Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp

Abstract

Cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, occupying a key position at the base of marine food webs. The cynS gene that encodes cyanase was identified among bacterial, fungal, and plant sequences in public databases, and the gene was particularly prevalent among cyanobacteria, including numerous Prochlorococcus and Synechococcus strains. Phylogenetic analysis of cynS sequences retrieved from the Global Ocean Survey database identified >60% as belonging to unicellular marine cyanobacteria, suggesting an important role for cyanase in their nitrogen metabolism. We demonstrate here that marine cyanobacteria have a functionally active cyanase, the transcriptional regulation of which varies among strains and reflects the genomic context of cynS. In Prochlorococcus sp. strain MED4, cynS was presumably transcribed as part of the cynABDS operon, implying cyanase involvement in cyanate utilization. In Synechococcus sp. strain WH8102, expression was not related to nitrogen stress responses and here cyanase presumably serves in the detoxification of cyanate resulting from intracellular urea and/or carbamoyl phosphate decomposition. Lastly, we report on a cyanase activity encoded by cynH, a novel gene found in marine cyanobacteria only. The presence of dual cyanase genes in the genomes of seven marine Synechococcus strains and their respective roles in nitrogen metabolism remain to be clarified.

FIG. 1.

Typical example of RT-PCR amplification of cynS cDNA for Synechococcus sp. WH7803. Samples (50 μl) were run for 30 cycles, and 6-μl subsamples were collected at five-cycle intervals. PCR products from each cycle set were run on a 1.5% agarose gel and visualized by staining with ethidium bromide. The band density was determined for the set in which subsamples had clearly not reached saturation phase (arrow), assuming that they most closely resembled the phase of exponential amplification.

FIG. 2.

Alignment of the amino acid sequences for 17 putative cyanase genes in marine Prochlorococcus (P.) sp. and Synechococcus (S.) sp. strains, along with that of the characterized E. coli cyanase. Fully conserved residues are labeled with “*”, conserved replacements are labeled with “:”, and functional similarity are labeled with “.”. The proposed active-site residues Arg-96, Glu-99, and Ser-122 are shown in boldface against a gray background. The GenBank accession numbers for cyanase sequences in alignment are as follows: Synechococcus sp. strains WH8102 (NP_898579), WH7803 (YP_001226218), WH7805 (ZP_01124911), WH8109 (ZP_05789360), RS9916 (ZP_01471501), RS9917 (ZP_01079240), RCC307 (YP_001228741), CC9902 (YP_378288), CC9605 (YP_382939), CC9311 (YP_732088), BL107 (ZP_01469110), PCC7335 (ZP_05037924), PCC7001 (ZP_05043889, and YP_001733904); Prochlorococcus sp. strains MED4 (NP_892492), NATL1A (YP_001013899), and NATL2A (YP_292581); and E. coli strain K-12 (NP_414874) (4).

FIG. 3.

Tree topology resulting from Bayesian phylogenetic analysis of translated environmental cynS sequences (135 amino acids) derived from the Global Ocean Survey database, along with CynS sequences retrieved from GenBank. Detailed relationships among cyanobacterial clades are shown, while bacterial and fungal branches are collapsed for the purpose of presentation. The scale bar provides a distance measure for two substitutions per 100 nucleotides between sequences. Posterior probabilities are given at the nodes using a scale from 0 to 1. The denomination “JCVI PEP” has been omitted from all environmental sequences in order to improve the presentation.

FIG. 4.

Schematic representation of the genome regions that contain the cyanate utilization genes in Prochlorococcus sp. strain MED4 and Synechococcus spp. strains WH8102, WH7803, and WH7805. Vertical red arrows indicate location of putative NtcA binding sites shown above the arrows, and numbers indicate the nucleotide distance between two adjacent genes. Horizontal arrows indicate cynABD genes encoding for the cyanate transporter (brown), nirA (COG0155, dark blue); focA (COG2116, orange), cynS (bright yellow), cynH (pale yellow), trpD (COG0547E, blue), proP (COG2814G, purple), ppk (COG0855P, green), and rpoD (COG0568K, gray) and ORFs encoding unidentified proteins (white).

FIG. 5.

Transcript accumulation of cynA (A and D), ntcA (C) and cynS (B, E, and F), in Prochlorococcus sp. MED4 (A to C), Synechococcus sp. WH8102 (D and E), and Synechococcus sp. WH7803 (F) cells grown on ammonium or on alternative N sources or deprived of combined N for 3, 6, 9, and >12 h after resuspension in fresh medium. The data are log₂ of median values of three replicates, normalized to their initial transcription level, with 25th to 75th percentiles.

FIG. 6.

Median log-normalized values of cynA and cynS transcription for Prochlorococcus sp. MED4 (A) and Synechococcus sp. WH8102 (B) cultures grown on alternative N sources or with no N for 3, 6, and 9 h after medium replacement. The error bars represent 25th to 75th percentiles. Linear regression values and R² values for cynA (continuous) and cynS (dashed) accumulation are presented above and below the respective trend line.

FIG. 7.

(A) IPTG-induced expression of CynS and its accumulation in soluble (lane S) and particulate (lane P) fractions of E. coli cell lysates. (B and C) Purification of CynS (B) and CynH (C) recombinant proteins using amylose resin-based affinity chromatography. M, molecular weight markers; E1 to E3, elution fractions.

FIG. 8.

Characterization of cyanase activity of CynS and CynH fusion constructs at different temperatures and their sensitivity to Na-azide (200 μmol liter⁻¹) addition. Freshly prepared sodium cyanate (2 mmol liter⁻¹) was added to the reaction mix (50-mmol liter⁻¹ PBS buffer [pH 7.6], 3 mmol of sodium bicarbonate liter⁻¹) complemented with aliquots of recombinant protein (20 μg) and incubated at the desired temperature for 10 min. Ammonium accumulation was determined with Nessler reagent. The activities shown are averages from at least nine replicates from three independent experiments.

FIG. 9.

(A and B) Amino acid alignments for catalytic site regions of cyanases for 17 CynS sequences (A) and 9 full-length CynH sequences (B) found in marine Synechococcus (S.) and in a single metagenomic clone GRIST19. Identical residues are presented in boldface and labeled “*”, conserved residue replacements are labeled “:”, and functionally similar residues are labeled “.”. The proposed active-site residues Arg (R), Glu (E), and Ser (S) are shown in boldface against a gray background. The bottom line presents the consensus secondary structure predicted by the Jpred prediction server that identifies randomly coiled region (C), α-helix (H), and β-sheet (E) motifs. In panel B, the aligned sequences are cyanate hydratase (ZP_01124909) in Synechococcus sp. WH7805, RS9916_37357 (ZP_01471502) in Synechococcus sp. RS9916, Syncc9902_2288 (YP_378289) in Synechococcus sp. CC9902, sync_2840 and sync_2903 (YP_732028 and YP_732090) in Synechococcus sp. CC9311, SynWH7803_2496 (YP_001226219) in Synechococcus sp. WH7803, SH8109_0550 (ZP_05789530) in Synechococcus sp. WH8109, non-annotated (reverse strand 1870903-1871064) in Synechococcus sp. BL107 (NZ_AATZ00000000), and metagenomic clone GRIST19 (EU795157).