Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

Thorough intestinal adhesion and colonization greatly promote the probiotic properties of lactic acid bacteria (LAB). Labeling and tracing with fluorescent proteins are effective and reliable for studying the in vivo physiological activities of LAB including localization, adhesion, and colonization. Lactiplantibacillus plantarum WCFS1 was successfully traced with a red fluorescent protein (RFP), which was expressed by the bacteria-carrying recombinant plasmids. In this study, we aimed to construct a stable RFP mCherry expression system, whose encoding gene was integrated into the bacterial chromosome via double-crossed homologous recombination, and use it for labeling WCFS1 with the goal of avoiding the potential loss of non-chromosomal plasmids along with intestinal growth. First, the constitutive expression of the mCherry protein was improved after adjusting the length of the spacer between the promoter and the gene start codon. Then, the optimized mCherry gene expression cassette was integrated into the chromosome of WCFS1. The resulting strain had normal unimpaired growth and strong fluorescent signals, even after 100 generations, indicating its stability. Furthermore, quantitative polymerase chain reaction (PCR) results revealed a strong positive correlation between the fluorescence intensity of the strain and the number of viable cells, demonstrating its potential usage for the quantification of in vivo WCFS1 cells. Finally, the increased adhesion ability of WCFS1 due to the recombinant expression of the bsh gene was visualized and evaluated using fluorescence intensity, the results of which were consistent with those obtained using the previously established quantification methods. These results suggest that the chromosomal-integrated mCherry labeling system can be extensively used to examine the distribution, colonization, and survival of LAB in vivo in order to determine the mechanism of its probiotic function.