Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock

Abstract

In mammals, multiple physiological, metabolic, and behavioral processes are subject to circadian rhythms, adapting to changing light in the environment. Here we analyzed circadian rhythms in the fecal microbiota of mice using deep sequencing, and found that the absolute amount of fecal bacteria and the abundance of Bacteroidetes exhibited circadian rhythmicity, which was more pronounced in female mice. Disruption of the host circadian clock by deletion of Bmal1, a gene encoding a core molecular clock component, abolished rhythmicity in the fecal microbiota composition in both genders. Bmal1 deletion also induced alterations in bacterial abundances in feces, with differential effects based on sex. Thus, although host behavior, such as time of feeding, is of recognized importance, here we show that sex interacts with the host circadian clock, and they collectively shape the circadian rhythmicity and composition of the fecal microbiota in mice.

Keywords: Bmal1 gene; circadian rhythm; gender differences; microbiome.

Fig. 1.

Diurnal oscillation of intestinal microbiota composition in C57BL/6 mice. (A and B) Fecal bacterial load oscillates diurnally in mice both when combined (A) and separated by sex (B), as indicated by 16S rRNA copy numbers normalized to sample weight. JTK_ CYCLE revealed P = 0.00012 for mice of combined genders, P = 0.032 for male mice, and P = 2.8E-06 for female mice. (C) Bar graphs of average relative abundances of bacterial phyla in fecal microbial communities of combined genders at different time-of-day. Bacteroidetes (red) and Firmicutes (green) proportions are labeled. (D–I) The relative abundance (left y axis) and inferred absolute abundance (right y axis) of Bacteroidetes of combined genders (D, p_r = 0.00012 and p_a = 1.7E-06), male and female sex (E, p_r = 0.025 and p_a = 0.0029 for male mice; p_r = 0.0006 and p_a = 9.5E-10 for female mice); Firmicutes of combined genders (F, p_r = 6.3E-05 and p_a = 1), male and female sex (G, p_r= 0.049 and p_a = 1 for male mice; p_r = 0.0011 and p_a = 1 for female mice); and Proteobacteria of combined genders (H, p_r = 1 and p_a = 0.00065), male and female sex (I, p_r= 0.69 and p_a= 0.0047 for male mice; p_r = 1 and p_a = 0.13 for female mice). N_male = 7, N_female = 7. M, male; F, female. r, relative abundance; a, inferred absolute abundance. The dark phase is indicated by gray shading.

Fig. S1.

Diurnal oscillation of genus abundances in C57BL/6 mice. The relative abundance (black, left y axis) and inferred absolute abundance (red, right y axis) at genus level. Bonferroni-adjusted P values from JTK_CYCLE are indicated on each figure (black, P value for relative abundance; red, P value for inferred absolute abundance). S24-7 spp., Clostridiales spp., and Turicibacter exhibited significant diurnal oscillation in both relative and inferred absolute abundance. Sutterella and Clostridiaceae spp. exhibit significant diurnal oscillation in inferred absolute abundance, but not in relative abundance. Ruminococcaceae spp., Clostridia spp., Anaeroplasma, Oscillospira, and Lachnospiraceae spp. exhibit significant diurnal oscillation in relative abundance, but not in inferred absolute abundance. n = 14. Mean ± SEM shown.

Fig. 2.

Deletion of Bmal1 abolishes the diurnal oscillation of intestinal microbiota composition in mice. (A) Fecal bacterial load in Bmal1 KO mice (red) was slightly lower than that in WT mice (blue) but showed a similar fluctuating pattern during the light–dark cycle. (B) Bar graphs of average relative abundances of bacterial phyla in fecal microbial communities of WT (Left) and Bmal1 KO (Right) mice at the indicated times of day. Bacteroidetes (red) and Firmicutes (green) proportions are labeled. (C–E) The relative abundance (left y axis) and inferred absolute abundance (right y axis) of Bacteroidetes (C) of WT (p_r = 0.0075 and p_a = 0.39) and Bmal1 KO (p_r = 1 and p_a = 1) mice; Firmicutes (D) of WT (p_r = 0.017 and p_a = 0.093) and Bmal1 KO (p_r = 1 and p_a = 1) mice; and Proteobacteria (E) of WT (p_r = 0.52 and p_a = 1) and Bmal1 KO (p_r = 1 and p_a = 1) mice. N_WT = 5, N_{Bmal1 KO} = 6. r, relative abundance; a, inferred absolute abundance. The dark phase is indicated by gray shading.

Fig. S2.

Effect of Bmal1 deletion on the rhythmicity of bacterial genera in mice. The relative abundance (left y axis) and inferred absolute abundance right y axis) in WT (blue) and Bmal1 KO mice (red) at genus level. Bonferroni-adjusted P values from JTK_CYCLE are indicated on each figure (blue, P value for WT mice; red, P value for Bmal1 KO mice). Bacteroides and Lacotobacillaceae spp. oscillate significantly in WT mice in both relative and inferred absolute abundance, but not in Bmal1 KO mice. S24-7 spp. and Clostridia spp. relative abundances oscillate in WT mice but not in Bmal1 KO mice; whereas its absolute abundance didn’t oscillate. N_WT = 5, N_{Bmal1 KO} = 6. Mean ± SEM shown.

Fig. 3.

