In vitrogut modeling as a tool for adaptive evolutionary engineering ofLactiplantibacillus plantarum

Abstract

Research and marketing of probiotics demand for holistic strain improvement considering both, the biotic and abiotic gut environment. Here we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donors gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205 and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization in colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence.

Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota

“…Thereafter, L. plantarum strains that lost their aggregation capacity upon consecutive cultivation in MRS medium were grown in a minimal medium supplemented with different carbon sources and in EMS, a medium that simulates the abiotic environment of the reactor gut microbiota [ 40 ]. L. plantarum strains IA01, PA4_02 and PA1.2_01 lost their auto-aggregation capacity in repeated MRS cultures but recovered this capacity when regrown in EMS medium and in minimal medium containing sucrose or maltose, but not in the same medium with glucose as sole carbon source.…”

“…Indeed, an alternative junction was identified in all samples of the aggregating strains IA01 t0 and PA4_02, exactly at the repeat in the promoter of msa . This new junction was not identified in the transcriptome samples from the wild type, IA01 t4 , nor in the samples of a previously constructed non-aggregating lamC knockout strain L. plantarum ΔLP_RS14990 [ 40 ] (Isenring, unpublished results).…”

“…L. plantarum NZ3400, a WCFS1 derivative harboring a chloramphenicol resistance cassette in a neutral locus on the chromosome, was used as wild type strain [ 72 ]. NZ3400 derivative strains were recovered from in vitro human colonic microbiota after cultivation for up to 100 generations as described previously [ 40 ] (Table 1 ).…”

“…Glucose, sucrose, maltose and fructose were added as individual carbon source at 1% (w/v). In addition, the previously designed Effluent-MacFarlane-Sugar (EMS) medium [ 40 ], mimicking the abiotic environment of in vitro colonic microbiota, was used to test the effect on auto-aggregation in absence of microbiota. EMS medium consisted of sterile filtered effluent from the in vitro colonic microbiota, MacFarlane medium [ 74 ], which mimics the chyme entering the colon (ratio 9:1), and glucose (0.75%, w/v).…”

“…However, there is no description of a WCFS1 auto-aggregation phenotype yet. Recently, we supplemented L. plantarum NZ3400, a derivative strain of WCFS1, to different in vitro human colonic microbiota, continuously cultivated in the PolyFermS model in the context of an evolutionary engineering experiment [ 40 ]. L. plantarum NZ3400 derivatives were recovered from the microbiota and phenotypically and genotypically characterized.…”