Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling

Abstract

Lactobacillus rhamnosus is a bacterial species commonly colonizing the gastrointestinal (GI) tract of humans and also frequently used in food products. While some strains have been studied extensively, physiological variability among isolates of the species found in healthy humans or their diet is largely unexplored. The aim of this study was to characterize the diversity of carbohydrate utilization capabilities of human isolates and food-derived strains of L. rhamnosus in relation to their niche of isolation and genotype. We investigated the genotypic and phenotypic diversity of 25 out of 65 L. rhamnosus strains from various niches, mainly human feces and fermented dairy products. Genetic fingerprinting of the strains by amplified fragment length polymorphism (AFLP) identified 11 distinct subgroups at 70% similarity and suggested niche enrichment within particular genetic clades. High-resolution carbohydrate utilization profiling (OmniLog) identified 14 carbon sources that could be used by all of the strains tested for growth, while the utilization of 58 carbon sources differed significantly between strains, enabling the stratification of L. rhamnosus strains into three metabolic clusters that partially correlate with the genotypic clades but appear uncorrelated with the strain's origin of isolation. Draft genome sequences of 8 strains were generated and employed in a gene-trait matching (GTM) analysis together with the publicly available genomes of L. rhamnosus GG (ATCC 53103) and HN001 for several carbohydrates that were distinct for the different metabolic clusters: l-rhamnose, cellobiose, l-sorbose, and α-methyl-d-glucoside. From the analysis, candidate genes were identified that correlate with l-sorbose and α-methyl-d-glucoside utilization, and the proposed function of these genes could be confirmed by heterologous expression in a strain lacking the genes. This study expands our insight into the phenotypic and genotypic diversity of the species L. rhamnosus and explores the relationships between specific carbohydrate utilization capacities and genotype and/or niche adaptation of this species.

FIG 1

Dendrogram based on the analysis of AFLP patterns of the primer combination E01-T13 with visualization of the banding patterns. The strain origin and clustering (at a similarity level of 70%) are represented in the columns. The 25 strains selected for OmniLog growth experiments are highlighted with boxes.

FIG 2

Heat map and clustering of L. rhamnosus strains and their substrates based on growth endpoint data (Phenotype MicroArray Biolog data) for carbohydrates in clusters 1 and 2. The shading of the heat map refers to the growth level: black, >100 OU; dark gray, 100 to 20 OU; light gray, <20 OU. The two-way clustering of the strains and substrates was created in PAST using the Euclidian distance matrix based on UPGMA clustering. Origins of isolates and AFLP grouping of the strains are displayed below the strain's dendrogram. The origin abbreviations are as follows: A, adult feces; B, baby feces; G, goat feces; D, dairy; S, saliva; Va, vagina; Ve, vegetable; U, unknown. The type of carbohydrate is displayed to the left of the heat map: monosaccharide (M), disaccharide (D), trisaccharide (T), glycoside (G), fatty acid (FA), nucleoside (N), sugar alcohol (SA), carboxylic acid (C), amino acids (AA), and amide (AD). The bottom rows display the numbers of carbohydrates above 20 OU and above 100 OU for each L. rhamnosus strain, and the highest and lowest values are marked with an asterisk.

FIG 3

Growth of the wild-type GG strain (dark gray) and expression strains M12 (transformed with pSOR253 containing the l-sorbose operon) (white) and M17 (transformed with pAMG253 containing the α-methyl-d-glucoside operon) (light gray) on l-sorbose and α-methyl-d-glucoside in batch cultures. The numbers represent OD values per milliliter for the cultures in sugar-free MRS medium supplemented with either l-sorbose or α-methyl-d-glucoside. ***, a P value of <0.001 using Student's t test.