Negative Regulation of Expression of the Nitrate Assimilation

Transcription

1 JB Accepts, published online ahead of print on 26 March 2010 J. Bacteriol. doi: /jb Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Negative Regulation of Expression of the Nitrate Assimilation nira Operon in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120 José Enrique Frías and Enrique Flores* Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, Centro de Investigaciones Científicas Isla de la Cartuja, E-41092, Seville, Spain. *Corresponding author. Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Avenida Américo Vespucio 49, E-41092, Seville, Spain. Tel.: ; fax: address: eflores@ibvf.csic.es (E. Flores) Running title: Regulation of the nira operon Keywords: Anabaena, cyanobacteria, nitrate assimilation, regulation 1

2 Abstract In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nira operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positiveaction proteins that are required for expression of the nitrate assimilation nira operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nira operon promoter, CnaT acts by an as-yet-unknown mechanism. In the genome of this cyanobacterium, ORF all0605 (the nirb gene) is found between the nira (encoding nitrite reductase) and ntcb genes. A nirb mutant was able to grow at the expenses of nitrate as a nitrogen source and showed abnormally high levels of nira operon mrna both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium when the nirb mutant was grown in the presence of nitrate. A deletion-in-frame nira mutant also showed a phenotype of increased expression of the nira operon in the absence of ammonium independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nira operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate-regulation of the expression of the nira operon in Anabaena sp. strain PCC

3 Assimilatory nitrate reduction is carried out by many plants, algae, fungi and bacteria. It involves the uptake of nitrate into the cell and its two-step reduction via nitrite to ammonium, which is incorporated into carbon skeletons. In bacteria, uptake is carried out by ABC-type or MFS transporters, and reduction involves the direct transfer of electrons to nitrate and nitrite via nitrate reductase and nitrite reductase, respectively, from iron-sulfur or flavin-containing donor proteins (28). Expression of the nitrate assimilation system is frequently subjected to a dual regulation: repression by ammonium and induction by nitrate (or nitrite). Whereas repression is exerted by the general nitrogen control system of the bacterium, a variety of different mechanisms appear to exist that mediate induction (28). Cyanobacteria are photoautotrophs that carry out oxygenic photosynthesis. Nitrate and ammonium are excellent sources of nitrogen for cyanobacteria in general, and many strains are able to use urea or to fix atmospheric nitrogen (13, 15). In cyanobacteria, reduction of nitrate to ammonium is catalyzed by two ferredoxindependent enzymes, nitrate reductase and nitrite reductase. Genes encoding nitrite reductase (nira), an ABC-type nitrate/nitrite uptake transporter (nrtabcd), and nitrate reductase (narb) are clustered together constituting the nira operon (nira-nrtabcdnarb) in the genomes of Synechococcus elongatus strain PCC 7942 (hereafter referred to as S. elongatus) and Anabaena sp. strain PCC 7120(13). Several genes involved in the biosynthesis of the nitrate reductase molybdenum cofactor (molybdopterin guanine dinucleotide) and two additional genes, narm and nirb, that affect nitrate reductase and nitrite reductase activity levels, respectively, have also been identified in S. elongatus (13). The nirb gene has been shown to be required for attaining maximum levels of nitrite reductase, and its inactivation provokes an imbalance between nitrate and nitrite reduction resulting in release of nitrite to the external medium (36). 3

4 Nitrate reductase and nitrite reductase activities are lower in ammonium-grown than in nitrate-grown cyanobacterial cells (13, 15). Expression of these enzyme activities takes place at appreciable levels in the absence of nitrate or nitrite in some cyanobacteria such as S. elongatus, but not in the heterocyst-forming, N 2 -fixing cyanobacteria such as Anabaena sp. strain PCC Thus, in the non-n 2 -fixing cyanobacteria the nitrate assimilation system is mainly subjected to ammoniumpromoted repression, whereas in the N 2 -fixing cyanobacteria, in addition to repression by ammonium, induction by nitrate or nitrite is also required for attaining high levels of expression, giving rise to a nitrate effect in this type of cyanobacteria (13, 15). Expression of the nira operon upon ammonium withdrawal is promoted by the NtcA protein, a CAP-family transcription factor that is widespread among cyanobacteria (25). NtcA activity is enhanced by 2-oxoglutarate, a putative C to N balance signal in the cyanobacterial cell (17, 30) that can act on NtcA both directly (3, 33, 37-39) and indirectly via the signal-transduction protein P II (13, 33). In addition to NtcA, a route-specific, LysR-type trancriptional regulator, NtcB, is involved in the regulation of nira operon expression (1, 2, 18, 27). In contrast to NtcA that is strictly necessary for expression of the nira operon in all investigated cyanobacterial strains, NtcB is involved in regulation with different stringency levels depending on the cyanobacterial strain. In the case of Anabaena sp. strain PCC 7120, the NtcB protein is strictly required for expression of the nira operon and for growth at the expense of nitrate, and expression of ntcb itself takes place from an NtcA-dependent promoter (18). A third positive regulatory element of nira operon expression in Anabaena sp. strain PCC 7120 is the CnaT protein (20), which shows overall sequence similarity to glycosyl transferases. An Anabaena cnat insertional mutant is unable to use nitrate as a nitrogen source due to a defect in activation of transcription of the nira operon. However, CnaT 4

5 does not appear to be a DNA-binding protein and, consequently, the effect of CnaT on nira operon expression could be indirect. Four open reading frames (ORFs all0603, all0604, all0605 and all0606) are located in the genome of Anabaena sp. strain PCC 7120 between the nira and ntcb genes, with the same orientation as ntcb (Fig. 1). all0603 would encode a 101-amino acid transcriptional regulator of the XRE-family. all0604 would encode a 119-amino acid polypeptide that shows no homology with any protein of known function. all0605 (previously designated orf398 (19) encodes a protein with sequence similarity to several proteins previously characterized in S. elongatus: the phycocyanobilin lyase alpha subunit CpcE (26 % identity in a 163 amino acid overlap), the NblB polypeptide involved in phycobilisome degradation (22 % identity in a N-terminal 180 amino acid overlap plus 23 % identity in a C-terminal 148 amino acid overlap), and the NirB protein (23 % identity in a 331 amino acid overlap). all0606 (previously designated orf136 (19) would encode a protein similar to the cytochrome b 6 f-complex iron-sulfur protein PetC. Besides all0606, three other Anabaena sp. strain PCC 7120 ORFs, all2453, all4511, and all1512, show overall homology to the cytochrome b 6 f-complex iron-sulfur protein PetC (26). In this study, we show that, in addition to the positive elements described above, the expression of the Anabaena nira operon is subjected to the action of two negative elements, the products of ORF all0605 (the Anabaena nirb gene) and nira, which repress the expression of the nira operon when nitrate is absent from the culture medium giving rise to the aforementioned nitrate effect in Anabaena sp. strain PCC

