Functional Analysis Of Alr1390 And Alr3938 In Anabaena Sp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous gram-negative cyanobacterium that can form 5%-10% specialized nitrogen-fixing heterocysts along the filament in combined nitrogen starvation condition. NtcA is a global transcriptional regulator in nitrogen metabolism and heterocysts differentiation.The function of two genes alr1390 and alr3938 were investigated using the methods of molecular biology and microbial genetics in this study.Vectors for gene deletion and disruption of alr1390 and alr3938 were constructed for the homologous recombination in vivo in Anabaena sp. PCC 7120. The single recombinant strain for alr1390 was successfully obtained through triparental conjugation. Overexpression plasmids were also constructed and the strain overexpressing alr3938, named oe3938, was obtained.Bioinformatic analysis suggested that there was a conservative NtcA binding site upstream the putative tsp position of alr1390. Real-time RT-PCR results showed that the transcriptional level of alr1390 remained constant before and after combined nitrogen step-down in wild type Anabaena sp. PCC 7120. However, in the ntcA mutant, there was an increase in the transcriptional level of alr1390 after 12h of combined nitrogen step-down, which indicated that alr1390 may be subject to the regulation of NtcA. But this supposition was contradicted by the EMSA result which indicated no binding activity between NtcA and the predicted NtcA binding site of alr1390 in vitro.There was also a conservative NtcA-binding box upstream the putative tsp position of alr3938. Real-time RT-PCR results showed that the transcriptional level of alr3938 also remained constant before and after combined nitrogen deficiency in wild type Anabaena sp. PCC 7120, and there was an abruptly marked up-regulation of the transcriptional level of alr3938 after 12h of nitrogen deficiency in the ntcA mutant. EMSA result confirmed that in vitro purified NtcA protein was able to bind upon a fragment from the alr3938 upstream region containing the NtcA-binding box. These results indicated that alr3938 might be regulated by NtcA directly. The sequence analysis of alr3938 showed that this gene might encode an iron binding protein. Iron deficiency may result in oxidative stress, and the strain overexpressing alr3938 exhibited more resistance in the sensitivity assay to H₂O₂ and MV compared with the wild type. Thus, we speculate that alr3938 might be involved in both nitrogen metabolism and iron metabolism.