Polymorphisms, Chromosomal Rearrangements, and Mutator Phenotype Development during Experimental Evolution of Lactobacillus rhamnosus GG

Abstract

Lactobacillus rhamnosus GG is a lactic acid bacterium widely marketed by the food industry. Its genomic analysis led to the identification of a gene cluster encoding mucus-binding SpaCBA pili, which is located in a genomic island enriched in insertion sequence (IS) elements. In the present study, we analyzed by genome-wide resequencing the genomic integrity of L. rhamnosus GG in four distinct evolutionary experiments conducted for approximately 1,000 generations under conditions of no stress or salt, bile, and repetitive-shearing stress. Under both stress-free and salt-induced stress conditions, the GG population (excluding the mutator lineage in the stress-free series [see below]) accumulated only a few single nucleotide polymorphisms (SNPs) and no frequent chromosomal rearrangements. In contrast, in the presence of bile salts or repetitive shearing stress, some IS elements were found to be activated, resulting in the deletion of large chromosomal segments that include the spaCBA-srtC1 pilus gene cluster. Remarkably, a high number of SNPs were found in three strains obtained after 900 generations of stress-free growth. Detailed analysis showed that these three strains derived from a founder mutant with an altered DNA polymerase subunit that resulted in a mutator phenotype. The present work confirms the stability of the pilus production phenotype in L. rhamnosus GG under stress-free conditions, highlights the possible evolutionary scenarios that may occur when this probiotic strain is extensively cultured, and identifies external factors that affect the chromosomal integrity of GG. The results provide mechanistic insights into the stability of GG in regard to its extensive use in probiotic and other functional food products. IMPORTANCELactobacillus rhamnosus GG is a widely marketed probiotic strain that has been used in numerous clinical studies to assess its health-promoting properties. Hence, the stability of the probiotic functions of L. rhamnosus GG is of importance, and here we studied the impact of external stresses on the genomic integrity of L. rhamnosus GG. We studied three different stresses that are relevant for understanding its robustness and integrity under both ex vivo conditions, i.e., industrial manufacturing conditions, and in vivo conditions, i.e., intestinal tract-associated stress. Overall, our findings contribute to predicting the genomic stability of L. rhamnosus GG and its ecological performance. Lactobacillus rhamnosus GG is a human intestinal isolate (1) that has been extensively studied over the last 2 decades for its probiotic properties and impact on human health (2, 3). In numerous clinical trials, L. rhamnosus was shown to display beneficial properties in humans, such as the reduction of diarrhea (4-7), atopic eczema (2), and respiratory infections (8, 9). Its implementation in a large variety of applications, food products, and clinical trials was possible, thanks to the remarkably high adaptability and resilience of L. rhamnosus GG to adverse culturing, handling, or stora...

The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement

“…If selective forces govern genomic architecture, then some rearrangements likely carry fitness effects, including possible benefits. Rearrangement-mediated adaptation has been observed during laboratory evolution experiments, including recombination between IS elements (44,45). Perhaps recently observed population sweeps have actually been driven by selection in favor of genomic structure, not nucleotide sequence.…”

Mutator genomes decay, despite sustained fitness gains, in a long-term experiment with bacteria

“…genes that encode the proteins involved in DNA replication, maintenance, and repair (6). Bacterial populations in laboratory evolution experiments often evolve hypermutable phenotypes (20)(21)(22). Hypermutators are also frequently observed among clinical isolates (23,24), where they are considered a risk factor for drug therapy failure (25,26).…”

Lactobacillus spp. belonging to the Casei group display a variety of adhesins

“…Isolates L. paracasei HI1 and L. casei HI7 show slight adhesion to the intestinal mucus, despite expression of the SpaCBA complex, which may have been caused by changes in the sequence of the SpaC subunit responsible for mucus binding. Instability of the coding complex subunit sequence was shown in a previous study (Markowicz et al, 2014) and it has also been reported by other researchers (Douillard et al, 2016;Sybesma et al, 2013). The lack of mucus binding may also be caused by factors hindering access of fimbrae formed by the SpaCBA complex to the mucus and the epithelium, such as exopolysaccharides produced by certain Lactobacillus strains (Dertli et al, 2015).…”