Research On Function Of A Novel Second Messenger C-di-GMP In Anabaena PCC7120

Cyclic diguanylate(c-di-GMP) is a novel second messenger in bacteria,but not in any Archaea or Eukarya.It is now established that this nucleotide is almost ubiquitous in bacteria,and it regulates a range of signaling pathways such as bacterial motility,biofilm formation,virulence and so on.Diguanylate cyclase(DGC) and phosphodiesterase(PDE) control cellular c-di-GMP level together.The conserved GGDEF domain of DGC synthesizes c-di-GMP,whereas the conserved EAL and HD-GYP domains of PDE are involved in c-di-GMP hydrolysis.These domains always couple with other signal input domains to regulate bacterial physiology.Since 6 vectors for inactive mutants had been obtained in previous works,16 putative c-di-GMP-associated genes in Anabaena PCC7120 genome were found after searching in cyanobase database,and they covered three domains which regulated c-di-GMP level:GGDEF,EAL and HD-GYP.Blasted of amino acid sequences of these GGDEF proteins with known PleD showed that the proteins’s identity exceeded 30%.11 of 14 proteins are of this kind.And there were 9 proteins that had allosteric regulated RXXD motif.Nevertheness,input domains such as RR,PAS were coupled with these domains.Since 6 vectors for inactive mutants had been obtained in previous works,other 9 vectors for deletion mutants were constructed in this study.Triparental mating was performed,then 2 deletion mutants were obtained.There were no significant differences between wild type and mutants after nitrogen depletion.But one mutant showed sensitive to Na-Salt.At the same time,c-di-GMP-assotiated genes which were fused with different promoters were overexpressed in Anabaena PCC7120.14 recombinant plasmids fused with their own promoters and 1 petE promoter fused plasmid were constructed successfully.By Triparental mating,13 overexpression strains recombined with their own promoters were obtained.And two overexpression strains grew slower than WT strain.C-di-GMP-associated gene expression vectors were also constructed,and 12 vectors were obtained.A high level of expression of fusion proteins in E.coli were detected after IPTG induction and 2 purified proteins were obtained successfully,and the sizes were consistent with what were expected.These studies provide a molecular biology basis and significant materials for further research on c-di-GMP in caynobacteria.