Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M311518200 on December 16, 2003

J. Biol. Chem., Vol. 279, Issue 12, 11582-11592, March 19, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/12/11582    most recent
M311518200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Picossi, S.
Right arrow Articles by Herrero, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Picossi, S.
Right arrow Articles by Herrero, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Nitrogen-regulated Genes for the Metabolism of Cyanophycin, a Bacterial Nitrogen Reserve Polymer

EXPRESSION AND MUTATIONAL ANALYSIS OF TWO CYANOPHYCIN SYNTHETASE AND CYANOPHYCINASE GENE CLUSTERS IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA SP. PCC 7120*

Silvia Picossi, Ana Valladares, Enrique Flores, and Antonia Herrero{ddagger}

From the Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas-Universidad de Sevilla, E-41092 Seville, Spain

Received for publication, October 21, 2003 , and in revised form, November 26, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N2 as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N2, but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyanobacteria are oxygenic photoautotrophs that make an important contribution to primary productivity in our planet. They fix CO2 through the reductive penthose phosphate pathway and preferentially assimilate inorganic sources of nitrogen. Many cyanobacteria are able to carry out the fixation of atmospheric nitrogen, and, as a way to protect the nitrogen fixation machinery from oxygen, some filamentous strains differentiate cells called heterocysts where nitrogenase is confined (1). Nitrogen fixed in the heterocysts is donated, probably in the form of amino acids, to the rest of cells in the filament by a mechanism not yet understood that may involve the operation of amino acid uptake permeases (2) and that might be related to the accumulation of cyanophycin (see below) in the heterocyst. The differentiation of heterocysts and the assimilation of nitrogen in cyanobacteria are subjected to nutritional repression by ammonium (3). In the absence of ammonium, the transcriptional regulator NtcA activates expression of many genes encoding elements of the pathways for the assimilation of nitrogen or involved in the differentiation and function of the heterocyst (4). NtcA is homologous to transcriptional regulators of the CAP family and directly binds to the promoter regions of nitrogen-regulated genes at specific DNA sites with the consensus sequence GTAN8TAC frequently centered at -41.5 and located upstream from a {sigma}70-consensus -10 box in the form TAN3T (4).

Cyanophycin is a nonribosomically synthesized peptide composed of multi-L-arginyl-poly-L-aspartate ({alpha}-amino groups of arginine residues linked to {beta}-carboxyl groups of a polyaspartate backbone) that until very recently was considered exclusive of cyanobacteria. In these organisms, cyanophycin accumulates in the cytoplasm in the form of granules mainly under unbalanced growth conditions (e.g. under stationary phase or under starvation conditions that do not involve nitrogen starvation) (5, 6). It is considered that cyanophycin can represent a dynamic reserve of nitrogen; e.g. in Cyanothece sp. ATCC 51142, which temporarily separates N2 fixation and photosynthesis, cyanophycin is degraded during the light period and is formed during the period of darkness, when N2 fixation is operative (7). In the N2-fixing heterocyst, cyanophycin accumulates, forming conspicuous deposits in the cellular poles adjacent to the vegetative cells (e.g. see Ref. 8).

A cyanophycin-synthesizing enzyme, called cyanophycin synthetase, has been identified that could add both L-aspartic acid and L-arginine to a cyanophycin primer (9). Recently, cyanophycin synthetase has been purified from the heterocystforming Anabaena variabilis ATCC 29413 and characterized as a homodimer of a 100-kDa subunit (10). Based on the amino acid sequence from this protein, the corresponding gene, cphA, has been identified in the genomic sequence of the unicellular cyanobacterium Synechocystis sp. PCC 6803 and then in Anabaena variabilis (10). On the other hand, based on analysis of the products of cyanophycin degradation by crude or fractionated cell extracts of different cyanobacteria, two different enzymatic activities have been implicated in cyanophycin degradation: an exopeptidase, called cyanophycinase, that would produce {beta}-Asp-Arg dipeptides (11) and a peptidase that would hydrolyze this peptide. Recently, the gene cphB encoding a cyanophycinase has been identified in the genomic sequence of Synechocystis sp. PCC 6803 (12), and ORFs1 of both Synechocystis sp. and Anabaena sp. have been described to encode plant-type asparaginases able to hydrolyze {beta}-Asp-Arg bonds and that, thus, may be responsible for the last step of cyanophycin degradation (13). The cphB and cphA genes have also been identified in whole genome sequence projects of several cyanobacteria.

Putative cyanophycin synthetase genes have recently been identified in the genomic sequences of a number of eubacteria different from cyanobacteria (14), and those from Acinetobacter sp. strain DSM 587 (14) and Desulfitobacterium hafniense (15) have proven able to direct the synthesis of cyanophycin-like polymers when expressed in Escherichia coli. Moreover, 11 eubacteria different from cyanobacteria have been found to be able to utilize cyanophycin as a carbon source for growth based on the action of extracellular cyanophycinases. The product of the so-called cphE gene from Pseudomonas anguilliseptica strain BI, although exhibiting only 27–28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria, catalyzes a very specific degradation of cyanophycin to {beta}-Asp-Arg dipeptides (16). Thus, cyanophycin appears to occur in a wide range of bacteria that make use of diverse metabolic options including phototrophy, aerobic and anaerobic respiration, fermentation, and chemolithoautotrophy, where it can serve, perhaps among other functions, as a nitrogen reserve or an external carbon source. Therefore, cyanophycin seems to have a role in the biology of bacteria much wider than previously recognized.

The present work deals with the genetic systems for cyanophycin synthesis and degradation in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, which is amenable to genetic manipulation and whose entire genomic sequence is available (17). The regulation of the expression of cyanophycin metabolism genes with regard to the nitrogen regime and cell type in the diazotrophic filament has been studied, and the involvement of cyanophycin in diazotrophic metabolism has been investigated through the generation of cyanophycin metabolism gene mutants.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—This study was carried out with the heterocyst forming cyanobacterium Anabaena sp. PCC 7120 and an insertional mutant of the ntcA gene, strain CSE2 (18). Anabaena sp. PCC 7120 was grown axenically in BG11 medium, which contains 17.6 mM NaNO3 (19), in BG110 (nitrogen-free) medium or in BG110 medium supplemented with 8 mM NH4Cl and 16 mM TES-NaOH buffer (pH 7.5). Strain CSE2 was grown in ammonium-containing medium in the presence of 2 µg ml-1 of streptomycin and 2 µg ml-1 of spectinomycin. For plates, the medium was solidified with 1% separately autoclaved agar (Difco). Liquid cultures were incubated at 30 °C in the light (75 microeinsteins m-2 s-1), with shaking (80–90 rpm). Anabaena mutants carrying gene cassette C.K3 (20) were routinely grown in medium supplemented with 5–25 µg ml-1 neomycin, mutants carrying cassette C.S3 (21) were grown in medium supplemented with 2–5 µgml-1 Sm and Sp, and mutants carrying cat and erm genes, encoding for CmR and EmR, respectively, (SalI-XhoI fragment from plasmid pRL271) (22) were grown in medium supplemented with 5 µgml-1 Em. Growth rates were estimated from the increase in protein concentration of the cultures, determined by a modified Lowry procedure (23) in 0.2-ml aliquots periodically withdrawn from the cultures. The growth rate constant (µ) corresponded to ln 2/td, where td represents the doubling time.