6 MATERIALS AND METHODS Strains and growth conditions. Anabaena sp. (also known as Nostoc sp.) strain PCC 7120 was routinely grown photoautotrophically at 30 C under white light (about 25 µe s -1 m -2 ), with shaking for liquid cultures. Media used for growth were BG11 + (NaNO 3 as the nitrogen source)(34), BG11 0 (BG11 without nitrate) or BG11 0 NH 4 (BG11 0 supplemented with 2 mm NH 4 Cl and 4 mm N-tris(hydroxymethyl)methyl-2- aminoethane sulfonic acid (TES)-NaOH buffer, ph 7.5). For growth on plates, medium solidified with separately autoclaved 1% agar (Difco) was used. When appropriate, antibiotics were added to plates at the following final concentrations: streptomycin (Sm), 5 µg/ml; spectinomycin (Sp), 5 µg/ml; and neomycin (Nm), 30 µg/ml. In liquid cultures, antibiotic concentrations used were as follows: Sm, 2 µg/ml; Sp, 2 µg/ml; and Nm, 5 µg/ml. Strains CSE17 (19), CSE23, CSE271 and CSE272 were routinely grown in BG11 0 NH + 4 medium supplemented with Sm and Sp. Strains EF116B and CSE27B were routinely grown in BG11 0 NH + 4 medium supplemented with Nm. CSE27 and CSE172 were routinely grown in BG11 0 NH + 4 medium. For derepression experiments, cells grown in BG11 0 NH + 4 (BG11 0 supplemented with 4 mm NH 4 Cl and 8 mm TES-NaOH buffer, ph 7.5) medium bubbled with a mixture of air and CO 2 (1% of CO 2, v/v) at 30 C in the light (75 to 100 µe s -1 m -2 ) were harvested by filtration and washed with BG11 0 medium, resuspended in the media indicated in each experiment and incubated under the same conditions. All media used for derepression experiments were supplemented with 12 mm NaHCO 3. Escherichia coli DH5α, HB101, XL1-Blue and ED8654 were grown in Luria- Bertani medium as described previously (4). Generation of mutant strains. The method of sacb-mediated positive selection for double recombinants in Anabaena sp. (7) was used to generate mutant strains 6

7 EF116B (nrtb::c.k1), CSE27 ( nira), CSE27B ( nira nrtb::c.k1) and CSE23 (all0604::c.s3), and to complement mutant strain CSE17 (all0605::c.s3). Plasmids pcse111b (for CSE23), pcse129 (for CSE172), pcse142b (for CSE27), pcse149 (for CSE271 and CSE272) and pcse152b (for EF116B and CSE27B) were transferred to the cyanobacterial parental strain by conjugation (11). See Table 1 for a description of strains and plasmids. Plasmid prl623 was used as helper plasmid in conjugations, except for the generation of strain CSE23 for which plasmids prl528 and prl591- W45 were used as helper plasmids. In all cases plasmid prl443 was used as conjugative plasmid. For generation of strains CSE27 and CSE172, some sucrosesensitive exconjugants (Sm r Sp r for CSE27; Sm r Sp r Nm r for CSE172) were grown in BG11 0 NH + 4 liquid medium without antibiotics. These cultures were sonicated in a cleaning bath and plated on BG11 0 NH + 4 solid medium containing 5% sucrose. Double recombinants were identified by their sucrose-resistant antibiotic-sensitive phenotype. In CSE27, a 666-bp internal fragment of nira, corresponding to nucleotides of the 1611-nucleotide-long coding region, was deleted from the genome, and as a consequence of the in-frame deletion of nucleotides, the modified nira should encode a protein of 304 amino acid residues. In all cases the genomic structure of the resultant Anabaena mutant strain was checked by Southern analysis or PCR analysis to confirm the absence of parental chromosomes in the resultant Anabaena strains. DNA isolation and Southern blot analysis. Isolation of DNA from Anabaena sp. was performed as previously described (7). For Southern blots, restriction endonuclease-digested DNA was subjected to electrophoresis in agarose gels and transferred to Hybond -N + membranes following the instructions of the manufacturer. Labeling of probes with 32 P and hybridization was performed as described previously (9, 21). 7

8 RNA isolation and analysis. RNA from Anabaena sp. was prepared as described previously (29). The resulting RNA preparations were treated with RNasefree DNase I to eliminate contaminating DNA. For Northern blot analysis, RNA (approximately µg) was subjected to electrophoresis in denaturing formaldehyde gels, transferred to Hybond -N + membranes, and subjected to hybridization at 65 C as described previously (9). DNA probes (see Table 2) used in Northern experiments were: nira probe, PCR-generated DNA fragment using primers nir and nir (probe a), nir and nir (probe b), or nir and nir (probe c); narb probe, PCR-generated DNA fragment using primers N-narB-7120 and C-narB-7120; ntca probe, NcoI/SalI DNA fragment from pcsam61 (29); ntcb probe, PCR-generated DNA fragment using primers Nc-ntcB and ntcb-3 (18); cnat probe, HincII/BstXI DNA fragment from pcse118 (20) bearing most of the Anabaena cnat gene; all0603 probe, PCR-generated DNA fragment using primers all and all0603-2; all0604 probe, PCR-generated DNA fragment using primers all and all0604-2; and all0605 (nirb) probe, PCR-generated DNA fragment using primers orf398-2 and nir For PCR-generated probes, Anabaena chromosomal DNA, plasmid pcse21 (19) or plasmid pcse95 (18) was used as a template. Primer extension experiments were performed as described elsewhere (4), using µg of RNA and primer nir-1 (19). Results were visualized and quantified with a Cyclone storage phosphor system and OptiQuant image analysis software (Packard). Nitrate uptake. Nitrate uptake assays were performed as previously described (14). Ammonium-grown cells (4 to 5 µg of chlorophyll a/ml) were derepressed by incubation for 4 h in BG11 medium. Cells were then harvested by filtration, washed with 10 mm Tricine-NaOH buffer (ph 8.1), resuspended in the same buffer to 10 µg of chlorophyll a/ml, and incubated under culture conditions. Uptake assays were started by 8

