The Functional Analysis Of The Histidine Kinase Gene Alr0428 And Response Regulator Gene Alr0429 In Anabaena Sp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous cyanobacterium capable of differentiating heterocysts, which account for 5 to 10% of the cells along each filament, upon limitation of a combined nitrogen source in the growth medium. Heterocysts provide an environment for nitrogenase to fix N₂ and support nitrogen source for vegetative cells.The genome of Anabaena sp. strain PCC 7120 possesses 211 genes encoding proteins belonging to two-component signaling systems, which contain 131 genes encoding histidine kinase and 80 genes encoding response regulator. In this study, the functions of histidine kinase alr0428 and response regulator alr0429 were analyzed.alr0428 encodes a histidine kinase with a length of 637AA, and alr0429 encodes a 276AA response regulator. They have the same transcriptional orientation and their intergenic noncoding sequence is 130bp. Our results showed that alr0428 and alr0429 are co-transcription, and their expression is very low under the normal BG11 medium.The interruption constructs for alr0428 and alr0429 were used to construct the interruption Anabaena strains contains an antibiotic fragment in the target gene by conjugation. The mutant strain carrying an antibiotic fragment in alr0429 was assigned as M29. The overexpression mutant strain Oe29 was isolated by conjugating a replication plasmid with alr0429 induced by petE promoter. The plasmid for expression His-tagged Alr0429 protein was constructed, and the anti-serum was generated by injection the His-tagged alr0429 protein into rabbit.The functional analysis of response regulator alr0429 showed that the inactivated mutant M29 and overexpression mutant Oe29 grew at the same rate as the wide type in BG11 medium. Upon nitrogen limitation, the inactivated mutant M29 and overexpression mutant Oe29 also grew at the same rate as the wide type in BG11 medium and can differentiate normal heterocysts to fix N₂. Real-Time RT-PCR results showed that the expression of alr0428 and alr0429 had no significant changes upon nitrogen depletion. At iron depletion, the inactivated mutant M29 grew at a same rate as the wide type, but the inactivated mutant M29 grew much slower under both the iron and nitrogen limitations. The growth of inactivated mutant M29, overexpression mutant Oe29 and wide type share an identical rate upon the high-salt stress. The thermal tolerance of inactivated mutant M29, overexpression mutant Oe29 and wide type were also tested, and the results showed that no significant growth difference among these strains. However, the pigment constitution of these strains had some difference. The sensitivity test of inactivated mutant M29, overexpression mutant Oe29 and wide type to oxidant H₂O₂ and MV were also examined. The wide type can survive at 1.5mM H₂O₂ and 3.5mM MV treatment respectively. But the inactivated mutant M29 can only tolerate 0.5mM H₂O₂ and 0.5mM MV respectively.