Mutant Construction—To inactivate gene cphB1, a 1.63-kb DNA fragment from the cph1 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CB1–1 (corresponding to nucleotides +8 to +29 with respect to the translation start of cphB1) and CA1–3 (complementary to nucleotides +536 to +519 relative to the translation start of cphA1) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in vector pGEM-T (Promega) to generate plasmid pCSS37. The C.K3 gene cassette excised with SmaI (24) was inserted into the BclI site that is present in the Anabaena DNA insert of pCSS37 to generate plasmid pCSS38. The SpeI-ScaI fragment from pCSS38, filled in with Klenow enzyme, was ligated to the sacB-containing vector pRL277 (22) linearized with EcoRV, rendering plasmid pCSS39. The sacB gene determines sensitivity to sucrose and can be counterselected for in Anabaena sp., allowing positive selection for double recombinants (25).

To inactivate gene cphA1, a 2-kb DNA fragment from the cph1 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CA1–1 (corresponding to nucleotides +43 to +63 relative to the translation start of cphA1) and CA1–2 (complementary to nucleotides +2029 to +2009 relative to the translation start of cphA1) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in vector pGEM-T to generate plasmid pCSS33. A 15-bp DNA fragment from pCSS33 was excised with EcoRV and substituted by the C.S3 gene-cassette excised with HindIII (24) and filled in with Klenow enzyme, rendering plasmid pCSS34. The SalI-NcoI fragment from pCSS34, filled in with Klenow enzyme, was ligated to the sacB-containing vector pRL278 (22) digested with NruI, rendering plasmid pCSS35.

To inactivate gene cphB2, a 2-kb DNA fragment from the cph2 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CB2-1 (complementary to nucleotides +1654 to +1636 with respect to the translation start of cphB2) and CB2-2 (corresponding to nucleotides -386 to -366 with respect to the translation start of cphB2) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in vector pGEM-T to generate plasmid pCSS50. The C.S3 gene cassette excised with HindIII was inserted into the HindIII site that is present in the Anabaena DNA insert of pCSS50, rendering plasmid pCSS51. The PvuII fragment from pCSS51 was ligated to the sacB-containing vector pRL271 (22), digested with BglII, and filled in with Klenow enzyme, rendering plasmid pCSS52.

To inactivate gene cphA2, a 1.7-kb DNA fragment from the cph2 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CA2–2-XhoI (complementary to nucleotides +1783 to +1762 relative to the translation start of cphA2 and ended with a XhoI restriction site) and CA2-3-XhoI (corresponding to nucleotides +60 to +77 relative to the translation start of cphA2 and ended with a XhoI restriction site). PCR products were cloned in vector pGEM-T to generate plasmid pCSS53. The EmR/CmR gene cassette (containing erm and cat genes) excised with XbaI from plasmid pCSE52, which contains the SalI-XhoI fragment from pRL271 cloned in vector pIC20R, and filled with Klenow enzyme was inserted into the Eco47III site that is present in the Anabaena DNA insert of pCSS53, rendering plasmid pCSS54. The XhoI fragment from pCSS54 was ligated to the sacB-containing vector pRL278 digested with XhoI, rendering plasmid pCSS55.

Constructs generated in vitro bearing a gene cassette inserted into cphB1, cphA1, cphB2, or cphA2 and cloned in sacB-containing vectors were transferred by conjugation (26) to Anabaena sp. to generate strains bearing mutations in the cph1 or/and cph2 genomic regions. For generation of strains CSS7, CSS13, CSS21, and CSS25, E. coli strain HB101 containing plasmid pCSS35, pCSS39, pCSS52, or pCSS55 and helper plasmids pRL528 (26) and pRL591-W45 (27) or pRL623 (28) was mixed with E. coli ED8654 carrying the conjugative plasmid pRL443 and thereafter with Anabaena sp. For the generation of double mutants, plasmids pCSS39 and pCSS55 were transferred to strain CSS7 to generate strains CSS35 and CSS27, respectively, plasmid pCSS52 was transferred to strain CSS13 to generate strain CSS23, and plasmid pCSS55 was transferred to strain CSS21 to generate strain CSS36. Exconjugants were isolated (26), and double recombinants were identified as clones resistant to the antibiotic for which resistance was encoded in the inserted gene cassette, resistant to sucrose, and sensitive to the antibiotic for which the resistance determinant was present in the vector portion of the transferred plasmid and were confirmed by PCR analysis. Homozygous mutant clones were selected for this study.

DNA Isolation and Analysis—Total DNA from Anabaena sp. PCC 7120 and its derivatives was isolated as previously described (25). For sequencing ladders used in primer extension analysis, sequencing was carried out by the dideoxy chain termination method, using a T7-SequencingTM kit (Amersham Biosciences) and [{alpha}-35S]thio-dATP. DNA fragments were purified from agarose gels with the Geneclean II kit (BIO 101). Southern blot analysis, plasmid isolation from E. coli, transformation of E. coli, digestion of DNA with restriction endonucleases, ligation with T4 ligase, and PCR were performed by standard procedures (29).

Band Shift Assays—DNA fragments to be used in electrophoretic mobility shift assays were obtained by PCR amplification. Oligonucleotides CB1–4 (corresponding to positions -697 to -679 relative to the translation start of cphB1), CB1–5 (complementary to positions -250 to -272 relative to the translation start of cphB1), CB1–7 (complementary to positions -458 to -477 relative to the translation start of cphB1), and CB1–9 (corresponding to positions -485 to -465 relative to the translation start of cphB1) and plasmid pCSS68, containing the cphB1 promoter sequence (cloned by PCR with oligonucleotides CB1–4 and CB1–5 in vector pGEM-T), were used to obtain DNA fragments of the cphB1 upstream region. Oligonucleotides CA1–6 (corresponding to positions -214 to -196 relative to the translation start of cphA1) and CA1–5 (complementary to positions +16 to -3 relative to the translation start of cphA1) and plasmid pCSS37 as a template were used for PCR amplification of the cphA1 upstream region. For competition tests, a DNA fragment from the region upstream from glnA containing an NtcA-binding site (18) was used as a specific binding fragment, and a DNA fragment of the pBluescript vector was used as a nonspecific fragment. In the case of the glnA upstream region, oligonucleotides GA3 (corresponding to positions -238 to -215 relative to the translation start of glnA) and GA6 (complementary to positions -70 to -87 relative to the translation start of glnA) and plasmid pAN503 (30) were used for PCR. In the case of the pBluescript DNA fragment, oligonucleotides Forward and Reverse were used, rendering a DNA fragment of 220 nucleotides. DNA fragments were end-labeled with T4 polynucleotide kinase (Roche Applied Science) and [{gamma}-32P]dATP as described previously (29). Assays were carried out as described previously (31) with 1 fmol of labeled fragment and 0.1–3 µM of NtcA purified from E. coli strain DH5{alpha} (pCSAM61) bearing the strain PCC 7120 ntcA gene cloned in pTrc99A vector (Amersham Biosciences) and thus expressed from the trc promoter. Images of radioactive gels were obtained using a Cyclone storage phosphor system (Packard).

RNA Isolation and Analysis—Cells used for RNA isolation were exponentially growing in the light (75 microeinsteins m-2 s-1) in liquid BG11 or BG110 media (19) or in medium BG110 containing 8 mM NH4Cl and 16 mM TES-NaOH buffer (pH 7.5), supplemented with 10 mM of NaHCO3 and bubbled with air and CO2 (1%, v/v). Alternatively, filaments growing in ammonium-containing medium were harvested at room temperature and either used directly or washed with and resuspended in BG110 medium and further incubated under culture conditions for the number of hours indicated in each experiment. For the isolation of RNA from heterocysts, cells were grown in ammonium-containing medium until they reached a chlorophyll a concentration of 3–5 µg ml-1. Filaments were then washed with and resuspended in nitrogen-free medium (BG110) and further incubated until mature heterocysts were observed (19 h). Heterocysts were then isolated as described (32).