9 addition of NaNO 3 (100 to 110 µm, final concentration). Nitrate disappearance was determined by estimating the concentration of nitrate in the medium in aliquots of the cell suspensions after removal of the cells by filtration through Millipore HA 0.45-mmpore-size filters. Nitrate concentration was determinated by HPLC using a Partisil 10 SAX WCS Analytical Column (4.6 mm x 250 mm; 10 µm) from Whatman International Ltd (England). Enzyme activities. Nitrate reductase (23) and nitrite reductase (24) were measured with dithionite-reduced methyl viologen as the reductant in cells made permeable with mixed alkyltrimethylammonium bromide. The amount of cells added to an enzymatic assay for nitrate reductase and nitrite reductase contained 5 and 25 µg of chlorophyll a, respectively. Activity units correspond to µmol of nitrite produced (nitrate reductase) or removed (nitrite reductase) per minute. Downloaded from on March 8, 2019 by guest 9

10 RESULTS AND DISCUSSION Verification of strain CSE17. Construction of mutant strain CSE17 (all0605::c.s3 or nirb::c.s3) has been previously described (19). When ammoniumgrown cells of this mutant were incubated in the absence of ammonium in cultures bubbled with CO 2 -enriched air, they showed abnormally low levels of nitrite reductase activity in nitrate-containing medium and abnormally high levels of nitrate reductase activity in medium containing no combined nitrogen (see below). This data confirm previously reported data (19). To verify that the phenotype shown by strain CSE17 results from inactivation of all0605 (nirb) and not from any additional mutation elsewhere in the genome, the mutated version of nirb in CSE17 was replaced by a wild-type version of the gene (see Materials and Methods for details). In strain CSE172, the genomic structure in the nirb region was identical to that of the wild-type strain PCC 7120 (not shown). Nitrate and nitrite reductase levels in strain CSE172 were similar to those of the wild type rather than to those of its parental strain CSE17 showing that the phenotype of strain CSE17 results from the inactivation of nirb. Two small ORFs, all0604 and all0603, whose coding sequences overlap by 3 nucleotides, are located downstream of all0605 (Fig. 1). The gene-cassette inserted into all0605 in strain CSE17, C.S3 (12), contains transcriptional terminators that are effective in Anabaena sp. strain PCC 7120 (see, e. g., 19). To verify that the phenotype of strain CSE17 results from inactivation of all0605 and not from a polar effect on a downstream gene, the expression of all0604-all0603 was investigated and a mutant of all0604 was constructed with the same cassette, C.S3 (see Fig. 1 and Materials and Methods for details). Mutant strain CSE23 (all0604::c.s3) was able to grow in media containing ammonium, nitrate or no combined nitrogen and exhibited nitrate reductase 10

11 and nitrite reductase activity levels that were similar (within 10 %) to those of the wildtype strain. Additionally, the expression of nira and narb in mutant strain CSE23, investigated by Northern analysis, showed no appreciable difference to that observed in the wild-type strain PCC 7120 (not shown). To investigate the expression of all0604 and all0603, Northern analysis was performed with probes of each of these ORFs and RNA isolated from strains PCC 7120, CSE17 and CSE172. The all0604 probe hybridized to bands of about 0.33 kb (likely corresponding to an all0604 transcript) and 0.87 kb (likely corresponding to a transcript covering both all0604 and all0603) (Fig. 2). Only the latter band was obtained using an all0603 probe and no expression of all0603 was observed in mutant strain CSE23 (all0604::c.s3) (not shown). Thus, a bicistronic transcript is produced, but a monocistronic transcript is also observed that may result from premature transcription termination downstream of all0604. The 0.87-kb bicistronic transcript accumulated at an increased level after 24 h incubation in the absence of combined nitrogen. No significant differences in the expression profiles of all0603 and all0604 were observed between strains PCC 7120, CSE17, and CSE172 (Fig. 2). The results of analysis of the nirb mutant (all0605::c.s3) and of expression of all0604-all0603 both indicate that the phenotype of CSE17 cannot be ascribed to a polar effect on the expression of downstream genes. Effect of inactivation of nirb on the expression of nitrate assimilation enzyme activities and genes. The development of the nitrate and nitrite reductase activities was analyzed in cultures of wild-type strain PCC 7120 and mutant strain CSE17 during a 24-h induction experiment in nitrate-containing media. In the wild type (Fig. 3A), both enzymatic activities reached a plateau after 6 h of incubation in nitratecontaining media, with activities of about 89 mu (mg of protein) -1 for nitrate reductase 11

12 and about 23 mu (mg of protein) -1 for nitrite reductase. In the mutant (Fig. 3B), the nitrite reductase activity also reached a plateau at 6 h, showing an activity of about 11 mu (mg of protein) -1, whereas the nitrate reductase activity increased during the course of the experiment to reach an activity of about 131 mu (mg of protein) -1 at 24 h. An accumulation of nitrite in the culture medium was observed specifically for strain CSE17 indicating that its nitrite reductase is unable to reduce all the nitrite produced by nitrate reductase, the excess nitrite being excreted. The development of nitrate reductase in strains PCC 7120, CSE17 and CSE172 was also studied in media lacking combined nitrogen (Fig. 4). Expression was observed, and after reaching a peak shortly after the beginning of the incubation, the activity decayed in the wild type but much less in strain CSE17. Strain CSE172 behaved like the wild type. The level of nitrate reductase activity after 24 h in the absence of nitrate was about 15-fold higher in mutant CSE17 than in the wild type. These results show that inactivation of nirb has profound effects on the enzyme activities in the nitrate assimilation system resulting in low levels of nitrite reductase and, especially when the cells are incubated in the absence of nitrate, abnormally high levels of nitrate reductase. To test whether the abnormal levels of nitrate and nitrite reductase activities shown by strain CSE17 resulted from an altered expression of the nira operon, Northern analyses were carried out with probes of the nira and narb genes. Hybridizations were performed with total RNA isolated from cells of strains PCC 7120, CSE17, and CSE172 incubated under different nitrogen regimes. As previously reported for the nira operon (19), which produces a long transcript of close to 10 kb that is unstable, only a smear of degraded RNA products could be detected with both the nira and narb probes (Fig. 5). In strains PCC 7120 and CSE172, a high level of expression of narb took place only in media without ammonium in the presence of nitrate. In the 12

