Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria

Abstract

Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production.

Inhibition of anaerobic digestion processes: Applications of molecular tools

“…Anaerobic GFP (AnGFP) was developed to overcome this challenge and is capable of producing fluorescence in both aerobic and anaerobic environments (Drepper et al, 2007). AnGFP has been used to study microbes in the human gut (Landete et al, 2014) and lactic acid producing bacteria (Landete et al, 2015). Another reporter system used in anaerobic systems is the proprietary SNAP-tag™ system, which uses a modified protein derived from human DNA to tag a protein.…”

Investigating the influence of spore maturation and sporulation conditions on MalS expression during Bacillus subtilis spore germination

“…The increase in MalS fluorescence intensity may be linked with proper folding of the GFP or its maturation. GFP maturation requires molecular oxygen 25 . Interestingly, in Bacillus megaterium spores previous experimental work has shown that heat activation increases the respiration rate and thereby likely promotes the occurrence of higher molecular oxygen levels in spores 26 .…”

Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius

“…The GFP reporter was found to be more favorable in the bacteriocinproducing backgrounds than either the RFP or the Lux system. Interestingly, a very recent study has reported the development of an optimized GFP variant suitable for anaerobic environments (39); the strains expressing GFP in the current study were analyzed under microaerobic conditions to facilitate fluorescence detection. Both the RFP and Lux systems were also investigated due to their usefulness in live tissues.…”