Total RNA from whole filaments or from isolated heterocysts was isolated in the presence of ribonucleoside-vanadyl complex as previously described (33). For Northern analysis, 40–70 µg of RNA were loaded per lane and subjected to electrophoresis in 1% agarose denaturating formaldehyde gels. Transfer and fixation to Hybond-N+ membranes (Amersham Biosciences) were carried out using 0.1 M NaOH. Hybridization was performed at 65 °C according to the recommendations of the manufacturer of the membranes.

The cph probes were internal fragments of these genes amplified by PCR, using plasmid pCSS33 as a template and oligonucleotides CA1–1 and CA1–2 (see above) in the case of the cphA1 probe; plasmid pCSS37 as a template and oligonucleotides CB1–1 (see above) and CB1–2 (complementary to nucleotides +885 to +864 with respect to the translation start of cphB1) for the cphB1 probe; plasmid pCSS53 as a template and oligonucleotides CA2–2 and CA2–3 (see above) for the cphA2 probe; and plasmid pCSS50 as a template and oligonucleotides CB2–3 (complementary to nucleotides +858 to +840 with respect to the translation start of cphB2) and CB2–4 (corresponding to nucleotides +76 to +95 with respect to the translation start of cphB2) in the case of the cphB2 probe. All probes were 32P-labeled with a Ready to GoTM DNA labeling kit (Amersham Biosciences) using [{alpha}-32P]dCTP. Images of radioactive filters and gels were obtained with a Cyclone storage phosphor system and OptiQuant image analysis software (Packard). The rnpB gene, which encodes a stable RNA (34), was used as an RNA loading and transfer control.

Primer extension analysis of the cph transcripts was carried out as previously described (35) with 32P-labeled oligonucleotides CB1–6 (complementary to nucleotides -198 to -220 with respect to the translation start of cphB1) and CB1–5 (see above) for the case of cphB1 and CA1–4 (complementary to nucleotides +74 to +55 with respect to the translation start of cphA1) and CA1–5 (see above) for the case of cphA1.

Cyanophycin Measurements—Cells subjected to a treatment to force cyanophycin accumulation were used. Cells of 150-ml liquid cultures in BG11 medium supplemented with 10 mM NaHCO3 and bubbled with air and CO2 (1%) were harvested in the exponential growth phase (2–5 µg of Chl ml-1), washed twice with BG110 medium, and used to inoculate 150-ml cultures in BG110 medium supplemented with bicarbonate and bubbled with air and CO2 (1%). These cultures were then incubated for about 8 h under culture conditions. After this incubation, NH4NO3 was added at 4 mM final concentration, and the cultures were incubated under dim light (5 microeinsteins m-2 s-1) for 12–14 h. Cells from 100 ml of these cultures were collected at room temperature, washed twice with, and resuspended in, milliQ-purified and autoclaved H2O, and disrupted with a French Press (twice at 20,000 p.s.i.). After measuring the obtained volume of cell extract, chlorophyll a was determined in a 100-µl sample. The remnant of each extract was centrifuged for 15 min at 15,000 rpm in a SS34 rotor, the resulting supernatants were discarded, and the pellets were washed twice with 11 ml of milliQ-purified, autoclaved H2O and resuspended in 1 ml of 0.1 M HCl. After 2–4 h of incubation at room temperature and centrifugation under the same conditions, the resulting supernatants were stored. The pellets were resuspended in 1 ml of 0.1 M HCl, incubated overnight at room temperature, and again subjected to centrifugation. The obtained supernatants were combined with those obtained after the first centrifugation, stored at 4 °C, for no longer than 1 week, and used for arginine determination, which was carried out by the Sakaguchi method as modified by Messineo (36).

Microscopy—Cells grown during 7–10 days in shaken BG110 liquid medium were observed and photographed with a Zeiss Axioscop microscope equipped with an MC 80 camera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Two Clusters of Cyanophycin Metabolism Genes—In the genomic sequence of Anabaena sp. PCC 7120 (17), two clusters of ORFs showing homology to cyanophycin synthetase or cyanophycinase-encoding genes could be identified (Fig. 1). We have named cluster 1 to that containing genes more similar to those identified in other cyanobacteria (e.g. see Refs. 10, 12, and 37). Gene cphB1 would encode a cyanophycinase of 298 amino acids showing 99% identity to CphB from A. variabilis (12) and 60% identity in a 269-amino acid overlap to cyanophycinase from Synechocystis sp. PCC 6803 (37). Gene cphA1 would encode a cyanophycin synthetase of 901 amino acids showing 99% identity to the product of cphA from A. variabilis (10) and 70% identity in an 870-amino acid overlap to CphA from Synechocystis sp. PCC 6803 (37). In Anabaena sp. PCC 7120, cphB is located upstream from cphA, and both genes show the same orientation (Fig. 1).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1.
Schematic representation of two gene clusters found in the genome of Anabaena sp. PCC 7120 that contain genes homologous to cph genes (drawn from data available on the World Wide Web at www.kazusa.or.jp/cyanobase/Anabaena/). The ORF name, length in bp, and proposed gene name are indicated. Flanking ORFs are also indicated. Double lines indicate probes used for Northern analysis (see "Experimental Procedures"). The insertion sites of the C.K3, C.S3, or EmR gene cassettes to generate mutants of each gene are shown (see "Experimental Procedures" for details).

 
Gene cphB2 would encode a 293-amino acid product showing 43% identity in a 259-amino acid overlap to cyanophycinase from A. variabilis and 40% (248-amino acid overlap) to CphB from Synechocystis sp. PCC 6803. Gene cphA2 from Anabaena sp. PCC 7120 would encode a 636-amino acid product showing 33% identity in a 423-amino acid overlap to CphA from strain PCC 6803 and 30% (421-amino acid overlap) to the protein from Anabaena variabilis. In this gene cluster of strain PCC 7120, the cphB2 and cphA2 genes are arranged in opposite orientations separated by 914 bp that include an ORF named asl0572 that would encode a 75-amino acid product showing no significant similarity to any protein in the data bases (Fig. 1).

Expression of cphB1 and cphA1—When expression of the cphB1 gene was analyzed by RNA/DNA hybridization, transcripts of up to ~5 kb could be detected that were present at higher levels in cultures growing diazotrophically than in those growing with combined nitrogen, either nitrate or ammonium (Fig. 2a). Because the length from the beginning of cphB1 to the end of cphA1 is 3,774 bp, the ~5-kb transcript could encompass the message of the two genes. Expression of cphB1 was also tested with RNA from isolated heterocysts, with which only hybridization signals corresponding to smaller transcripts could be detected (Fig. 2a).



View larger version (70K):
[in this window]
[in a new window]
  		FIG. 2.
Expression analysis of the cph1 gene cluster in Anabaena sp. PCC 7120. a and b, RNA was prepared from whole filaments grown with ammonium (A), nitrate (N), or N2 ({Phi}) as the nitrogen source or from isolated heterocysts (H) and probed with DNA fragments of cphB1 (a), cphA1 (b), rnpB (loading and transfer control), nifH (control of the quality of heterocyst RNA), or rbcL (control of the presence of vegetative cell mRNA) amplified by PCR as indicated under "Experimental Procedures." c, RNA was isolated from filaments of strain PCC 7120 (wild type; WT) or CSS13 (cphB1::C.K3) grown with ammonium and incubated during the indicated number of hours in the absence of combined nitrogen and hybridized with a cphA1 probe (upper panel) or an rnpB probe. The positions of some size markers are indicated.