13 nirb mutant (strain CSE17), however, a high level of expression was observed in the absence of ammonium both in the presence and absence of nitrate (Fig. 5A). A similar expression profile was obtained with the nira probe (Fig. 5C). Levels of the 5 region of the nira operon transcript in strain CSE17 were also analyzed by primer extension. The obtained data (Fig. 6) corroborated those obtained by Northern experiments, suggesting that the increased nira operon transcript levels observed in strain CSE17 in the absence of combined nitrogen corresponded to a higher utilization of the nira operon promoter in this strain. To test whether the expression of the positive regulatory elements NtcA, NtcB and CnaT was affected in mutant strain CSE17, Northern analysis was performed. The expression patterns observed for the ntca, ntcb and cnat genes under the different nitrogen regimes tested were similar in strains PCC 7120, CSE17 and CSE172 (Fig. 5) and similar to those previously reported (18, 20, 29). These results indicate that NirB is not involved in the regulation of transcription of these genes and, additionally, that the altered levels of nira operon expression observed in strain CSE17 do not result from an altered expression of the positive regulatory elements NtcA, NtcB and CnaT. Therefore, NirB appears to be a factor inhibiting the expression of the nira operon, especially in the absence of nitrate, in Anabaena sp. strain PCC The accumulation of nira operon transcripts in strain CSE17, observed with probes of nira and narb, explains the higher nitrate reductase activity observed in this mutant in both the presence and absence of nitrate; however, it does not explain the low levels of nitrite reductase activity observed in the presence of nitrate. A similar phenotype of expression of low levels of nitrite reductase activity has been observed for a nirb mutant of S. elongatus, although no effect of the corresponding mutation on the nira operon transcript level has been reported (35). It has been proposed that NirB is 13

14 required as a chaperone or scaffold for expression of maximum nitrite reductase activity (35). Similarity of NirB to CpcE and NblB is based on the presence in these proteins of a number of HEAT-like repeats, which consist of tandemly repeated amino acid modules that appear to function in protein-protein interactions and may have a scaffolding role (22). Anabaena NirB appears to have six such HEAT repeats. A role based on the presence of these structural motifs would explain why NirB from S. elongatus and Anabaena sp. strain PCC 7120, two homologous proteins that appear to have a similar function in nitrate reduction and are therefore orthologues, show such a low identity degree (23 %). If NirB works through protein-protein interactions, it is conceivable that NirB interacts with the nitrite reductase allowing it to reach a maximally active conformation for the development of its enzymatic activity. In turn, this raises the possibility of a regulation of the nira operon by nitrite reductase itself rather than by NirB. Study of a nira mutant. Different mutant strains in the Anabaena nira operon generated by lux-tagged Tn5 transposon mutagenesis have been described (6). One of those mutants, strain TLN10 (nira::tn5), shows a phenotype similar to that of the nirb mutant. When cells are transferred from ammonium-containing medium to medium lacking combined nitrogen, a luciferase activity develops in strain TLN10 that is very high as compared to that showed by the mutant strains TLN12 (nrtc::tn5) and TLN21 (Tn5 inserted in the nrtd-narb intergenic region) (6). Given that in both cases, the nirb mutant and strain TLN10, the nitrite reductase activity is affected (we assume that the Tn5 insertion inactivates nitrite reductase), we decided to mutate the nira gene in order to check whether the nitrite reductase is involved in the regulation of expression of the nira operon. 14

15 A nira mutant, strain CSE27, was constructed by deleting in frame a 666-bp internal fragment of nira, corresponding to nucleotides of the nucleotide-long coding region (see Fig. 1 and Materials and Methods for details). When nitrate was used as a nitrogen source, strain CSE27 was unable to grow and nitrite accumulated in the culture medium (not shown). The expression of nira in mutant strain CSE27 was investigated by Northern analysis using RNA isolated from strains PCC 7120, CSE17 and CSE27 (Fig. 7). As is the case for strain CSE17, a high expression of the nira operon in the absence of ammonium in both the presence and absence of nitrate was observed in strain CSE27, and the observed levels of expression were even higher than in strain CSE17. To verify that the phenotype shown by strain CSE27 resulted from inactivation of nira, a wild-type version of the nira gene bearing the complete nira operon promoter was introduced into strain CSE27. Plasmid pcse149 (see Table 1), a derivative of pcsel24 (31), can recombine with the nira region in the chromosome and with the nuca region in the Anabaena alpha megaplasmid. In the exconjugant strains CSE271 and CSE272, pcse149 was integrated into the alpha megaplasmid (Fig. 8A) and into the chromosome (Fig. 8B), respectively, in such a way that in both cases one copy of the nira operon was maintained as in CSE27 and an intact nira gene together with its promoter was additionally present. The genomic structure of these strains was confirmed by PCR analysis using different primer pairs (nui and nir ; nir and nrta ; nir and orf398-4; ntcs3 (19) and nrta ; ntcs3 and nrta ; see Table 2). Both strains were able to grow using nitrate as a nitrogen source. The expression of narb was investigated by Northern analysis using total RNA isolated from strains CSE271 and CSE272 and, as controls, PCC 7120 and CSE27 (Fig. 8C). In contrast to strain CSE27, strains CSE271 and CSE272 presented an 15

