Genome Biol Evol. 2014 Jun 19;6(7):1772-89. doi: 10.1093/gbe/evu137.
From prediction to function using evolutionary genomics: human-specific ecotypes of Lactobacillus reuteri have diverse probiotic functions.
Spinler JK1, Sontakke A2, Hollister EB2, Venable SF2, Oh PL3, Balderas MA4, Saulnier DM5, Mistretta TA2, Devaraj S2, Walter J6, Versalovic J7, Highlander SK8.
Texas Children's Microbiome Center, Department of Pathology, Texas Children's Hospital, Houston, TXDepartment of Pathology & Immunology, Baylor College of Medicine, Houston, TX spinler@bcm.edu.
Texas Children's Microbiome Center, Department of Pathology, Texas Children's Hospital, Houston, TXDepartment of Pathology & Immunology, Baylor College of Medicine, Houston, TX.
Department of Food Science and Technology, University of Nebraska, Lincoln.
Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX.
Texas Children's Microbiome Center, Department of Pathology, Texas Children's Hospital, Houston, TXDepartment of Pathology & Immunology, Baylor College of Medicine, Houston, TXPresent address: Department of Gastrointestinal Microbiology, German Institute of Human Nutrition, Nuthetal, Germany.
Department of Food Science and Technology, University of Nebraska, LincolnPresent address: Departments of Agricultural, Food, & Nutritional Science and Biological Sciences, University of Alberta, Edmonton, AB, Canada.
Texas Children's Microbiome Center, Department of Pathology, Texas Children's Hospital, Houston, TXDepartment of Pathology & Immunology, Baylor College of Medicine, Houston, TXDepartment of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX.
Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TXHuman Genome Sequencing Center, Baylor College of Medicine, Houston, TXPresent address: Genomic Medicine, J. Craig Venter Institute, La Jolla, CA.
The vertebrate gut symbiont Lactobacillus reuteri has diversified into separate clades reflecting host origin. Strains show evidence of host adaptation, but how host-microbe coevolution influences microbial-derived effects on hosts is poorly understood. Emphasizing human-derived strains of L. reuteri, we combined comparative genomic analyses with functional assays to examine variations in host interaction among genetically distinct ecotypes. Within clade II or VI, the genomes of human-derived L. reuteri strains are highly conserved in gene content and at the nucleotide level. Nevertheless, they share only 70-90% of total gene content, indicating differences in functional capacity. Human-associated lineages are distinguished by genes related to bacteriophages, vitamin biosynthesis, antimicrobial production, and immunomodulation. Differential production of reuterin, histamine, and folate by 23 clade II and VI strains was demonstrated. These strains also differed with respect to their ability to modulate human cytokine production (tumor necrosis factor, monocyte chemoattractant protein-1, interleukin [IL]-1β, IL-5, IL-7, IL-12, and IL-13) by myeloid cells. Microarray analysis of representative clade II and clade VI strains revealed global regulation of genes within the reuterin, vitamin B12, folate, and arginine catabolism gene clusters by the AraC family transcriptional regulator, PocR. Thus, human-derived L. reuteri clade II and VI strains are genetically distinct and their differences affect their functional repertoires and probiotic features. These findings highlight the biological impact of microbe:host coevolution and illustrate the functional significance of subspecies differences in the human microbiome. Consideration of host origin and functional differences at the subspecies level may have major impacts on probiotic strain selection and considerations of microbial ecology in mammalian species.
Phylogenetic analysis of Lactobacillus reuteri isolates derived from four hosts. The genealogy of 119 L. reuteri strains based on MLSA of sequences. Tree branches are color coded by host origin: Green = rodent, red = pig, blue = human, yellow = poultry. The 25 strains included in the functional analyses are indicated by closed black circles , and strains with sequenced genomes are indicated by a closed black circle with a yellow center . Reuterin production is designated as follows: high reuterin production, low reuterin production. Strains that produce high amounts of folate are indicated by closed green circles , and strains producing histamine are indicated by closed red circles .
Genome Biol Evol. 2014 Jul;6(7):1772-1789.
Genomic comparison of ten Lactobacillus reuteri genomes to clade II strain JCM 1112. Diagram represents BLASTn results of each genome against JCM 1112 with results rendered using the BRIG program (). Each genome is color coded as indicated by the legend. Relative shading density (from darker to lighter) within each circle represents relative levels of nucleotide homology. White regions indicate regions with no identity to the reference. Features of interest are annotated.
Ecological and genetic diversity within the Lactobacillus reuteri species. Conserved genes (y axis) versus average nucleotide evolutionary distance (x axis) plot for ten sequenced L. reuteri genomes isolated from three mammalian hosts. Each data point represents a whole-genome comparison between two genomes. (A) Pairwise comparisons between all ten genomes. Intraclade comparisons of human-derived strains are circled near the origin. (B) Pairwise comparisons of human-derived MLSA II and MLSA VI L. reuteri genomes.
The pdu–cbi–hem–cob gene cluster and clade-specific reuterin production. (A) CDS map of the 57 gene pdu–cbi–hem–cob cluster that encodes the PocR regulator (shown in red), genes pduCDE responsible for production of the antimicrobial reuterin, Pdu proteins required for 1,2-propanediol and glycerol utilization, and Cbi, Hem, and Cob proteins required for synthesis of vitamin B12, as per . Below the CDS map are heat maps representing percent amino acid identities of each protein within the pdu–cbi–hem–cob gene cluster of four MLSA II Lactobacillus reuteri genomes relative to the amino acid sequences of MLSA VI proteins from strains ATCC 55730 and CF48-3A. (B) Growth curves of strains ATCC 6475 (clade II) and DSM 17938 (clade VI) in MRS medium at 37 °C under anaerobic conditions (left); growth phase production of reuterin by strain ATCC 6475 (clade II) and DSM 17938 (clade VI) (right). (C) Reuterin production at stationary phase (12 h) from 25 human-derived L. reuteri strains. Strains with sequenced genomes are indicated in bold with an asterisk (*). Results are expressed as the mean ± SD, n = 3, and significant differences between group means as determined by one-way ANOVA (P < 0.05) are indicated by the placement of different letters above each bracketed group.
Relative production of histamine by human-derived Lactobacillus reuteri clades. (A) CDS map of the hdcP–hdcA–hdcB–hisRS2 gene cluster in clade II L. reuteri strains. All values are presented on a 0–1 scale, indicating a max/min normalization per analyte, and are displayed as heatmaps with red indicating values closer to the maximum (1) and green indicating values closer to minimum (0). (B) Histamine in conditioned media by 25 human-derived L. reuteri strains was measured by ELISA and compared with media control. Results of end-point PCR tests for the four histidine decarboxylase cluster genes are summarized: √ indicates correct PCR product observed; - indicates no PCR product observed.
Functional illustration of human-derived Lactobacillus reuteri ecotypes. Lactobacillus reuteri strains from both clades modulate acidic environments, produce the antimicrobial reuterin, synthesize essential vitamins, and generate immunomodulatory compounds that effect immune signaling in the host. The differences associated with each ecotype are illustrated here.