A novel universal nano-luciferase-involved reporter system for long-term probing food-borne probiotics and pathogenic bacteria in mice by in situ bioluminescence imaging.

Food-borne bacteria have received increasing attention due to their great impact on human health. Bioimaging makes it possible to monitor bacteria inside the living body in real time and in situ. Nano-luciferase (NLuc) as a new member of the luciferase family exhibits superior properties than the commonly used luciferases, including small size, high stability and improved luminescence. Herein, NLuc, CBRLuc and FLuc were well expressed in varied food-borne bacteria. Results showed that the signal intensity of E. coli-NLuc was about 41 times higher than E. coli-CBRLuc, L. plantarum-NLuc was nearly 227 times that of L. plantarum-FLuc in vitro. Moreover, NLuc was applied to trace L. plantarum and E. coli in vivo through the whole body and separated digestive tract imaging, as well as the feces bacterium counting and probing. The persistence of bioluminescent strains was predominantly localized in colon and cecum of mice after oral administration. The NLuc system showed its incomparable superiority, especially in the application of intestinal imaging and the universality for food-borne bacteria. We demonstrated that the NLuc system was a brilliant alternative for specific application of food-borne bacteria in vivo, aiming to collect more accurate and real-time information of food-borne bacteria from the living body for further investigation of their damage mechanism and nutrition effect.

Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies.

Based on the complete genome of Cyanothece ATCC 51142, the oriCs of both the circular and linear chromosomes in Cyanothece ATCC 51142 have been predicted by utilizing a web-based system Ori-Finder. Here, we provide experimental support for the results of Ori-Finder to identify the replication origins of Cyanothece ATCC 51142 and their interactions with the initiator protein, DnaA. The two replication origins are composed of three characteristically arranged DnaA boxes and an AT-rich stretch, and the oriC in the circular chromosome is followed by the dnaN gene. The dnaA gene is located downstream of the origin of the circular chromosome and it expresses a typical DnaA protein that is divided into four domains (I, II, III, IV), as with other members of the DnaA protein family. We purify DnaA (IV) and characterize the interaction of the purified protein with the replication origins, so as to offer experimental support for the prediction. The results of the electrophoretic mobility shift assay and DNase I footprint assay demonstrate that the C-terminal domain of the DnaA protein from Cyanothece ATCC 51142 specifically binds the oriCs of both the circular and linear chromosomes, and the DNase I footprint assay demonstrates that DnaA (IV) exhibits hypersensitive affinity with DnaA boxes in both oriCs.