Study Of The GAF Domain Of Protein Kinases Pkn41 Or Pkn42 In Anabaena Sp.PCC 7120 And Variation In The Copy Number Of PDU1-Based Plasmids

Anabaena sp.PCC 7120 is a filamentous Gram-negative cyanobacterium.The vegetative cells in the filament are photosynthetic,fixing CO2.When under nitrogen starvation,N2-fixing heterocysts differentiate from vegetative cells in a pattern,which there are about 15?20%heterocysts along each filament.Since Anabaena is able to perform several important biological functions,such as photosynthesis,nitrogen fixation,cell differentiation and cell-cell communication,an extensive signaling capability is demanded in order to respond to the changing environment.Protein phosphorylation catalyzed by protein kinases regulates a variety of cellular activities in both prokaryotes and eukaryotes and is essential for signal transduction.There are three major families of protein kinases involved in signal transduction,the family of His kinases,the family of Ser/Thr-and/or Tyr-specific protein kinases,and the family of Tyr-specific protein kinases.His kinases were typically found in two-component signaling systems in prokaryotes,while Ser/Thr kinases were thought to exist mainly in eukaryotes.A family of protein kinases,represented by HstK from Anabaena sp.PCC 7120 possesses both a His kinase domain and a Ser/Thr kinase domain,between which arranges at least one GAF domain.The cotranscripted hstK family members,pkn41 and pkn42,are regulated by the global nitrogen metabolism transcription regulator NtcA.And lost of either,or both of them,impairs iron acquisition.These suggest that Pkn41 and Pkn42 might participate in both nitrogen metabolism and iron metabolism.The GAF domains are considered as signal sensors.We constructed overexpressing strains for the GAF domains of Pkn41 and Pkn42,which are two cotranscribed members of HstK family.The overexpression in Anabaena led to heterocyst formation even with nitrate as a combined nitrogen source.The results suggest that 41 GAF and 42GAF might combine a certain signal molecule and thus disturb the regulation of carbon/nitrogen metabolism and heterocyst formation.The phenotypes weakened during subculturing,which might be due to the selection of suppressor mutations.In addition,the phenotypes varied,depending on the variation of relative copy number of the pRL25T-derived plasmids.The vector pRL25T is based on pDU1,an endogenous plasmid in Nostoc sp.PCC 7524,which is used as the only cyanobacterial replicon for Anabaena studies.However,the relative copy numbers of pDUl-based plasmids in Anabaena sp.PCC 7120 are not well studied.We found that the relative plasmid copy number of pRL25T,varied widely,especially when the vector carried a recombinant insert,under different conditions,ranging from 0.53 to 1,812 per chromosome in different recombinant strains tested,either in the independent clones of the same strain,or in the same clone under different growth conditions.The phenotypes caused by the pRL25T-driven expression of green fluorescent protein or the GAF domain of Pkn41 or Pkn42 varied depending on the independent clones analysed.This phenotypic variation correlated with the relative plasmid copy number present in cells.