Bmal1 deletion alters bacterial abundances in fecal microbiota in mice. Fecal microbiota compositions from WT and Bmal1 KO mice at all time points across the day were compared. (A) Fecal microbiota composition from individual WT mice (blue) and Bmal1 KO mice (red) were clustered separately according to Principal Coordinate Analysis of Unweighted UniFrac (Left) and Weighted UniFrac (Right) distances. The percentages of variation explained by principal coordinates PC1 and PC2 are indicated on the x and y axes. (B) The relative abundance of Proteobacteria was decreased and that of TM7 was increased in Bmal1 KO mice, whereas those of Bacteroidetes and Firmicutes were not altered. (C) The inferred absolute abundance of Baceroidetes, Firmicutes, and Proteobacteria were decreased in Bmal1 KO mice, whereas that of TM7 was not altered. N_WT = 5, N_{Bmal1 KO} = 6. **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney test. All remained significant when adjusted to control for a false positive rate of 5%.

Fig. S3.

Bmal1 deletion alters relative abundances at genus level in fecal microbiota in mice. The relative abundances at genus level in WT mice (blue) and Bmal1 KO mice (red). The relative abundance of Bacteroidales spp., Rikenellaceae spp., F16 spp., Clostidiales spp., Clostridiaceae spp., Rikenella, Peptococaceae spp., and Ureaplasma were increased in Bmal1 KO mice. The relative abundance of S24-7 spp., Helicobacter, Allobaculum, Prevotella, Lactobacillaceae spp., and Sutterella were decreased in Bmal1 KO mice. N_WT = 5, N_{Bmal1 KO} = 6. Mean ± SEM shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney test. All remained significant when adjusted to control for a false positive rate of 5%.

Fig. S4.

Bmal1 deletion alters inferred absolute abundances at genus level in fecal microbiota in mice. The inferred absolute abundances at genus level in WT mice (blue) and Bmal1 KO mice (red). The inferred absolute abundance of Bacteroidales spp., Rikenellaceae spp., F16 spp., Rikenella, and Peptococaceae spp. were increased in Bmal1 KO mice. The inferred absolute abundance of S24-7 spp., Helicobacter, Allobaculum, Prevotella, Lactobacillaceae spp., and Sutterella were decreased in Bmal1 KO mice. N_WT = 5, N_{Bmal1 KO} = 6. Mean ± SEM shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney test. Ten out of 11 remained significant when adjusted to control for a false positive rate of 5%.

Fig. 4.

Intestinal microbiota compositions of male and female mice respond differently to Bmal1 deletion. Fecal microbiota profiles from individual mice at all time points across the day were compared based on sex and genotype. (A) Fecal microbiota composition of male WT mice (green), male Bmal1 KO mice (blue), female WT mice (orange), and female Bmal1 KO mice (red) were clustered according to Principal Coordinate Analysis of Unweighted UniFrac (left) and Weighted UniFrac (right) distances. Percentages of variation explained by principal coordinates PC1 and PC2 are indicated on the x and y axes. (B) The relative abundances at phylum level. (C) The inferred absolute abundances at phylum level. N_WT = 5, N_{Bmal1 KO} = 6. M, male; F, female. *P < 0.05, **P < 0.01, ****P < 0.0001 by Mann–Whitney test. Eleven out of 12 remained significant when adjusted to control for a false positive rate of 5%.

Fig. S5.

Bmal1 deletion differentially alters the relative abundances at genus level in female and male mice. The relative abundances at genus level in male WT mice (green), male Bmal1 KO mice (blue), female WT mice (orange), and female Bmal1 KO mice (red). The relative abundances of Bacteroidales spp., F16 spp., and Clostridiaceae spp. were increased in both male and female Bmal1 KO mice. The relative abundances of Helicobacter and Lactobacillaceae spp. were decreased in both male and female Bmal1 KO mice. The relative abundances of Prevotella and Turicibacter were decreased in male but increased in female Bmal1 KO mice. The relative abundances of Rikenellaceae spp., Bacteroidales(other), Clostridiales spp., and Rikenella were increased in male Bmal1 KO mice. The relative abundance of Allobaculum, S24-7 spp. and Sutterella were decreased in male Bmal1 KO mice. The relative abundance of Peptococaceae spp. and Ureaplasma were increased in female Bmal1 KO mice. N_WT = 5, N_{Bmal1 KO} = 6. Mean ± SEM shown. *P < 0.05, **P < 0.01, ****P < 0.0001 by Mann–Whitney test. Twenty two out of 23 remained significant when adjusted to control for a false positive rate of 5%.

Fig. S6.

Bmal1 deletion differentially alters the inferred absolute abundances at genus level in female and male mice. The inferred absolute abundances at genus level in male WT mice (green), male Bmal1 KO mice (blue), female WT mice (orange), and female Bmal1 KO mice (red). The inferred absolute abundances of Bacteroidales spp., and F16 spp. were increased in both male and female Bmal1 KO mice. The inferred absolute abundances of S24-7 spp., Helicobacter and Lactobacillaceae spp. were decreased in both male and female Bmal1 KO mice. The inferred absolute abundances of Prevotella and Turicibacter were decreased in male but increased in female Bmal1 KO mice. The inferred absolute abundances of Rikenellaceae spp. and Clostridiales spp. were increased in male but decreased in female Bmal1 KO mice. The inferred absolute abundances of Bacteroidales;Other, Clostridiaceae spp., and Rikenella were increased in male Bmal1 KO mice. The inferred absolute abundance of Allobaculum, and Sutterella were decreased in male Bmal1 KO mice. The inferred absolute abundance of Peptococaceae spp. and Ureaplasma were increased in female Bmal1 KO mice. N_WT = 5, N_{Bmal1 KO} = 6. Mean ± SEM shown. *P < 0.05, **P < 0.01, ****P < 0.0001 by Mann–Whitney test. Twenty four out of 25 remained significant when adjusted to control for a false positive rate of 5%.