16 expression of narb similar to that shown by strain PCC These results indicate that the introduction of a wild-type copy of nira into strain CSE27 restored a wild-type phenotype of nira operon expression at the mrna level. Using the DNA fragment deleted in CSE27 as a nira probe, we were also able to corroborate an ammonium- and nitrate-regulated expression of the wild-type copy of nira introduced in strains CSE271 and CSE272 (Fig. 8D). Interestingly, however, the nira copy in the alpha megaplasmid (strain CSE271) was expressed at higher levels than that present in the chromosome (strain CSE272). As described in the Introduction, a high level of expression of the nira operon in Anabaena sp. strain PCC 7120 requires both the absence of ammonium and the presence of nitrate or nitrite, which act as inducers, in the culture medium. The results described above suggest that nitrite reductase has, in addition to its role in nitrite reduction to ammonium during nitrate assimilation, a negative role in the expression of the nira operon when this cyanobacterium is incubated in the absence of both ammonium and nitrate in the culture medium. A simple interpretation of our results would be that in the nirb and nira mutants, which have low levels or lack nitrite reductase, respectively, nitrite accumulates in the cells up to levels that strongly induce the expression of the nira operon. However, whereas this interpretation would be evident for those cultures supplemented with nitrate, we observed similarly increased nira operon mrna levels in the nirb and nira mutants incubated in the absence as well as in the presence of added nitrate. The possibility remains, nevertheless, that traces of nitrate or nitrite present in the BG11 0 medium are concentrated within the cells to act as inducers. Mutants of the NrtABCD transporter. An Anabaena ABC-type nitrate transporter, NrtABCD, has been previously identified (19). To check whether nitrate 16

17 concentrated within the cells from traces present in the medium could be acting as an inducer, the nrtb gene, which encodes the transmembrane component of the nitrate transporter, was mutated in strain CSE27 and in its parental strain EF116 (see Table 1 for details). For this, the C.K1 cassette, which confers resistance to neomycin and does not bear transcriptional terminators, was introduced into the ScaI restriction site of nrtb in the same orientation as this gene (see Fig. 1 and Materials and Methods for details). Two mutants, strains EF116B (nrtb::c.k1) and CSE27B ( nira, nrtb::c.k1), were obtained that were defective in the active transport of nitrate (Fig. 9A). The expression of the nira gene was studied by Northern analysis, and both mutants presented an expression profile similar to that shown by their respective parental strains (Fig. 9B). The fact that similar expression levels were observed in the presence and absence of added nitrate in spite of the absence of an active transporter makes it unlikely the possibility of a role of traces of nitrate in regulation. The Anabaena NrtABCD transporter also mediates the uptake of nitrite into the cells (ref. 32 and our unpublished results), but nitrite can enter into the cyanobacterial cells also by means of diffusion of nitrous acid (16). Diffusion is not however a mechanism that could concentrate nitrite within the cells, and direct determination of nitrite in cell extracts from filaments incubated without added nitrate under the conditions described in this work rendered negligible levels of nitrite (not shown). Our results argue against accumulation of an inducer (nitrate or nitrite) as responsible for the increased levels of the expression of the nira operon observed in nirb and nira mutants. They suggest instead that NirA (the nitrite reductase) and NirB (a possible nitrite reductase chaperone) somehow inhibit the expression of the nira operon when the wildtype Anabaena filaments are incubated in the absence of both ammonium and nitrate. 17

18 Concluding remarks. As mentioned above, both Anabaena mutants, nira and nirb, have lost the nitrate effect on expression of the nira operon (see Fig. 7). We do not know whether the observed phenotype of the nirb mutant just results from the lack of NirB protein or is related to the possible role of NirB as scaffolding protein in the maturation of the nitrite reductase. If the latter were the case, the nitrite reductase would be the key element in the negative regulation of the nira operon in the absence of ammonium. How nitrite reductase could exert this negative role remains to be elucidated. A plausible hypothesis is that the nitrite reductase acts as a modulator of a transcription factor. Because NtcA is a general N-control transcription factor and NtcB is a nitrate assimilation pathway-specific transcriptional activator, NtcB could be favored as a candidate target for nitrite reductase. Indeed, a role of NtcB mediating a nitrite effect in the expression of the nira operon in S. elongatus has been suggested (1). The nitrite reductase could switch from being an inhibitor of the NtcB transcriptional activator in the absence of nitrate or nitrite to acting as an enzyme in the presence of these nitrogen sources. It has recently been found that some enzymes, termed trigger enzymes, can control gene expression in response to the availability of their substrates through different mechanisms: by binding to either DNA or RNA or by modulating of the activity of transcription factors through either covalent modification or proteinprotein interactions (8). Our results suggest that nitrite reductase from Anabaena sp. strain PCC 7120 is a trigger enzyme that, in addition to its role in nitrite reduction to ammonium, exerts a regulatory role on the nitrate assimilation system. 18

19 ACKNOWLEDGMENTS We thank A. M. Muro-Pastor and A. Valladares for their help during this work and A. Herrero for discussion. Use of DNA sequences from the DOE-Joint Genome Institute (USA) and Kazusa DNA Research Institute (Japan) databases is acknowledged. This work was supported by grant numbers BFU and BFU from the Ministerio de Ciencia y Tecnología (Spain). 19

20 REFERENCES 1. Aichi, M., and T. Omata Involvement of NtcB, a LysR family transcription factor, in nitrite activation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp. strain PCC J. Bacteriol. 179: Aichi, M., N. Takatani, and T. Omata Role of NtcB in activation of nitrate assimilation genes in the cyanobacterium Synechocystis sp. strain PCC J. Bacteriol. 183: Aldehni, M. F., J. Sauer, C. Spielhaupter, R. Schmid, and K. Forchhammer Signal transduction protein P(II) is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC J. Bacteriol. 185: Ausubel, F. M., R. Brent, R. E. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl Current protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York. 5. Black, T. A., Y. Cai, and C. P. Wolk Spatial expression and autoregulation of hetr, a gene involved in the control of heterocyst development in Anabaena. Mol. Microbiol. 9: Cai, Y., and C. P. Wolk Nitrogen deprivation of Anabaena sp. strain PCC 7120 elicits rapid activation of a gene cluster that is essential for uptake and utilization of nitrate. J. Bacteriol. 179: Cai, Y. P., and C. P. Wolk Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J. Bacteriol. 172:

21 8. Commichau, F. M., and J. Stülke Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression. Mol. Microbiol. 67: Church, G. M., and W. Gilbert Genomic sequencing. Proc. Natl. Acad. Sci. U S A 81: Elhai, J., A. Vepritskiy, A. M. Muro-Pastor, E. Flores, and C. P. Wolk Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC J. Bacteriol. 179: Elhai, J., and C. P. Wolk Conjugal transfer of DNA to cyanobacteria. Methods Enzymol. 167: Elhai, J., and C. P. Wolk A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68: Flores, E., J. E. Frías, L. M. Rubio, and A. Herrero Photosynthetic nitrate assimilation in cyanobacteria. Photosynth. Res. 83: Flores, E., M. G. Guerrero, and M. Losada Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans. Biochim. Biophys. Acta. 722: Flores, E., and A. Herrero Assimilatory nitrogen metabolism and its regulation, p In D. A. Bryant (ed.), The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers, Dordrecht. 16. Flores, E., A. Herrero, and M. G. Guerrero Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans. Biochim. Biophys. Acta 896:

22 17. Forchhammer, K The P II protein in Synechococcus PCC 7942 senses and signals 2-oxoglutarate under ATP-replete conditions, p In P. GA, L. W, and G. Schmetterer (ed.), The Phototrophic Prokaryotes. Kluwer Academic/Plenum Publishers, New York. 18. Frías, J. E., E. Flores, and A. Herrero Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors. Mol. Microbiol. 38: Frías, J. E., E. Flores, and A. Herrero Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC J. Bacteriol. 179: Frías, J. E., A. Herrero, and E. Flores Open reading frame all0601 from Anabaena sp. strain PCC 7120 represents a novel gene, cnat, required for expression of the nitrate assimilation nir operon. J. Bacteriol. 185: Frías, J. E., A. Mérida, A. Herrero, J. Martín-Nieto, and E. Flores General distribution of the nitrogen control gene ntca in cyanobacteria. J. Bacteriol. 175: Groves, M. R., and D. Barford Topological characteristics of helical repeat proteins. Curr. Opin. Struct. Biol. 9: Herrero, A., E. Flores, and M. G. Guerrero Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp. FEMS Microbiol. Lett. 26: Herrero, A., and M. G. Guerrero Regulation of nitrite reductase in the cyanobacterium Anacystis nidulans. J. Gen. Microbiol. 132: Herrero, A., A. M. Muro-Pastor, and E. Flores Nitrogen control in cyanobacteria. J. Bacteriol. 183:

23 26. Kaneko, T., Y. Nakamura, C. P. Wolk, T. Kuritz, S. Sasamoto, A. Watanabe, M. Iriguchi, A. Ishikawa, K. Kawashima, T. Kimura, Y. Kishida, M. Kohara, M. Matsumoto, A. Matsuno, A. Muraki, N. Nakazaki, S. Shimpo, M. Sugimoto, M. Takazawa, M. Yamada, M. Yasuda, and S. Tabata Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC DNA Res. 8: Maeda, S., M. Okamura, M. Kobayashi, and T. Omata Nitrite-specific active transport system of the cyanobacterium Synechococcus sp. strain PCC J. Bacteriol. 180: Moreno-Vivián, C., and E. Flores Nitrate Assimilation in Bacteria, p In H. Bothe, S. J. Ferguson, and W. E. Newton (ed.), Biology of the Nitrogen Cycle. Elsevier B.V. 29. Muro-Pastor, A. M., A. Valladares, E. Flores, and A. Herrero Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development. Mol. Microbiol. 44: Muro-Pastor, M. I., J. C. Reyes, and F. J. Florencio Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J. Biol. Chem. 276: Olmedo-Verd, E., A. M. Muro-Pastor, E. Flores, and A. Herrero Localized induction of the ntca regulatory gene in developing heterocysts of Anabaena sp. strain PCC J. Bacteriol. 188: Paz-Yepes, J., E. Flores, and A. Herrero Expression and mutational analysis of the glnb genomic region in the heterocyst-forming Cyanobacterium Anabaena sp. strain PCC J. Bacteriol. 191:

24 33. Paz-Yepes, J., E. Flores, and A. Herrero Transcriptional effects of the signal transduction protein P(II) (glnb gene product) on NtcA-dependent genes in Synechococcus sp. PCC FEBS Lett. 543: Rippka, R., J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111: Suzuki, I., N. Horie, T. Sugiyama, and T. Omata Identification and characterization of two nitrogen-regulated genes of the cyanobacterium Synechococcus sp. strain PCC7942 required for maximum efficiency of nitrogen assimilation. J. Bacteriol. 177: Suzuki, I., H. Kikuchi, S. Nakanishi, Y. Fujita, T. Sugiyama, and T. Omata A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum. J. Bacteriol. 177: Tanigawa, R., M. Shirokane, S. Maeda Si, T. Omata, K. Tanaka, and H. Takahashi Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro. Proc. Natl. Acad. Sci. U S A 99: Valladares, A., E. Flores, and A. Herrero Transcription activation by NtcA and 2-oxoglutarate of three genes involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC J. Bacteriol. 190: Vázquez-Bermúdez, M. F., A. Herrero, and E. Flores Oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glna promoter. FEBS Lett. 512:

25 40. Vioque, A The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria. Nucleic Acids Res. 25: Wolk, C. P., Y. Cai, L. Cardemil, E. Flores, B. Hohn, M. Murry, G. Schmetterer, B. Schrautemeier, and R. Wilson Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen. J. Bacteriol. 170: Downloaded from on March 8, 2019 by guest 25

