the 3.0-kb genomic region carrying the @A coding sequence. Restriction enzymes are abbreviated as follows: H3, HindIII; HZ, HincII; X, XhoII, RI, EcoRI; Hp, HpaI; PI, PstI. (B) Nucleotide sequence of the glnA gene. Start and stop codons are indicated in bold type. The putative ribosome-binding site is underlined. Arrows indicate a palindromic sequence.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence Databases under accession number LO5609.

corresponds binding

to an open-reading

site (AGGAG)

frame of 471 amino

acids preceded by a putative r&some-

located 6bp upstream from the initiation codon AUG. The predicted

molecular mass of the protein is 52,290 daltons which is in agreement with the molecular mass estimated for the purified GS subunit (2). A hypothetical palindromic sequence could be drawn within the 3’flanking region of the glnA gene. This structure with a 10 nucleotides long stem and a loop of 6 nucleotides displays a thermodynamic stability of 15.3 kcal/mol, as calculated according to the method of Salser (24) modified by Cech et al. (25). DNA-RNA hybridization experiments were performed using total RNA extracted from Calothrix PCC 7601 cells grown in BG-11 medium under red or green light, and from cells grown under white light in a medium containing either nitrate or ammonia as a nitrogen source. As shown in figure 2, two transcripts of 1.6 and 1.8 kb are revealed with a DNA probe internal to the @A gene. While the relative abundance of the two mRNA species is approximately equivalent in Culorhrix

cells grown under either red or green light, the 1.6 kb species predominates in cells

grown in the presence of nitrate and the 1.8 kb species in cells grown in the presence of ammonia. Based on DNA-DNA hybridization experiments, it is very likely that there exists only one glnA gene copy in the Calothrix genome (data not shown). Thus, together the results indicate that &A is a monocistronic unit and suggest that transcription initiation may occur at two different promoters whose expression is modulated by the available exogenous source of nitrogen. This regulation of the expression of the gld gene according to the nitrogen status of the cells is 1298

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NO3 NO3

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w

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w

NO3 NH4

i: cn

1.81.6-

FIG. 2. Analysis by DNA-RNA hybridization of the glnA mRNAs. Total RNAs, extracted from Calothrix PCC 7601 cells grown under either red (R), green (G) or white (W) illumination in a culture medium containing NaN03 (N03) or NH&l (Nb) as nitrogen sources,were hybridized at 45°C in 50% formamide with a probe internal to the glnA genefrom Calothrix PCC 7601. Sizes are indicated in kilobases.

reminiscent of the nitrogen regulation system controlling the expression of the ,@A gene in enteric bacteria (14). However, the consensus sequences GG-NIo-CC, dependent promoter of glnA , and GCAC-N7-GTGC,

characteristic

of the NtrA-

specific of the binding site for the

transcriptional activator NtrC (26) in enteric bacteria, have not been found in the 374 nucleotidelong sequence upstream from glnA in Calothrix PCC 7601. In vivo and in vitro analyses have shown that, in cells from another cyanobacterium, Anabaena PCC 7 120, the glnA gene is mainly transcribed from two promoters, an E. coli-like (P2) and an atypical promoter (P4), located at nucleotides -155 and -273, respectively, while in cells grown under anaerobic nitrogen-fixing conditions, the major mRNA species starts at the @-type promoter (Pl) located at nucleotide -93 (9,27). Given the sizes of the glnA transcripts in Calothrix PCC 7601 and assuming that the 3’ end is located close to the stem and loop structure (nucleotides 1815 to 1840), transcription initiation signals must be present in the 5’flanking sequence shown in figure 1B. Moreover, the 374 nucleotide-long sequence upstream from the start site of translation of glnA from Calothrix PCC 7601 is 74% identical to the corresponding sequence from Anabaena PCC 7120. This suggests that the control of glnA expression very likely involves similar regulatory sequences in these two nitrogen-fixing cyanobacteria. The deduced polypeptide sequence of the glnA gene from Calothrix

has been compared

with the corresponding sequences from Anabaena PCC 7120 and from twelve other bacteria (fig. 3). The similarity coefficient (SAR) (28) calculated between the Calothrix PCC 7601 glnA gene product and each of the corresponding sequences from other micro-organisms showed that the highest value (0.89) is obtained between cyanobacterial sequences. In contrast, the SAR values drop to 0.59-0.35 with sequences from other bacteria, the lowest values being observed with Bacillus subtilis, Clostridium acetobutylicum and archaebacteria. Five regions, that are underlined in figure 3, are closely associated with the GS active site (29) and correspond to domains that are conserved across both the procaryotic and eucaryotic kingdoms (30). Similarly to all GS enzymes 1299

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R.l. A.6. S.C. T.f. B.S. c.a. T.lll. S.S. P.W.

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Cdl. Ana. E.C. s.t. A.V. R.l. A.b. s. c. T.f. B.S. C.d. T. m. S.S. P.W.

