Cloning And Characterization Of The Bidirectional Hydrogenase Genes And The Application In Phylogenic Study Of Arthrospira And Spirulina

The complete sequence of bi-directional hydrogenase was obtained by PCR and chromosome walking method, including diaphorase moiety (encoded by hoxEFU) and [NiFe]hydrogenase moiety (encoded by hoxYH). Primers were designed accoring to the published gene sequences of bi-directional hydrogenase. The sequence is totally 7081 bp, contaning hoxE 540bp, hoxF 1596bp, a putative ORF between hoxF and hoxU 1650bp, hoxU 732bp, hoxY 549bp, hoxH 1431bp and 349bp upstream of hoxE and the spacer sequences between the vicinal subunits.In the gene cluster of bi-directional hydrogenase, two putative promoter sequences were obtained, locating upstream of hoxE and between hoxF and hoxU respectively. The two sequences were inserted into pEGFP-1 and transformed into E.coli JM109 and Synechococcus sp. PCC6803. Green fluorescence proteins were detected in both of the recipient cells. The functions of the promoter sequences were detected.The hoxH is single copy in Arthrospira FACHB341 genome tested by southern blotting and the hoxH and hoxY are authentically transcribed verified by RT-PCR. Therefore the genes of [NiFe] hydrogenase are typical in non-N2-fixing cyanobacterium. The complete sequence of [NiFe] hydrogenase expressed in PBV220. High rate of expression could be detected in E.coli. The expression products of large and small subunits accounted for 12.1% and 21.0% respectively after 4 hours' inducing. The products can be seen even diluted by 10 times.Partial hoxH genes of 2 cyanobacterial genera, including 5 strains of Arthrospira and 2 strains of Spirulina were cloned and characterized. This gene encodes the large subunit of nickel-iron hydrogenase. The results show that these genes comprise 1349 nucleotides in Arthrospira and 1343 nucleotides in Spirulinarespectively. The GC contents of hoxHare 45.7%~47.3% in Arthrospira, while those of Spirulina are up to 50.4%~50.9%. The similarities of nucleotide sequences among the 5 strains of Arthrospira range from 95.7% to 99.8%, which are higher than those between Arthrospira and Spirulina (72.9~77.0%). However, similarity between the two Spirulina strains is only 72.5% slightly lower than those between the 2 genera. Phylogenetic tree was constructed based on hoxH. All the 5 strains of Arthrospira form a monophyletic clade, which is highly supported by bootstrap value.The partial hoxY genes were cloned and characterized from 2 cyanobacterial genera, including 5 Arthrospira strains and 1 Spirulina strain. The gene encodes the small subunit of nickel-iron hydrogenase with the same length of 479 bp in two genera. GC content ranges from 46.0% to 46.6% in Arthrospira and 43.5% in Spirulina, respectively. The similarities of nucleotide sequences among the 5 strains in Arthrospira (93.7-100%) are higher than those between Arthrospira and Spirulina (69.5-72.2%). The alignment of deduced amino acid sequence also indicates that S. subsalsa FACHB351 is phylogenetically distinct from Arthrospira strains. Phylogenetic tree was constructed by comparing with the corresponding sequence of other 6 cyanobacterial strains. All the 5 strains of Arthrospira form a monophyletic clade, which is highly supported by bootstrap values, while the Spirulina stain is in another clade.