 
Northern experiments performed with a probe of the cphA1 gene also showed hybridization to transcripts of up to ~5 kb in size (Fig. 2b), similar to those detected with the cphB1 probe (Fig. 2a). Hybridization signals were observed with RNA isolated from whole filaments grown under different conditions of nitrogen nutrition or from isolated heterocysts but were more abundant in the diazotrophic culture. Transcripts of ~3 kb were also detected with the cphA1 gene probe (Figs. 2, b and c) that could correspond to cphA1 monocistronic transcripts. To test this possibility, cphA1 expression was analyzed in strain CSS13, an insertional mutant that bears gene cassette C.K3 inserted into the cphB1 gene in the orientation opposite to that of the gene (see below and "Experimental Procedures" for details). Fig. 2c shows that only hybridization signals corresponding to ~3 kb and smaller could be detected in this mutant strain confirming independent transcription of the cphA1 gene.

Primer extension experiments with two different primers (see "Experimental Procedures") detected three 5' transcript ends upstream of cphB1 (Fig. 3a and not shown). The first 5' end, IB in Fig. 3a, was located at nucleotide -339 with respect to the cphB1 initiation codon, showed low relative abundance being preferentially detected with RNA from diazotrophic whole filaments and from isolated heterocysts, and its level increased upon combined nitrogen deprivation (see Fig. 4c). The second 5' end (IIB) was located at nucleotide -357 and appeared to be the most abundant in whole filaments, being detected at similar levels irrespective of the nitrogen source used for growth. Although at a lower level, it was also present in heterocyst RNA. Finally, 5' end IIIB, corresponding to nucleotide -499, was preferentially found with RNA from diazotrophic cultures and was also present in heterocyst RNA. Its cellular level also increased upon combined nitrogen deprivation (see Fig. 4c). (Although some other putative 5' transcript ends are observed in Fig. 3a, only those above mentioned were repeatedly detected with different oligonucleotides.)



View larger version (120K):
[in this window]
[in a new window]
  		FIG. 3.
Primer extension analysis of the expression of the cph1 gene cluster in Anabaena sp. PCC 7120. a and b, RNA was isolated from whole filaments grown with ammonium (A), nitrate (N), or N2 ({Phi}) as the nitrogen source or from isolated heterocysts (H). Oligonucleotides used as primers were CB1–6 (complementary to nucleotides -198 to -220 with respect to cphB1) (a) or CA1–5 (complementary to nucleotides +16 to -3 with respect to the translation start of cphA1) (b). c, RNA was isolated from whole filaments of strain PCC 7120 (WT) or CSS13 (cphB1) grown with ammonium and incubated for 24 h in the absence of combined nitrogen. Oligonucleotide used as primer was CA1–5. The putative 5' transcript ends are indicated. Sequencing ladders were generated with the corresponding oligonucleotides.

 



View larger version (93K):
[in this window]
[in a new window]
  		FIG. 4.
Expression analysis of the cph1 gene cluster in an ntcA mutant strain. a and b, RNA isolated from whole filaments of strain PCC 7120 (wild type; WT) or CSE2 (ntcA) grown with ammonium (0) or grown with ammonium and incubated for the indicated number of hours in the absence of combined nitrogen was subjected to Northern analysis with a probe of cphB1 (a), cphA1 (b), or rnpB as indicated in the legend to Fig. 2. The size of the larger transcript detected is indicated. c and d, RNA isolated from whole filaments of strain PCC 7120 (wild type) or CSE2 (ntcA) grown with ammonium (0) or grown with ammonium and incubated for the indicated number of hours in the absence of combined nitrogen was used in primer extension experiments with oligonucleotides CB1–6 to detect tsps upstream of cphB1 (c)orCA1–5 to detect tsps upstream of cphA1 (d). The putative 5' transcript ends are indicated. Sequencing ladders were generated with the corresponding oligonucleotides.

 
To test for the existence of possible functional promoters upstream from cphA1, primer extension experiments were performed with two different primers of the 5' region of this gene (Fig. 3b and not shown) using RNA from both the wild type and the cphB1 mutant strain CSS13. For the wild type, five different 5' RNA ends were detected corresponding to positions -89 (IA), -116 (IIA), -141 (IIIA), -191 (IVA), and -230 (VA) with respect to the cphA1 initiation codon. 5' RNA ends IVA and VA corresponded to nucleotides inside the cphB1 gene and were not detected in RNA from the cphB1 mutant; the corresponding RNA molecules could represent degradation products of transcripts initiated at promoters upstream from cphB1. 5' ends IA, IIA, and IIIA corresponded to positions located in the intergenic cphB1-cphA1 region. 5' ends IA and IIIA were detected with RNA from both the wild type and the cphB1 mutant strain (Fig. 3, b and c), and they were observed with similar intensity with RNA isolated from wild-type cells subjected to any tested nitrogen regime but were only barely detected with RNA from isolated heterocysts (Fig. 3b). On the other hand, 5' end IIA was detected with RNA from the wild type and, although more weakly, also with RNA from the cphB1 mutant (Fig. 3c), was specially represented in RNA isolated from diazotrophic cultures, and was also present in RNA from isolated wild-type heterocysts, where it was the more abundant species (Fig. 3b). Thus, 5' RNA ends IIIA and IIA (and possibly also IA) could represent true tsps of the cphA1 gene located in the intergenic region between cphB1 and cphA1. The distance from the IIIA and IIA positions to the end of cphA1 is 2,846 and 2,821 bp, respectively, conforming to the ~3-kb transcript size (Fig. 2c).

NtcA-dependent Expression of cphB1 and cphA1—Expression of the Anabaena cphB1 and cphA1 genes was tested by Northern and primer extension analysis in an insertional mutant of ntcA, strain CSE2 (18). Whereas, as described above, an increase in the levels of cphB1 (Fig. 4a) and cphA1 (Fig. 4b) transcripts could be observed in the wild-type strain when the cells were subjected to combined nitrogen deprivation, this increase was somewhat lower in the ntcA mutant. Regarding the 5' transcript ends upstream of cphB1, whereas abundance of IIB was similar in the wild type and in strain CSE2, induction of neither IB nor IIIB took place in this mutant (Fig. 4c). These results indicate a role of NtcA in the activation of expression of the two putative nitrogen-regulated promoters located upstream of cphB1. For the 5' ends upstream of cphA1, the abundance of IA, IIA, and IIIA in both the wild type and the ntcA mutant did not change significantly after 24 h of incubation in the absence of combined nitrogen (Fig. 4d). (Because strain CSE2 does not develop heterocysts, NtcA dependence of cph gene expression in these differentiated cells could not be assessed.)

To further analyze the basis for the dependence on NtcA of expression of cph1 genes, binding of NtcA to the DNA region upstream of cphB1 and to the cphB1-cphA1 intergenic region was tested. Mobility shift of a DNA fragment covering the region from -263 to -693 of the cphB1 gene (Fig. 5a) was specifically promoted by NtcA, producing two retarded bands (Fig. 5b). When any of two DNA fragments covering positions -250 to -477 (fragment B; Fig. 5c) and -458 to -697 (fragment C; Fig. 5d), respectively, were used, single retarded bands were observed in each case that were competed by an excess of an unlabeled DNA fragment containing a well characterized NtcA binding site but not by an excess of a DNA fragment containing no NtcA binding site. These results indicated that NtcA specifically binds to two different sites in the promoter region of cphB1. The dependence of NtcA binding on the concentration of NtcA showed that NtcA affinity for binding to fragment C (Kd = 1.77 µM) is higher than that for fragment B. Additionally, footprinting experiments (not shown) revealed that NtcA changed the sensitivity to DNase I degradation of a DNA stretch encompassing positions -380 to -625, thus confirming the presence in it of NtcA binding site(s). Mobility shift assays were also performed with a DNA fragment encompassing sequences from +16 to -214 of cphA1, showing specific binding of NtcA to this DNA region at a single site (Kd = 0.37 µM) (Fig. 6).