26 Figure legends FIG. 1. Genomic region of Anabaena sp. strain PCC 7120 bearing the nitrate assimilation gene cluster. Genes and ORFs are indicated by thick arrows, which also show the direction of transcription. Black arrows correspond to the ORFs investigated in this work. The location of the restriction sites into which gene-cassettes (C.S3 for strains CSE17 and CSE23; and C.K1 for strains EF116B and CSE27B) were inserted is indicated. The region deleted from nira in strain CSE27 is indicated as a ruled bar. Abbreviations for some restriction endonuclease sites: B, BglI; C, ClaI; E5, EcoRV; H, HindIII; P, PvuII; S, SpeI; Sc, ScaI; X, XbaI. Horizontal lines below the genes denote probes used for Northern analyses. FIG. 2. Northern analysis of the expression of all0604 in strains PCC 7120, CSE17, and CSE172. Hybridization assays were carried out using RNA isolated from cells grown with ammonium (NH + 4 ) or grown with ammonium and incubated for 4 or 24 h in medium containing nitrate (NO - 3 ) or no combined nitrogen (-N). Hybridization to rnpb (40) served as a loading and transfer control (lower panel). The position and size (indicated in kb) of the detected transcripts is shown on the left. FIG. 3. Changes in levels of nitrate and nitrite reductase activities, and nitrite accumulation in the culture medium after transfer of ammonium-grown cells to nitratecontaining medium. (A) Strain PCC 7120 (WT); (B) strain CSE17. Nitrate reductase activity (diamonds); nitrite reductase activity (squares); nitrite concentration in the medium (triangles). 26

27 FIG. 4. Changes in levels of nitrate reductase activity after transfer of ammonium-grown cells to a medium containing no combined nitrogen. Strain PCC 7120 (circles); strain CSE17 (squares); strain CSE172 (triangles). FIG. 5. Northern analysis of the expression of the nira operon and of some regulatory genes in strains PCC 7120, CSE17, and CSE172. Hybridization assays were carried out using RNA isolated from cells grown with ammonium (NH + 4 ) or grown with ammonium and incubated for 4 or 24 h in medium containing nitrate (NO - 3 ) or no combined nitrogen (-N). The hybridization probes used (see Materials and Methods for details) corresponded to narb (A), cnat (B), nira (C; fragment a in Fig. 1), ntca (D) or ntcb (E). Hybridization to rnpb (40) served as a loading and transfer control for each of the two filters used. The position of some size markers (A, C) or identified transcripts (B, D, E) is shown, indicated in kb, on the left. FIG. 6. Primer extension analysis of the expression of the nira gene in strains PCC 7120 and CSE17. Primer extension assays were carried out using oligonucleotide nir-1 as a primer and RNA isolated from cells grown with ammonium (NH + 4 ) or grown with ammonium and incubated for 4 h in medium containing nitrate (NO - 3 ) or no combined nitrogen (-N). The arrowhead points to the extension product identifying the Anabaena nira operon tsp. The sequencing ladders presented were generated with the same primer used in the primer extension reactions and plasmid pcse26 as template. FIG. 7. Northern analysis of the expression of nira in strains PCC 7120, CSE17, and CSE27. Hybridization assays were carried out using RNA isolated from cells grown with ammonium (NH 4 + ) or grown with ammonium and incubated for 4 in medium 27

28 containing nitrate (NO - 3 ) or no combined nitrogen (-N). Fragment b in Fig. 1 was used as nira probe (see Materials and Methods for details). Hybridization to rnpb (40) served as a loading and transfer control (lower panel). The position and size (indicated in kb) of some size standards is shown on the left. The small changes in the pattern of hybridization bands in strain CSE27 as compared to strains CSE17 and PCC 7120 result from the deletion of 666 bp from nira in CSE27. FIG. 8. Genomic structure of nira complementing constructs in strains CSE271 and CSE272 and Northern analysis of the expression of narb and nira in strains PCC 7120, CSE27, CSE271 and CSE272. The complementing constructs included ORF all0606, the nira operon promoter region and the nira gene incorporated into the nucanuia region of the alpha megaplasmid (A) or in the chromosomal nira region (B). Hybridization assays were carried out using RNA isolated from cells of the indicated strains grown with ammonium (NH + 4 ) or grown with ammonium and incubated for 4 in medium containing nitrate (NO - 3 ) or no combined nitrogen (-N). The hybridization probes used (see Materials and Methods) corresponded to narb (C) and nira (D; fragment c in Fig. 1). Hybridization to rnpb (40) served as a loading and transfer control. The position and size (indicated in kb) of some size standards is shown on the left. FIG. 9. Nitrate uptake and Northern analysis of the expression of nira in strains PCC 7120, EF116B, CSE27 and CSE27B. (A) Ammonium-grown cells of strains PCC7120 (closed diamonds), EF116B (open diamonds), CSE27 (closed triangles) and CSE27B (open triangles) were washed, resuspended in BG11 medium (17,6 mm NaNO 3 ), and incubated for 4 h as indicated in Materials and Methods for derepression 28

29 of the nitrate assimilation system. Cells were then harvested and used for nitrate uptake assays (see Materials and Methods). At the indicated times, nitrate was measured by HPLC using aliquots withdrawn from the assay mixture. (B) Hybridization assays were carried out using RNA isolated from cells of the indicated strains grown with ammonium (NH + 4 ) or grown with ammonium and incubated for 4 in medium containing nitrate (NO - 3 ) or no combined nitrogen (-N). Fragment b in Fig. 1 was used as nira probe (see Materials and Methods). Hybridization to rnpb (40) served as a loading and transfer control (lower panel). The position and size (indicated in kb) of some size standards is shown on the left. Downloaded from on March 8, 2019 by guest 29

