Anaerobic green fluorescent protein as a marker of Bifidobacterium strains

• First reporting a reliable method to track fluorescently labeled bifidobacteria
• Real-time reporter system for Bifidobacterium under aerobic/anaerobic conditions
• Marked bifidobacteria were distinguishable from lactic cultures or fecal microbiota

Some strains of Bifidobacterium are considered as probiotics and are being added as adjunct culture in food products due to their potential in maintaining a healthy intestinal microbial balance. However, despite these benefits, bifidobacteria still remain poorly understood at the genetic level compared with other microorganisms of industrial interest. In this work, we have developed a non-invasive green fluorescent based reporter system for real-time tracking of Bifidobacterium species in vivo. The reporter vector pNZ:Tu-GFPana is based on the pNZ8048 plasmid harboring a bifidobacterial promoter (elongation factor Tu from Bifidobacterium longum CECT 4551) and a fluorescent protein containing a flavin-mono-nucleotide-based cofactor (evoglow-Pp1) which is fluorescent under both aerobic and anaerobic conditions. pNZ:Tu-GFPana was constructed and found to stably replicate in B. longum CECT 4551 and in the intestinal strain Bifidobacterium breve INIA P734. The subsequent analysis of these strains allowed us to assess the functionality of this plasmid.Our results demonstrate the potential of pNZ:Tu-GFPana as a real-time reporter system for Bifidobacterium in order to track the behavior of this probiotic species in complex environments like food or intestinal microbiota, and to estimate their competition and colonization potential.