100 120 SO 140 DPNTAWIDPFMKEPTL--SIICSIKEPRTGEWYNRCPRVIAQKAIDYLVSTGLGDTAFFGPEAEFFIFDDARF ttttt*ttttt~~*.'~~**~******.*****.*************...*.*****".**********~*** l D l D l *SWSKR*K***R***IA**VL""P"'L*'*I'* *AS**V"**FADS**--I*R*D*L**G*LQG l E l **RA**IA**vL****p***L***~** +A~“V”“FAD~*t--~*~*~*~**~*~Q~.~t~t~~**~~ .~~“~~‘.‘~~““~~~‘V’D’I”S’MPG’D’D”A’t~~t~~tt~ttt~ttttttttt~ttt.tt~~~~ ‘TE’~~M**‘FAQ~*~--~~~t~.~~~~~**~~..~t*~~*~~*E~**~~~*~t*.~*~~*”**..~***~~ **T**VM***SAQ***--N*L*pJy **s**Q~*A****G**KA*EK*M~*~**~**~~********~~**~~* l LS**RV***RRDK**--N*NFF*HD . I .ttQ’~‘D”NV”~“EA’tA”‘IA”“*“““YV’t~~tt **A**QG*E*D*~SV*~R*EI'*K**K****~**~*****~~**~**~~~~ l *DS*VL****D*T**--LLR'DVI *L**FV*F*WTA'KGKVARF**D*YN*D-*TPFEGD **NNLKRILKEMEDL'FS*-FNL'**P**'L=KLD-NLDSFV*F*WRPQQGKVARL**DVYK*D-*TPFEGD**HVLKR*N~~EL*Y--*MNV'**C**~L~ETD-VLD*FAVL*WTVDGAKSARV**DVYT*D-*KPFEGD**YRLRRMMEKAEQL'Y--'PYA'**M***'LPIN-V*E'MTLI*WSPG---VARVL*KVFWGGGKGRFE*D**F**~E*EK*QTEQ*Y--*SYY***L***M**KVKL l *D*YVEV*WDN------VARVYGFIYKDNKP*GAD **G*LKR*LEE*EKE*Y--K*YI***P**YL*K----

Cal. Ana. E.c. s.t. A.V. R.l. A.b. S.C. T.f. B.S. C.a. T.Sl. S.S. P.W.

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Cal. Ana.

E.c. s.t. A.V.

Cal. Ana. E.C. s.t. A.V.

R.l. A.b. s. c. T.f. B.S.

C.a. T.m. S.S. P.W.

FIG. 3. Comparison of the amino acid sequences for glutamine synthetase. Micro-organisms and references are as follows: Cal., refers to the deduced amino acid sequence presented in this paper; Ana., Anabaena PCC 7120 (9); E. c., Escherichia coli (31); S. t., Salmonella typhimurium (32); A. v., Azotobacter vinelandii (33); R. l., Rhizobium leguminosarum (34); A. b., Azospirillum brasilense (35); S. c., Streptomyces coelicolor (36); T.f., Thiobacillus ferrooxidans (37); B. s., Bacillus subtilis (38); C. a., Clostridium acetobutylicum (39); T. m., Thermotoga maritima (40); S. s., Sulfolobus solfataricus (30); P. w., Pyrococcus woesei (40). Stars indicate identical residues. SAB is defined as twice the number of identical amino acids in A and B divided by the total number of amino acids in A + the total number of amino acids in B (28).

sequenced so far, with the exception of Clostridium acetobutylicum, the Calothrix sequence possesses a tyrosine residue in position 399 that corresponds to the tyrosine residue 398 known to be the site of GS adenylylation in E. coli and in other bacteria. However, the activity of 1300

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Cdl. Ana. E.C. s.t. A.V. R.l. A.b. S.C.

T.f. B.S. C.S. T.Rl. S.S. P.W. Gil. AIM. E.C. s.t. A.V. R.l. A.b. S.C. T.f. B.S. C.S. T.m. S.S. P.K.

Cal. AItS. E.C. s.t. A.V. R.l. A.b.

S.C. T.f. B.S. C.S. T.m. S.S. P.W.

FIG.

3 - Continued

the Calofhrix enzyme is likely not controlled by adenylylation, since a strict correlation between the amount of GS protein and its activity has been observed(2). Moreover, the activity of the Anabaena GS has been shown not to be controlled by adenylylation (3,12), and its amino acid sequenceis strongly homologousto that of the Culothrir enzyme. Acknowledements: We thank Douglas Campbell for critical reading of this manuscript. This work was supportedby the Institut Pasteurand by the CNRS (URA 1129).K. E. was supported by a fellowship from the Singer-PolignacFoundationand S. L. by a fellowship from the Minis&e de la Rechercheet de la Technologie. REFERENCES

::

Florencio, F. J., and Ramos, J. L. (1985) B&him. Biophys. Acta 838, 39-48. Merida, A., Leurentop,L., Candau,P., and Florencio, F. J. (1990) J. Bacterial. 172, 47324735.

3.

4.

Grr, J., Keefer, L. M., Keim, P., Nguyen, T. D., Wellems, T., Heinrikson, R. L., and Haselkom, R. (1981) J. Biol. Chem. 256, 13091-13098. Sampaio, M. J. A.M., Rowell, P., and Stewart, W. D. P. (1979) J. Gen. Microbial. 111, 181-191. 1301

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