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 5.
Mobility shift assay of the binding of NtcA to DNA sequences upstream of gene cphB1. a, schematic representation of the DNA region upstream of the cphB1 gene showing the location of putative tsps and an NtcA-binding site (double thick vertical lines) (see "Experimental Procedures"). The DNA fragments used (A, B, or C) amplified by PCR with the indicated oligonucleotides (see "Experimental Procedures"), are also represented. b–d, band shift assays were performed with 1 fmol of labeled DNA fragment A (b), B (c), or C (d) in the absence (-) or presence of increasing amounts (from 0.1 to 3 µM) of NtcA protein purified as specified under "Experimental Procedures." For competition tests, shown to the right in Fig. 5, c and d, 1 fmol of the labeled DNA fragment from the cphB1 promoter region was used in the absence (lane 2) or presence of 25 fmol of an unlabeled DNA fragment from the promoter region of the glnA gene containing an NtcA-binding site (lane 3) or from pBluescript plasmid (lane 4) (see "Experimental Procedures" for details). NtcA protein was present at 2 µM in lanes 2–4 of Fig. 5c and at 0.5 µM in lanes 2–4 of Fig. 5d. The assays shown in lanes 1 contained no NtcA protein. Positions reached by the retarded labeled fragment are indicated.

 



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 6.
Mobility shift assay of the binding of NtcA to DNA sequences of the intergenic region of the cphBA1 operon. a, schematic representation of the intergenic DNA region of the cphBA1 operon showing the location of putative tsps, 5' transcript ends, and an NtcA-binding site (double thick vertical lines) (see "Experimental Procedures"). The DNA fragment used, amplified by PCR with the indicated oligonucleotides (see "Experimental Procedures"), is also represented. b, band shift assay was performed with 1 fmol of the labeled DNA fragment shown in a in the absence (-) or presence of increasing amounts (from 0.1 to 3 µM) of NtcA protein purified as specified under "Experimental Procedures." For the competition test, shown to the right of Fig. 5b, 1 fmol of the labeled DNA fragment from the cphBA1 intergenic region was used in the absence (lane 2) or in the presence of 25 fmol of an unlabeled DNA fragment from the promoter region of the glnA gene containing an NtcA-binding site (lane 3) or from plasmid pBluescript (lane 4). NtcA protein present was 0.1 µM except for the assay shown in lane 1 that contained no NtcA protein. The position reached by the retarded labeled fragment is indicated.

 
Expression of cphB2 and cphA2—Expression of cphB2 and cphA2 was tested by Northern analysis (Fig. 7). When the cphB2 probe was used, a hybridization band corresponding to a transcript of ~0.9 kb was detected (Fig. 7a), and total hybridization signals were more abundant with RNA from cultures maintained in the absence of ammonium. A 0.9-kb mRNA would correspond to a monocistronic transcript of the cphB2 gene that is 881 bp long. The cphA2 probe showed a transcript of ~2.1 kb that was also more abundant in the absence than in the presence of ammonium (Fig. 7b). Cellular levels of this transcript were observed to increase when ammonium-grown cultures were subjected to combined nitrogen deprivation (Fig. 7c). A 2.1-kb mRNA would correspond to a monocistronic transcript of the cphA2 gene that is 1,910 bp long. It should be noted that hybridization to the cphB2 gene probe was difficult to detect, probably reflecting a lower expression level of the cphB2 than of the cphA2 gene.



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 7.
Expression analysis of the cph2 gene cluster in Anabaena sp. PCC 7120. a and b, RNA was prepared from whole filaments grown with ammonium (A), nitrate (N), or N2 ({Phi}) as the nitrogen source and probed with DNA fragments of cphB2 (a), cphA2 (b), or rnpB amplified by PCR as indicated under "Experimental Procedures." c, RNA isolated from whole filaments grown with ammonium and incubated during the indicated number of hours in the absence of combined nitrogen was hybridized with a cphA2 probe (upper panel) or an rnpB probe. The sizes of detected transcripts are indicated in kb.

 
Mutant Strains Bearing Inactivated cph Genes—To analyze the involvement of each cph gene cluster in the accumulation of cyanophycin in Anabaena filaments as well as the putative role of cyanophycin in diazotrophic growth, mutant strains that carry inactivated versions of cph genes were constructed. In vitro generated constructs including antibiotic resistance-encoding gene cassettes inserted into a cph gene or gene fragment were transferred to strain PCC 7120. The mutagenic constructs were inserted into the Anabaena genome by double recombination, and clones bearing only the mutant version of the transferred gene were selected in each case (see "Experimental Procedures" for details). Single mutants were generated that bear an inactivated cphB1 (strain CSS13), cphA1 (strain CSS7), cphB2 (strain CSS21), or cphA2 (strain CSS25) gene (Table I). Double mutants cphA1 cphB1 (strain CSS35), cphA2 cphB2 (strain CSS36), cphA1 cphA2 (strain CSS27), and cphB1 cphB2 (strain CSS23) were also generated.


View this table:
[in this window]
[in a new window]
  		TABLE I
Cyanophycin content and diazotrophic growth rate of Anabaena sp. PCC 7120 and mutants impaired in cph genes

 
Cyanophycin accumulation was tested in cells of each of those mutants grown on nitrate, subjected to an 8-h period of combined nitrogen deprivation, and finally incubated overnight under low light with ammonium nitrate (see "Experimental Procedures"). Results obtained are shown in Table I. Mutation of cphA1 rendered a strain (CSS7) with negligible levels of cyanophycin, similar to those of the negative control Synechococcus sp. PCC 7942, which does not synthesize cyanophycin (38). In contrast, mutation of the cphA2 gene had only a minor effect on cyanophycin accumulation (cyanophycin content in strain CSS25 was ~78% that of the wild type). Cyanophycin content in the double mutant cphA1 cphA2 (strain CSS27) was similar to that of the single mutant cphA1. It can be concluded that, under our experimental conditions, CphA1 contributes the bulk of total cyanophycin synthesis in strain PCC 7120, although cphA2 also produces an active synthetase.

Mutation of cphB1 (see strain CSS13) rendered cyanophycin accumulation at levels ~2.4-fold those of the wild-type strain. Mutation of cphB2 had little effect on cyanophycin accumulation (levels in strain CSS21 were ~1.3-fold those of the wild type). Cyanophycin accumulation levels in the double mutant cphB1 cphB2 (strain CSS23) were similar to those of the single mutant cphB1. Thus, CphB1 contribution to total cyanophycin degradation under our experimental conditions was much bigger than that of CphB2. The double mutant of cluster 1, strain CSS35, showed cyanophycin levels ~22% higher than those of the cphA1 mutant, and the double mutant of cluster 2, strain CSS36, had a cyanophycin content ~12% lower than that of the wild-type strain. These results confirm a low contribution of cphA2 to total cyanophycin synthesis in strain PCC 7120.

The ability of cph mutants to grow diazotrophically was also investigated (Table I). Whereas the strains mutated only in cyanophycin synthetase genes showed growth rates on N2 that were only slightly lower than those of strain PCC 7120 (percentage of the wild-type growth rate constant was 90, 94, and 86% for the cphA1, the cphA2 and the cphA1 cphA2 mutants, respectively), the strains impaired only in cyanophycinase gene cphB1 or in cphB1 and cphB2 showed more pronounced defects in diazotrophic growth, growth rates being 64 and 62% of the wild-type value for the single mutant cphB1 and the double mutant cphB1 cphB2, respectively. Strain CSS21, impaired only in cphB2, showed no noticeable impairment in diazotrophic growth.