30 Table 1. Cyanobacterial strains and plasmids used in this study. Strain or Relevant characteristics Reference plasmid Strains PCC 7120 Wild-type Anabaena strain (34) EF116 Derivative of Anabaena sp. strain PCC 7120 unable to fix nitrogen under aerobic conditions (41) EF116B Nm r derivative of strain EF116; nrtb::c.k1 This work CSE17 Sm r Sp r derivative of strain PCC 7120; all0605::c.s3 (19) CSE172 Sm s Sp s derivative of strain CSE17; all0605::c.s3 substituted by a wild-type version of This work all0605 CSE23 Sm r Sp r derivative of strain PCC 7120; all0604::c.s3 This work CSE27 Derivative of strain EF116; nira This work CSE27B Nm r derivative of strain CSE27; nira nrtb::c.k1 This work CSE271 Sm r Sp r derivative of strain CSE27; pcse149 plasmid integrated into nuca region of the This work alpha megaplasmid CSE272 Sm r Sp r derivative of strain CSE27; pcse149 plasmid integrated into nira region of the This work Anabaena chromosome Plasmids pcse111b 2.79-kb SpeI fragment from pcse73 (19), bearing AccI-ended gene-cassette C.S3 (12) This work inserted into the site ClaI of all0604, cloned in prl278; used to generate mutant strain CSE23 pcse kb XbaI/HindIII fragment from pcse26 (19), that contains part of all0605 and the This work whole all0606, cloned in prl278; used to complement mutant strain CSE17 pcse bp product of PCR with primers nir , nir , nir and nrta This work 3, with pcse26 as template; presents a deletion of a 666-bp internal segment of nira, corresponding to nucleotides of the 1611-nucleotide-long coding region pcse142b 1.3-kb PvuII/SalI fragment from pcse142 between the sites NruI/XhoI of prl277; used to This work generate mutant strain CSE27 pcse152b 2.9-kb ClaI/XbaI fragment from pcse2 (19), bearing HincII-ended gene-cassette C.K1 (12) This work inserted into the ScaI site of nrtb (in the same direction as nrtb), cloned in prl277; used to generate mutant strains EF116B, CSE17B and CSE27B pcse kb EcoRV/PvuII fragment from pcse78b (19) inserted between the sites EcoRV/NruI This work of pcsel24(31); used to complement mutant strain CSE27 prl277 Sm r Sp r, sacb-carrying, mobilizable vector (5) prl278 Nm r, sacb-carrying, mobilizable vector (5) prl443 Km s derivative of conjugative plasmid RP4 (11) prl528 Mobilization-helper; encodes M.AvaI and M.Eco47II. (11) prl591-w45 Mobilization-helper; encodes M.EcoT22I (10) prl623 Mobilization-helper; encodes M.AvaI, M.Eco47II, and M.EcoT22I (10) Downloaded from on March 8, 2019 by guest 30

31 Table 2. Oligodeoxynucleotide primers used in this work Primer Sequence (5-3 ) nir GGT GTT GGT CGT GGG TAC nir GCA AGC GAT CGC ACT GCC nir GCA ACA GAC CGA GAT CAT CG nir CCC CAT TCA TCA ATT AGC C nir CTA CCC CCA AAG CCA GCC TC nir GAG GGC GAA CGC ATG AAC TGA ATT ATC CC nir ACG TTC ATG CGT TCG CCC TCA TCG AAA CC nir CGG GAT AAT TCA GTT CAT GC nir TTC CGG CAC GGG CGC ACA ATT TGG CAA CTT CG nir GGA AAT CAA CGA TTT AGC CTT TGT TCC nir GTT GCA AAA TTG TGC GCC CGT G orf398-2 CAT AGT GCA GAT GAT TTG TC orf398-4 TCA GTG CAG CGA TCG CAT GG nrta TCC AAT CTT GCC GCA TAC nrta TCT AGA GGA AGT ACA GCC ATG TAC C N-narB-7120 GGA GCG AAG CGA CGT GAC C-narB-7120 GGT CAG TTG GGT AAA CTC all CGA AGC CAT TTG ATG AAC all CAA TCG AAC TGA GAA ATC AC all AAT GAC CTG GGA GGT AGA G all TCA TAG GAA CCC CTC TG nui ATG AGT GAG TCT GAA TAC CC Introduced restriction enzyme cutting sites are shown in bold face. 31

32 Fig.1 (Frías and Flores) (CSE23) C.S3 S C H C.S3 (CSE17) CSE27 S P B X E5 C.K1 (EF116B/CSE27B) Sc 1 kb Downloaded from cnat ntcb all0603 all0605 all0606 nira nrta nrtb nrtc nrtd narb all0604 (nirb) (c) (b) (a) on March 8, 2019 by guest

33 (rnpb) (all0604) NH h NO h NO h -N 24 h -N NH h NO h NO h -N 24 h -N NH h NO h NO h -N 24 h -N PCC 7120 CSE17 CSE172 Fig. 2 (Frías and Flores)

34 Fig. 3 (Frías and Flores) A B Enzymatic activity (mu/mg prot) Enzymatic activity (mu/mg prot) WT CSE time (h) nitrite (µm) nitrite (µm)

35 Fig. 4 (Frías and Flores) Enzymatic activity (mu/mg prot) time (h)

36 Fig. 5 (Frías and Flores) 24 h -N NH4+ 4 h NO324 h NO34 h -N CSE h -N 24 h -N CSE17 NH4+ 4 h NO324 h NO34 h -N NH4+ 4 h NO324 h NO34 h -N PCC 7120 A (narb) 0.5 B (cnat) 1.1 (rnpb) C (nira) D (ntca) 1.5 E (ntcb) 0.95 (rnpb)

37 Fig. 6 (Frías and Flores) A C G T PCC 7120 NH + 4 NO - 3 -N CSE17 NH + 4 NO - 3 -N Downloaded from on March 8, 2019 by guest

38 Fig. 7 (Frías and Flores) (nira) PCC 7120 CSE17 NO 3 - -N NH + 4 NO - 3 -N NH 4 + NO 3 - CSE27 -N NH 4 + Downloaded from (rnpb) on March 8, 2019 by guest

39 Fig. 8 (Frías and Flores) X E5 B 6 (Alpha megaplasmid) 1 kb 7 nuia nuca nira all0606 nuia nuca C.S3 B CSE27 8 P B X E5 B 1 (Chromosome) E5 X 3 D (narb) (nira) (rnpb) narb NH4+ CSE272 NO3- NH4+ CSE271 NO3- CSE27 nrtd -N nrtc -N NH4+ NO3- C (rnpb) nrtb PCC 7120 NH4+ -N CSE272 5 nrta NH4+ nira -N all0606 NO3- NH4+ -N CSE271 NO3- NH4+ NO3- CSE27 -N NH4+ NO3- -N PCC 7120 nuca nuia NO3-4 nira -N 2 all0605 all0606 C.S3 A

40 Fig. 9 (Frías and Flores) A B Nitrate (µm) (nira) Time (min) PCC 7120 EF116B CSE27 CSE27B NO 3 - -N NH 4 + NO 3 - -N NH 4 + NO 3 - -N NH 4 + NO 3 - -N NH 4 + (rnpb)