During the last 10 years increased scientific attention has been given to probiotics due to their promising therapeutic benefits in enteric diseases. In this sense, Lactobacillus reuteri 6475 has emerged as a potential probiotic strain that has demonstrated various probiotic therapeutic features. These characteristics include the ability of this strain to produce and secrete the antimicrobial compound reuterin and anti-inflammatory factors. Because the main probiotic effects of L. reuteri 6475 that have been identified thus far are secreted, we were interested in developing a mutant library consisting of disruptions of genes that encode for secreted or cell-wall proteins. To complete this task we employed two different strategies: the identification of secretome protein sequences by predictive mathematical methods and the disruption of the protein coding sequences by single-stranded DNA recombineering. As a result, we have developed a mutant library of 127 secretome genes that would drive the elucidation of important L. reuteri probiotic mechanisms of action. To demonstrate the utility of this library, I screened all 127 mutants for the ability to produce reuterin and found 11 genes that increase reuterin secretion and 3 that eliminate reuterin secretion when disrupted. Future characterization of these genes will further elucidate the bacterial pathways that are critical for reuterin production.
Read