Filaments from diazotrophic cultures of cph mutant strains were microscopically observed and photographed (Fig. 8). Mutants of the cph2 gene cluster, namely strains CSS21 (cphB2), CSS25 (cphA2), and CSS36 (cphB2 cphA2), looked quite similar to the wild-type strain PCC 7120 showing cyanophycin granules at the poles of the heterocysts. In contrast, strains CSS7 (cphA1) and CSS35 (cphB1 cphA1) showed no apparent polar granules in the heterocysts, whereas strain CSS13 (cphB1) showed extensive granulation that could correspond to cyanophycin granules both in vegetative cells and heterocysts. Interestingly, in contrast to the wild type, in strain CSS13 granules were not located at the heterocyst poles. The double mutant of the cyanophycin synthetase genes (strain CSS27) looked much like that of cphA1 (strain CSS7), and the double mutant of the cyanophycinase genes (strain CSS23) looked much like that of cphB1 (strain CSS13).



View larger version (143K):
[in this window]
[in a new window]
  		FIG. 8.
Microscopic examination of mutants derived from Anabaena sp. PCC 7120 affected in cyanophycin metabolism genes. The photographs show filaments grown in BG110 (combined nitrogen-free) medium. The genotype of each strain is indicated. The arrowheads point to some heterocysts. Magnification was x 700.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Anabaena sp. PCC 7120 genome contains two clusters of cyanophycin metabolism-like genes that could be identified by similarity to the corresponding sequences of other cyanobacteria. Genes in cluster 1 show higher similarity to previously identified cyanophycinase and cyanophycin synthetase genes than those of cluster 2. The study of the phenotype of mutant strains derived from Anabaena sp. PCC 7120 bearing inactivated versions of cph genes (Table I, Fig. 8) showed that, at least under the conditions used in this work, gene cphA1 is responsible for most of the cyanophycin synthetase activity, and cphB1 is responsible for the bulk of cyanophycin degradation. This appears to be the case in vegetative cells and heterocysts. Nonetheless, cphA2 and cphB2 genes also make some contribution to cyanophycin accumulation and degradation, respectively.

Both cphB1 and cphA1 genes are expressed in whole filaments growing with ammonium, nitrate, or N2 as nitrogen sources, but expression is higher in the combined nitrogen-free medium. In diazotrophic cultures, once N2-fixing heterocysts have developed, cphA1 and cphB1 are expressed in these differentiated cells and, taking into account the magnitude of the hybridization signals (Fig. 2) and the heterocyst frequency that is about one-tenth of the total cells of the filament, probably also in vegetative cells. It also appears that in heterocysts, the cphA1 gene is expressed more strongly than cphB1 (Fig. 2, a and b). Whereas cphB1 shows hybridization to heterocyst transcripts of up to ~5 kb, similar to those hybridizing to cphB1 and cphA1 in whole filaments, cphA1 shows hybridization preferably to heterocyst transcripts of smaller sizes, ~3 kb, similar to those found to hybridize with the cphA1 probe in whole diazotrophic filaments of cphB1 mutant strain CSS13 (Fig. 2c). Collectively, these observations suggest that cphB1 and cphA1 can be cotranscribed in vegetative cells and heterocysts, although in these differentiated cells cphA1 is expressed preferably as a monocistronic message resulting in a higher expression than that of cphB1. This interpretation is consistent with the finding of mRNA 5' ends localized both upstream of cphB1 and in the intergenic region between cphB1 and cphA1 (Fig. 3).

Upstream of cphB1 three different 5' transcript ends were found. RNA molecules with 5' ends IB and IIIB would be generated from nitrogen-regulated promoters that are induced upon transfer from ammonium-containing cultures to media lacking combined nitrogen. Whereas full activation of promoter PcphB1-3 appears to take place early upon the transference, activation of PcphB1-1 seems maximal only at later times, when fully developed heterocysts are present in the culture (Figs. 3a and 4c). Thus, whereas PcphB1-3 may be used in vegetative cells of filaments incubated in the absence of combined nitrogen, PcphB1-1 seems to be the main cphB1 promoter used in heterocysts (Fig. 3a). Activation of both PcphB1-1 and PcphB1-3 is dependent on the global nitrogen control transcriptional regulator NtcA (Fig. 4c), and indeed specific NtcA binding to DNA fragments encompassing the regions upstream of the tsp of each of these promoters is observed in vitro (Fig. 5). Both of these tsps are preceded by sequences conforming to the -10 box of NtcA-activated promoters (see Ref. 4), although none of them shows a canonical NtcA-binding site in the most frequent position (~22 nucleotides upstream of the -10 box) (Fig. 9a). The observed binding of NtcA to DNA fragment B (Fig. 5c) probably takes place at the canonical NtcA-binding sequence GTATCTAAAAGTAC centered at position -92.5 from tsp IB (Fig. 9a). This is a position fully compatible with a transcription activation mechanism similar to that of the homologous regulator CAP at Class I activated promoters (39). On the other hand, because no canonical NtcA binding box can be found in fragment C, binding of NtcA at promoter PcphB1-3 (Fig. 5d) should take place at a sequence imperfect with regard to the consensus of NtcA binding sites. Indeed, NtcA has been shown to bind to some Anabaena promoters with putative NtcA binding sites that resemble but do not match the consensual NtcA-binding box (4).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 9.
Promoters in the DNA regions upstream of the cphB1 (a) and cphA1 (b) genes of Anabaena sp. PCC 7120. In the promoter region schemes (sizes not to scale), the black boxes represent constitutive promoters mainly used in vegetative cells, open boxes represent N-regulated promoters, and the hatched box denotes the dual (constitutive and N-regulated) PcphA1-2 promoter. The relative affinity of NtcA and the cell type in which a regulated promoter is mainly used are indicated. In the DNA sequences, the ATG translation start of genes is underlined. Putative tsps are indicated by solid triangles, and the locations of putative 5' mRNA ends proposed to correspond to degradation products are shown by open triangles. Putative promoter -10 boxes are indicated by boldface underlined letters, -35 boxes are shown by gray boxes, UP element is shown by a discontinuous underline, and NtcA-binding sites are shown by open boxes.

 
Because the NtcA binding site of promoter PcphB1-1 is downstream from tsp IIIB (Fig. 9a), it is possible that binding of NtcA to this site has a negative effect on transcription initiated at tsp IIIB. On the other hand, affinity of NtcA for the PcphB1-3 promoter appears to be higher than for PcphB1-1. NtcA-regulated transcription of the cphBA operon would thus consist of activation of PcphB1-3 upon combined nitrogen deprivation, long before heterocysts have completed differentiation, consistent with the presence of 5' transcript end IIIB at early times after combined nitrogen deprivation, followed by activation of PcphB1-1 in heterocysts, consistent with the presence of 5' transcript end IB only at later times after combined nitrogen deprivation (Fig. 4c). Binding of NtcA to PcphB1-1 would be possible given the high levels of active NtcA protein probably present in heterocysts, and it could in turn inhibit transcription from PcphB1-3 in heterocysts, consistent with the fact that 5' transcript end IB was the most abundant in these cells. Additionally, the putative tsp corresponding to 5' RNA end IIB is preceded by sequences matching the -10 box and resembling the -35 box of {sigma}70 consensus-like promoters (Fig. 9a), which would be responsible for its constitutive expression in whole filaments. Given the low relative abundance of this transcript end in RNA preparations from heterocysts, it could be speculated that operation of PcphB1-2 is also inhibited by the operation of PcphB1-1.

