Genomic Analysis Of Cyanobacteria: A Case Study On Heterocyst Evolution And A 3', 5'-Bisphosphate Nucleotidase

Cyanobactera are among the most ancient organisms on the Earth.Some species of filamentous cyanobacteria can fix the atmosphere N₂ into ammonium in heterocysts,cells differentiated form vegetative cells upon nitrogen deficiency.Heterocyst differentiation is thought to having originated between 2,450 and 2,100 Ma based on geological data and fossil records,and thus it could be one of the earliest developmental phenomena.The mechanism of heterocyst differentiation is an attractive subject in the research areas of cyanobacteria.In recent years,many important genes involved in heterocyst development(het genes) have been identified,such as ntcA(global regulator in nitrogen metabolism),hetR(regulator for initiation of heterocyst differentiation),nifHDK (nitrogenase),hepA and hglE(required for the formation of heterocyst envelope),etc. Some small moleculars such as 2-oxoglutarate and calcium,are also shown to be important signals for heterocyst development.We have known much about the mechanism of heterocyst differentiation;however,how did heterocyst evolve during evolution?Here we studied the evolution history of the regulatory network involved in heterocyst differentiation by comparative genomics.Many het genes in Anabaena PCC7120 were found to be present in the genome of other cyanobacteria as well, including those non-heterocystous strains.According to their distribution,het genes can be assigned into several catalogs:1) genes in most strains,2) genes exclusively in filamentous strains,3) genes exclusively in diazotrophic strains,4) genes exclusively in heterocystous strains;5) other genes.The sequence information and distribution of het genes were used to rebuild their evolutionary history.The result shows that 1) het genes were obtained during the whole evolution history of cyanobacteria,2) most of het genes appeared before the occurrence of heterocyst,3) lateral gene transfer(LGT) played very important roles in the evolution of heterocyst development.In heterocystous cyanobacterium Anabaena PCC7120,HetR and PatS interact and act as the central regulators for the initiation of heterocyst differentiation and location of heterocysts in filaments.The hetR gene was found to be present in all examined filamentous cyanobacteria.We detected the expression of HetR in each tested filamentous cyanobacterium.We also found the ORF of the patS gene in five genomes of filamentous cyanobacteria(including non-heterocystous strains Trichodesmium IMS101 and Arthrospira platensis).The expression of HetR in Arthrospira platensis increased upon nitrogen deficiency.The hetR gene and patS gene from Arthropira platensis (arhetR and arpatS,respectively) stimulated and repressed heterocyst differentiation when expressed in Anabaena PCC7120,respectively.The results showed that arhetR and arpatS could regulate heterocyst development in heterocystous strains.Combining the bioinformatie analysis and experimental work in this study,we reconstructed the evolution history of the regulatory network for heterocyst development. A basic network for nitrogen metabolism consisting of many key regulators for heterocyst development probably existed much earlier before the occurrence of heterocyst,and the process of heterocyst evolution is actually a process of recruiting more and more het genes to perfect this network,for effectively utilizing nitrogen nutrition in changing environments.Our results will provide clues to the ancient events in life evolution,and be helpful for understanding the developmental mechanism in higher eukaryotic organisms.3'-Phosphoadenosine-5'-phosphatase(PAPase) is required for the removal of toxic 3'-phosphoadenosine-5'-phosphate(PAP) produced during sulfur assimilation in various eukaryotie organisms.This enzyme is a well-known as the target of lithium and sodium toxicity and as the limiting factor of salt-resistance of its host.One gene,halA,which could encode a protein closely related to the PAPases of yeasts and plants,was identified from the cyanobacterium Arthrospira(Spirulina) platensis.Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades,suggesting multiple origins of PAPases in cyanobacteria.The HalA polypeptide from A.platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties.HalA was dependent on Mg²⁺ for its activity and could use PAP or 3'-phosphoadenosine-5'-phospbosulfate (PAPS) as a substrate.HalA is sensitive to Li⁺(50%inhibitory concentration[IC₅₀]≈3.6 mmol/L) but only slightly sensitive to Na⁺(IC₅₀≈600 mmol/L).The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts,which are much more sensitive to both Li⁺ and Na⁺.Based on the structure of Hal2p,the PAPase in yeast,we modeled the structure of HalA.The major difference between them was in their substrate-binding sites.The putative substrate-binding motif of HalA has a proline residue(P254) which might lower its affinity with PAP and contribute its tolerance to Li⁺ and Na⁺.The E.coli strain expressing halA gene grew much better in the medium with high[Li⁺],suggesting PAPase also was the target of lithium and sodium toxicity in bacteria as well.HalA is the known PAPase that has best tolerance to Na⁺,so it is a potential way to improve the sodium-tolerance of various organisms by expressing halA gene.Here we identified and characterized HalA,a PApase from prokaryote for the first time.The results will help us to understand the sulfur-assimilating pathway and the mechanism of sodium-resistance in bacteria.