Roles Of The Second Messenger C-di-GMP In Bacillus Cereus 0-9

In recent years,the second messenger bis-(3'–5')-cyclic dimeric guanosine monophosphate(c-di-GMP)has emerged as one of the most common and important bacterial second messenger.The synthesis and breakdown of c-di-GMP are regulated by the diguanylate cyclase(DGC)and c-di-GMP-specific phosphodiesterase(PDE),respectively.The central domains in all DGCs and PDEs are GGDEF and EAL/HD-GYP,respectively.The proteins that regulate the metabolism of the c-di-GMP often contain a tandem arrangement of the GGDEF and EAL domains,and also possess other structures such as PAS domains,PAC domains,and transmembrane domains.The GGDEF and EAL domains are widely distributed in the organisms,and they have many differences in the quantities and species,generally speaking,the numbers and species in Gram-positive bacteria tend to have fewer than the Gram-negative bacteria.C-di-GMP has been shown to play key roles in lifestyle changes of many bacterias,including transition from the motile to the sessile state,and it also has been shown to regulate the cell cycle,differentiation,virulence,biofilm,and other processes.Research on the second messenger c-di-GMP have been focused on the Salmonella.spp,Pseudomonas aeruginosa,Caulobacter crescentus and other Gram-negative bacteria.Roles of signal transduction mechanisms of c-di-GMP in the Gram-positive bacteria Bacillus cereus has not been reported up to now.In order to determine whether the enzymes that controlling the metabolism of c-di-GMP presenced in the Bacillus cereus 0-9,in this study,we use the bioinformatics methods to analyze the genome of 0-9,and then to constructe 11 gene knockout strains so as to understand the roles of the 11 genes in 0-9 after analyzingphenotypes of different gene knockout strains.In this study,we extracted and re-sequencing the genome of 0-9,used the DNAman,Primer Premier5.0,SMART and other bioinformatics soft wares to analyse and compare the genome of 0-9.The results revealed that,in the genome of Bacillus cereus,the gene dgc1,dgc3,dgc4,dgc5,dgc6,dgc7,dgc12,dgc14,dgc16,dgc19 and eal were connected with the metabolism of c-di-GMP.Of all the 11 genes,dgc1,dgc3,dgc16,dgc19 had a purified GGDEF domain,eal had an EAL domain,and other proteins had a tandem arrangement of the GGDEF and EAL domains.In order to analysised the physiological effects of c-di-GMP in B.cereus systematic,we constructed 11 gene knockout strains through molecular methods,and compared the differences between the 11 gene knockout strains and wide type 0-9,which fouced on growing states,the production of extracellular polysaccharide,extracellular enzymes,the toleration of vitamine K(a kind of antioxidant),moveability,biofilm formation,and the production of total proteins.The results revealed that the gene knockout strains had more or less differences with the wild type 0-9.Disruption of dgc1 and dgc3 in B.cereus caused the colonies compact and smooth on the Coomassie brilliant bule plates,dgc14 and dgc19 also can caused the colonies smooth.The disruption of gene dgc12 lead to the capacity of secretion protease declined.The disruption of gene dgc1 and dgc3 caused the reduction in hemolysis enzyme formation.A certain concentration of vitamine K could inhibit the growth of the 0-9 strains.Disruption of gene dgc1,dgc3,dgc5,dgc6,dgc7,dgc12,dgc14,dgc16 enhanced the tolerance of vitamine K compared to the wild type 0-9.Disruption of dgc19 reduced the tolerance of vitamine K.Disruption of dgc1,dgc3,dgc12 caused the move abilityweakened.In the aspect of biofilm formation,the disruption of dgc1,dgc3,dgc12 gene improved the quantity of biofilm,and the disruption of dgc14,eal caused the reduction of biofilm formation.All these knockout strains had differences in the production of total proteins;the most obvious differences were 24 kDa and 39 kDa.All these results laid a foundation of the researches of mechanisms about each gene,the signal transduction mechanisms of c-di-GMP in 0-9,and the relations between the biological control of 0-9 and c-di-GMP.