In the intergenic region between cphB1 and cphA1, three 5' transcript ends were identified. The 5' ends IA and IIIA are found with similar abundance in whole filaments under the different nitrogen conditions tested (Fig. 3b). Whereas the putative tsp IIIA is preceded by sequences matching the -10 box and resembling the -35 box of {sigma}70-consensus promoters, only sequences matching the -10 box could be recognized upstream of IA, although in this case sequences that could represent an UP element are also found (Fig. 9b). Thus, 5' ends IA and IIIA seem to represent true constitutive promoters, although the similar pattern of occurrence of these ends makes it conceivable that IA might originate from the same transcript producing IIIA. The abundance of 5' transcript IIA is higher in established diazotrophic cultures than in those using combined nitrogen and is the most abundant in heterocysts (Fig. 3b). Specific NtcA binding to the cphB1-cphA1 intergenic DNA region has been observed, NtcA showing higher affinity for this region than for any of the sites upstream of cphB1 (Fig. 6). In this intergenic region, NtcA binding can take place at a sequence, GTACCTGAGGTTAG, centered at nucleotide -40.5 from tsp IIA (Fig. 9b) that resembles the canonical NtcA binding sequence. This putative NtcA-binding site is separated by 21 bp from a putative -10 box (Fig. 9b), thus conforming to the canonical structure of the more common (Class II) NtcA-activated promoters. These results suggest that the promoter corresponding to IIA (PcphA1-2), which represents the main promoter for monocistronic expression of cphA1 in heterocysts (Fig. 3b), is activated by NtcA. The location of the PcphA1-2 promoter NtcA-binding box overlapping the -10 box of PcphA1-3 (Fig. 9b) could lead to a negative effect of NtcA binding on transcription from PcphA1-3, consistent with the observation that the abundance of 5' transcript end IIIB is low in heterocysts (Fig. 3b). In addition, tsp IIA is preceded by sequences that resemble the -35 box of {sigma}70 consensus promoters (Fig. 9b) that, together with the -10 box that would be shared by the NtcA-activated promoter, could also promote transcription starting at the same tsp. This -35 box would be used preferably in vegetative cells and would thus be responsible for the presence of 5' end IIA in whole filaments grown under any nitrogen condition as well as for its apparent independence of the ntcA mutation (Fig. 4d).

In summary, besides cotranscription of cphB1 and cphA1 genes, monocistronic transcription of cphA1 can take place in Anabaena sp. PCC 7120 both in vegetative cells, from the consensus PcphA1-3 promoter (and perhaps also from PcphA1-1) and from the bivalent PcphA1-2 promoter, and in heterocysts from an NtcA-dependent operation of PcphA1-2. The preferential operation in heterocysts of NtcA-activated promoters PcphB1-1 and PcphA1-2 could respond for the higher extractable activities of both cyanophycin synthetase and cyanophycinase that have been found in heterocysts with respect to vegetative cells (e.g. see Ref. 11). Based on the observation that cphB and cphA from Synechocystis sp. PCC 6803 can independently be expressed in E. coli when cloned in the two possible orientations, it has been suggested that the cphB-cphA gene cluster would not be cotranscribed in this cyanobacterium (12). Additionally, cyanophycin synthetase and cyanophycinase of different cyanobacteria can show different relative abundances under different physiological conditions. We have shown here that, in Anabaena sp. PCC 7120, these observations can be accounted for by differential regulation of different promoters directing the synthesis of cphB1-cphA1 polycistronic or cphA1 monocistronic messages under different physiological conditions and in different cell types in the diazotrophic filament. Nonetheless, it is worth stressing that the whole system appears to be set to ensure a good level of expression of both genes under all conditions tested.

A second cluster of cyanophycin-related genes has been identified in Anabaena sp. PCC 7120. In contrast to the situation with cluster cph1, genes in cluster cph2 are found in opposite orientations (Fig. 1) and are expressed as monocistronic messages in whole filaments grown with ammonium, nitrate, or N2, although expression seems also to be higher in the absence of ammonium (Fig. 7). However, expression of cph2 genes was lower than that of cph1 genes precluding their detailed analysis. Low expression of cph2 genes is also consistent with the low metabolic impact of cph2 gene products in strain PCC 7120. Organization of cph genes in bacteria studied to date conforms to three different structures: (i) the cphB-cphA disposition found in cyanobacteria, including the cph1 cluster of Anabaena sp. PCC 7120, and also found in Clostridium botulinum strain ATCC 3802 and D. hafniense strain DCB-2; (ii) a cphA'-cphA disposition found in Bordetella bronchiseptica strain RB-50, Bordetella pertussis strain Tohama I, Bordetella parapertussis strain 12822, and Nitrosomonas europaea strain ATCC 25978; and (iii) a cphA-cphI (in which cphI would encode a kind of fusion protein of two cyanobacterial-like CphB units) found in Acinetobacter sp. strain ADP1 (14). The organization of the Anabaena sp. PCC 7120 cph2 gene cluster represents a fourth model of cyanophycin metabolism gene organization in bacteria.

The ability to grow under diazotrophic conditions was also studied in the cph mutant strains of Anabaena sp. PCC 7120. In heterocysts, conspicuous accumulation of cyanophycin takes place at the poles that are adjacent to vegetative cells. Mutant strains impaired in cyanophycin synthetase genes (cphA1 and/or cphA2) exhibit growth rates on N2 only slightly lower than that of the wild-type strain. Consistent with these results are those of Ziegler et al. (40), showing that in a cyanophycin synthetase mutant of A. variabilis ATCC 29413 growth on N2 was somewhat reduced only under high, nonlimiting light conditions. In contrast, diazotrophic growth rates of mutants impaired in cyanophycinase activity (cphB1 and cphB1 cphB2 strains) are significantly lower than that of the wild type (Table I). Microscopic observation (Fig. 8) shows that cphA1 mutant strains exhibit no apparent polar granules in the heterocysts, whereas cphB1 mutants show extensive granulation both in the vegetative cells and in heterocysts. In these strains, heterocysts look clearly different from those of the wild type. Cyanophycin accumulated as polar granules in heterocysts could be formed at the expense of amino acids synthesized after N2 fixation, and it can constitute a dynamic reservoir of fixed nitrogen (e.g. see Ref. 8), although our results suggest that the path through cyanophycin of newly synthesized organic nitrogen in the heterocyst is a dispensable, rather than an obligatory, route (little effect of cyanophycin synthetase inactivation). In contrast, the accumulation of amino acids into cyanophycin that could not be easily degraded (in mutants exhibiting low cyanophycinase activity) probably represents a sink of fixed nitrogen that is detrimental for diazotrophic growth.


   FOOTNOTES
 
* This work was supported by Ministerio de Ciencia y Tecnología, Spain, Grant BMC2001-0509. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Antonia Herrero, Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, c/Américo Vespucio s/n, E-41092 Seville, Spain. Tel.: 34-95-4489522; Fax: 34-95-4460065; E-mail: herrero{at}cica.es.

