| Bacillus thuringiensis, which is widely distributed in soil, dead insects and hoards, is the most widely used microbial pesticides and is also of great research value gram-positive strain.Now, c-di-GMP mediated signal transduction system has an important significance on physiological and biochemical activities, which can regulate several physiological processes such as motility, bacterial aggregation, biosynthesis of biofilm and expression of numerous virulence factors through cross signal network. The intracellular level of c-di-GMP is up-regulated by guanylate cyclase (DGC) that contains GGDEF conserved domain and down-regulated by phosphodiesterase containing EAL or HD-GYP conserved domain.In B. thuringiensis BMB171, there are ten proteins containing GGDEF and EAL conserved domains, but what kinds of biological functions the proteins can play in B. thuringiensis and even in B. cereus group have never been reported so far.Based on the genomics of the B. thuringiensis BMB171, we found ten proteins associated with c-di-GMP, In which three proteins contain GGDEF domain, one protein contains EAL domain and six proteins contain GGDEF-EAL domain. This study was focused on the identification and fuctional analysis of the ten proteins. These genes were cloned, sequenced and expressed, respectively, and the purified combinant proteins were obtained. Using HPLC, we determinated whether the ten proteins possess the synthesis and degradation enzyme activity of c-di-GMP or not. The results showed that in addition to C3708 and C4901, we successfully got the rest of the proteins and demonstrated their enzymatic activities. We found C3661 and C5008 didn’t have synthetase activity; C3653 had strong degradative activity and four dual structure domain proteins, C0469, C0550, C3200 and C3420 only had degradative activity. Besides, C4944 neither had synthetase activity nor had degradative activity. So, for further research we obtained three single-deletion mutants △C0550, △C3653 and △C4944, two double-deletion mutants △C0550△C0469 and △C3653 △C0469, and a three-deletion mutant △C0550△ C0469△C3420, using homologous double-exchage and triparental hybird. Through the preliminary testing of the phenotypes of the strains, we found that △C3653 strain growed more slowly than BMB171 and its growth cycle delayed about 3 h in GYS medium containing 2 μg/mL nisin. Meanwhile, compared with BMB171, △C3653 strain had more stronger sensitivity to low temperature. These indicated that the cell membrane of strain △C3653 was hurt in a certain degree. Furthermore, compared with BMB171, we observed three strains △C0550△C0469, △C3653△C0469 and △C0550△C0469 △C3420 had obvious phenotypic differences compared to the initial strain BMB171. Their mobilities declined, bacterial aggregation rates quickened and biofilm formation quantities increased. These results suggested that these mutants could regulate the synthesis of extracellular matrix, mobility, bacterial aggregation and biofilm formation through altering the concentration of c-di-GMP in vivo.So, we inferred that c-di-GMP was a kind of functional signal molecule in B. thuringiensis. This is the first time to confirm that c-di-GMP can affect a variety of biological functions in B. thuringiensis. Morever, it has the vital significance to deeply analysis the regulatory mechanism about the c-di-GMP signal in gram-positive bacteria and these works lay the foundation for further study of c-di-GMP signaling system mechanism in B. thuringiensis and even B. cereus group. |
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