Alysis of select transcriptsThe abundance of the identified proteins Rosiglitazone was calculated using emPAI values to investigate their differential expression. Protein abundance was considered significantly upor downregulated when emPAI ratios between Cu and CK from two replicates were both higher than 1.25 or lower than 0.8. Results showed that Cu2+ upregulated 27 types of proteins (Table 2) and downregulated 25 types of proteins (Additional file 1: Table S4). Upregulated proteins included 3-oxoacyl-[acyl-carrier-protein] synthase 2 (KAS II), aldehyde dehydrogenase (ALDH), elongation factor G (EF-G), 3-hydroxybutyrate dehydrogenase (BDH), and PhaR protein. Downregulated proteins included succinate dehydrogenase subunit A (SHDA), ATP synthaseLiu et al. Microb Cell Fact (2015) 14:Page 8 ofFig. 7 Functional classification of the identified proteins obtained from the spore-release period in B. thuringiensis X022. a Functional classification of the total identified proteins, b subclassification of macromolecular metabolic proteins, and c subclassification of identified small molecular metabolic proteins. CK: proteins from the original medium; Cu: proteins from the medium to which 10-6 mol/L Cu2+ had been addedThe protein samples for 2D-LC S/MS analysis were extracted after 44 h after cultivation, during which cells are in the spore-release phase. In this period, specific metabolic-related proteins, such as SHDA, ATPS, and PrkA, have low transcriptional and translational levels but high degradation rates. As Cu2+ shortens the fermentation cycle and releases spores and crystals earlier (Fig. 6), thereby resulting in downregulation phenomena. RNA samples for qRT-PCR analysis were extracted after 31 h of cultivation, during which cells are in the spore-formation phase. In this period, ICP and metabolic-related proteins exhibit high transcriptional and translational levels as well as relatively low degradation rates. These differences may result in the noncorrelation of downregulatedproteins with changes at the translational and transcriptional levels. In addition, post-transcriptional regulation may also explain the noncorrelation observed.Mechanism of Cu2+ in improving crystal protein productionCu2+ brought about changes in fermentation parameters: a prolonged stationary phase, rapid pH rebound, a prolonged pH plateau phase, and lower pH in the plateau phase (Fig. 5). We propose that such differences are related to ICP upregulation. Prolonging the stationary phase may directly bring about increased time for ICP synthesis. pH affects nutrient ionization in the medium, influences nutrient absorption, conversion and utility of nutrients by regulatingLiu et al. Microb Cell Fact (2015) 14:Page 9 ofFig. 8 Fold changes of the functional categories of the proteins between Cu and CK. a Total identified proteins, b macromolecular metabolic proteins, and c small molecular metabolic proteins. CK represents proteins from the original medium; Cu represents proteins from the medium to which 10-6 mol/L Cu2+ had been addedthe activity of various enzymes. Thus, pH finally influences growth, sporulation, and ICP synthesis. If pH does not rebound after the logarithmic phase, B. thuringiensis would be unable to form spores and crystals [13]. The present study also showed that the initial pH of the medium influences ICP biosynthesis significantly (Fig. 2a, d). Upregulation of two PHB PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7500280 metabolism-related proteins, PhaR and BDH (Table 2), is an interesting phenomenon. PHB, a.