1 The abbreviations used are: ORF, open reading frame; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; tsp, transcription start point. Back


   ACKNOWLEDGMENTS
 
A. H. and E. F. thank Mary M. Allen (Wellesley College (Wellesley, MA)) for instruction on cyanophycin measurements.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wolk, C. P., Ernst, A., and Elhai, J. (1994) in The Molecular Biology of Cyanobacteria (Bryant, D. A., ed) pp. 769-823, Kluwer Academic Publishers, The Netherlands
  2. Montesinos, M. L., Herrero, A., and Flores, E. (1995) J. Bacteriol. 177, 3150-3157[Abstract/Free Full Text]
  3. Flores, E., and Herrero, A. (1994) in The Molecular Biology of Cyanobacteria (Bryant, D. A., ed) pp. 487-517, Kluwer Academic Publishers, Dordrecht, The Netherlands
  4. Herrero, A., Muro-Pastor, A. M., and Flores, E. (2001) J. Bacteriol. 183, 411-425[Free Full Text]
  5. Allen, M. M. (1984) Annu. Rev. Microbiol. 38, 1-25[CrossRef][Medline] [Order article via Infotrieve]
  6. Simon, R. D. (1987) in The Cyanobacteria (Fay, P., and van Baalen, C., eds) pp. 199-225, Elsevier, Amsterdam
  7. Sherman, L. A., Meunier, P., and Colón-López, M. S. (1998) Photosyn. Res. 58, 25-42[CrossRef]
  8. Sherman, D. M., Tucker, D., and Sherman, L. A. (2000) J. Phycol. 36, 932-941[CrossRef]
  9. Simon, R. D. (1976) Biochim. Biophys. Acta 422, 407-418[Medline] [Order article via Infotrieve]
  10. Ziegler, K., Diener, A., Herpin, C., Richter, R., Deutzmann, R., and Lockau, W. (1998) Eur. J. Biochem. 254, 154-159[Medline] [Order article via Infotrieve]
  11. Gupta, M., and Carr, N. G. (1981) J. Gen. Microbiol. 125, 17-23[Abstract/Free Full Text]
  12. Richter, R., Hejazi, M., Kraft, R., Ziegler, K., and Lockau, W. (1999) Eur. J. Biochem. 263, 163-169[Medline] [Order article via Infotrieve]
  13. Hejazi, M., Piotukh, K., Mattow, J., Deutzmann, R., Volkmer-Engert, R., and Lockau, W. (2002) Biochem. J. 364, 129-136[CrossRef][Medline] [Order article via Infotrieve]
  14. Krehenbrink, M., Oppermann-Sanio, F. B., and Steinbüchel, A. (2002) Arch. Microbiol. 177, 371-380[CrossRef][Medline] [Order article via Infotrieve]
  15. Ziegler, K., Deutzmann, R., and Lockau, W. (2002) Z. Naturforsch. C 57, 522-529[Medline] [Order article via Infotrieve]
  16. Obst, M., Oppermann-Sanio, F. B., Luftmann, H., and Steinbüchel, A. (2002) J. Biol. Chem. 277, 25096-25105[Abstract/Free Full Text]
  17. Kaneko, T., Nakamura, Y., Wolk, C. P., Kuritz, T., Sasamoto, S., Watanabe, A., Iriguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takazawa, M., Yamada, M., Yasuda, M., and Tabata, S. (2001) DNA Res. 8, 227-253[CrossRef]
  18. Frías, J. E., Flores, E., and Herrero, A. (1994) Mol. Microbiol. 14, 823-832[Medline] [Order article via Infotrieve]
  19. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and Stanier, R. Y. (1979) J. Gen. Microbiol. 111, 1-61[Abstract/Free Full Text]
  20. Mazodier, P., Cossart, P., Giraud, E., and Gasser, F. (1985) Nucleic Acids Res. 13, 195-205[Abstract/Free Full Text]
  21. Prentki, P., and Krisch, H. M. (1984) Gene (Amst.) 29, 303-313[CrossRef][Medline] [Order article via Infotrieve]
  22. Black, T. A., Cai, Y., and Wolk, C. P. (1993) Mol. Microbiol. 9, 77-84[Medline] [Order article via Infotrieve]
  23. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 87, 206-210[CrossRef][Medline] [Order article via Infotrieve]
  24. Elhai, J., and Wolk, C. P. (1988) Gene (Amst.) 68, 119-138[CrossRef][Medline] [Order article via Infotrieve]
  25. Cai, Y. P., and Wolk, C. P. (1990) J. Bacteriol. 172, 3138-3145[Abstract/Free Full Text]
  26. Elhai, J., and Wolk, C. P. (1988) Methods Enzymol. 167, 747-754[Medline] [Order article via Infotrieve]
  27. Elhai, J., Cai, Y., and Wolk, C. P. (1994) J. Bacteriol. 176, 5059-5067[Abstract/Free Full Text]
  28. Elhai, J., Vepritskiy, A., Muro-Pastor, A. M., Flores, E., and Wolk, C. P. (1997) J. Bacteriol. 179, 1998-2005[Abstract/Free Full Text]
  29. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., and Smith, J. A. (2003) Current Protocols in Molecular Biology, Green/Wiley-Interscience, New York
  30. Tumer, N. E., Robinson, S. J., and Haselkorn, R. (1983) Nature 306, 337-342[CrossRef]
  31. Luque, I., Flores, E., and Herrero, A. (1994) EMBO J. 13, 2862-2869[Medline] [Order article via Infotrieve]
  32. Golden, J. W., Whorff, L. L., and Wiest, D. R. (1991) J. Bacteriol. 173, 7098-7105[Abstract/Free Full Text]
  33. Muro-Pastor, A. M., Valladares, A., Flores, E., and Herrero, A. (2002) Mol. Microbiol. 44, 1377-1385[CrossRef][Medline] [Order article via Infotrieve]
  34. Vioque, A. (1997) Nucleic Acids Res. 25, 3471-3477[Abstract/Free Full Text]
  35. Muro-Pastor, A. M., Valladares, A., Flores, E., and Herrero, A. (1999) J. Bacteriol. 181, 6664-6669[Abstract/Free Full Text]
  36. Messineo, L. (1966) Arch. Biochem. Biophys. 117, 534-540[CrossRef]
  37. Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M., and Tabata, S. (1996) DNA Res. 3, 109-136[Abstract]
  38. Lawry, N. H., and Simon, R. D. (1982) J. Phycol. 18, 391-399[CrossRef]
  39. Busby, S., and Ebright, R. H. (1999) J. Mol. Biol. 293, 199-213[CrossRef][Medline] [Order article via Infotrieve]
  40. Ziegler, K., Stephan, D. P., Pistorius, E. K., Ruppel, H. G., and Lockau, W. (2001) FEMS Microbiol. Lett. 196, 13-18[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Bacteriol.Home page
J. Paz-Yepes, E. Flores, and A. Herrero
Expression and Mutational Analysis of the glnB Genomic Region in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120
J. Bacteriol., April 1, 2009; 191(7): 2353 - 2361.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
M. Maheswaran, K. Ziegler, W. Lockau, M. Hagemann, and K. Forchhammer
PII-Regulated Arginine Synthesis Controls Accumulation of Cyanophycin in Synechocystis sp. Strain PCC 6803.
J. Bacteriol., April 1, 2006; 188(7): 2730 - 2734.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
N. H. Kolodny, D. Bauer, K. Bryce, K. Klucevsek, A. Lane, L. Medeiros, W. Mercer, S. Moin, D. Park, J. Petersen, et al.
Effect of Nitrogen Source on Cyanophycin Synthesis in Synechocystis sp. Strain PCC 6308
J. Bacteriol., February 1, 2006; 188(3): 934 - 940.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
Y. Elbahloul and A. Steinbuchel
Engineering the Genotype of Acinetobacter sp. Strain ADP1 To Enhance Biosynthesis of Cyanophycin
Appl. Envir. Microbiol., February 1, 2006; 72(2): 1410 - 1419.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
Y. Elbahloul, K. Frey, J. Sanders, and A. Steinbuchel
Protamylasse, a Residual Compound of Industrial Starch Production, Provides a Suitable Medium for Large-Scale Cyanophycin Production
Appl. Envir. Microbiol., December 1, 2005; 71(12): 7759 - 7767.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
Y. Elbahloul, M. Krehenbrink, R. Reichelt, and A. Steinbuchel
Physiological Conditions Conducive to High Cyanophycin Content in Biomass of Acinetobacter calcoaceticus Strain ADP1
Appl. Envir. Microbiol., February 1, 2005; 71(2): 858 - 866.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/12/11582    most recent
M311518200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Picossi, S.
Right arrow Articles by Herrero, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Picossi, S.
Right arrow Articles by Herrero, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement