{'fraction_non_alphanumeric': 0.04778682016673283, 'fraction_numerical': 0.05329495831679238, 'mean_word_length': 4.414562063406771, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 2, 'https://': 2, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Syntrophy via interspecies H2 transfer between Christensenella and Methanobrevibacter underlies their global co-occurrence in the human gut

Across human populations, 16S rRNA gene-based surveys of gut microbiomes have revealed that the bacterial family Christensenellaceae and the archaeal family Methanobacteriaceae co-occur and are enriched in individuals with a lean, compared to an obese, BMI. Whether these association patterns reflect interactions between metabolic partners remains to be ascertained, as well as whether these associations play a role in the lean host phenotype with which they associate. Here, we validated previously reported co-occurrence patterns of the two families, and their association with a lean BMI, with a meta-analysis of 1,821 metagenomes derived from 10 independent studies. Furthermore, we report positive associations at the genus and species level between Christensenella spp. and Methanobrevibacter smithii, the most abundant methanogen of the human gut. By co-culturing three Christensenella spp. With M. smithii, we show that Christensenella spp. efficiently support the of M. smithii via H2 production, far better than Bacteroides thetaiotaomicron. C. minuta forms flocs colonized by M. smithii even when H2 is in excess. In culture with C. minuta, H2 consumption by M. smithii shifts the metabolic output of C. minuta’s fermentation towards acetate rather than butyrate. Together, these results indicate that the widespread co-occurrence of these microbiota is underpinned by both physical and metabolic interactions. Their combined metabolic activity may provide insights into their association with a lean host BMI. Importance The human gut microbiome is made of trillions of microbial cells, most of which are Bacteria, with a subset of Archaea. The bacterial family Christensenellaceae and the archaeal family Methanobacteriaceae are widespread in human guts. They correlate with each other and with a lean body type. Whether species of these two families interact, and how they affect the body type, are unanswered questions. Here, we showed that species within these families correlate with each other across people. We also demonstrated that particular species of these two families grow together in dense flocs, wherein the bacteria provide hydrogen gas to the archaea, which then make methane. When the archaea are present, the ratio of bacterial products (which are nutrients for humans) is changed. These observations indicate when these species grow together, their products have the potential to affect the physiology of their human host.


Introduction 59
Obesity was the first human disease phenotype to be associated with an altered 60

Results 115
Christensenella relative abundance is significantly correlated with leanness 116 across populations -Both the Christensenellaceae family and the genus 117 Christensenella had a very high prevalence, as they were present in more than 99% of 118 the 1,821 samples; both the family and the genus have a mean abundance of 0.07% ± 119 0.05 (Fig. 1b,d and Fig. S1). To correct for the influence of environmental factors on the 120 relative abundance of the Christensenellaceae family and of the Christensenella genus, 121 we first constructed null models in which we selected covariates (Appendix 1) that 122 explained a significant proportion of the variance of the transformed relative abundance 123 of the family Christensenellaceae, and in the same manner of the Christensenella 124 genus. BMI and age were significantly correlated to the transformed relative 125 abundances of Christensenellaceae and of Christensenella (designated with the suffix '-126 tra', i.e., Cf-tra and Cg-tra), and were retained in the null models (Cf-null and Cg-null). 127 BMI was negatively correlated to both Cf-tra (type II ANOVA, p-value = 0.0002 128 and F-value(339) = 14.46) and Cg-tra (type II ANOVA, p-value = 0.0002 and F-129 value(339) = 14.29), indicating that leaner individuals harbor higher relative abundances 130 of Christensenellaceae and Christensenella. Age was negatively correlated with Cf-tra 131 (type II ANOVA, p-value = 0.01 and F-value(1,468) = 6.56) and with Cg-tra (type II 132 ANOVA, p-value = 0.01 and F-value(1,468) = 6.53), indicating that younger subjects 133 carry a greater relative abundance of Christensellaceae and Christensenella. However, 134 the interaction term between BMI and age was not significantly correlated to the 135 transformed relative abundances (type II ANOVA, p-values > 0.1), indicating that their 136 effects are additive. These results show that regardless of their BMI, younger subjects 137 have higher levels of Christensenellaceae and Christensenella, and that the lower a 138 subject's BMI, the more of these microbes they harbor, regardless of their age. Methanobrevibacter genus also had a high prevalence with 92% and 89% of the people 143 harboring them respectively, with mean abundances of 0.48% ± 1.55 and 0.49% ± 1.54, 144 respectively. As above, we evaluated the association between the 145 Methanobacteriaceae family, and of the Methanobrevibacter genus, with BMI and age 146 was included in addition to BMI and age (model Cf-Mf). This allowed us to test whether 163 adding Mf-tra to the model improved its fit and if so, how much of the variance of Cf-tra 164 not explained by age and BMI could be explained by Mf-tra. We also evaluated the 165 interaction terms between Mf-tra and BMI, and between Mf-tra and age, to assess 166 whether the correlation between Cf-tra and Mf-tra was dependent on age and BMI. The 167 interaction term for BMI and Mf-tra was not significant and was removed from the 168 model; the interaction term for age and Mf-tra was significant and was retained (type I 169 ANOVA, F-value(339) = 8.30 and p-value = 0.0042). We compared the log-likelihoods 170 of the null and full models (Cf-null and Cf-Mf) to confirm that the relative abundances of the Methanobacteriaceae and Christensenellaceae families were significantly correlated 172 (χ 2 test, p-value = 1.78x10 -59 ). Furthermore, the model Cf-Mf showed that Mf-tra was 173 significantly positively correlated to Cf-tra (Fig. 1b; type I ANOVA, F-value(339) =  174 287.03, p-value < 0.0001) and that the interaction term between Mf-tra and age was 175 positively correlated to Cf-tra as well. These results indicate that the relative 176 abundances of the Christensenellaceae and Methanobacteriaceae families are 177 positively correlated across multiple populations/studies. In addition, although both 178 families are enriched in low-BMI people, they are correlated regardless of a subject's 179 BMI. Moreover, their association is stronger in older people, suggesting that although 180 elders are less likely to carry as much Christensenellaceae as youths, the more 181 Methanobacteriaceae they have, the more Christensenellaceae they have. 182 We performed a similar analysis using the abundances of the two most 183 prominent genera belonging to these families (Christensenella and Methanobrevibacter, 184 models Cg-null and Cg-Mg) and obtained equivalent results. First, the interaction term 185 between Mg-tra and age was positively correlated with Cg-tra (Fig. 1c; type I ANOVA, 186 F-value(339) = 10.19, p-value = 0.0015). Then, by comparing model Cg-null with the full 187 model Cg-Mg, we showed that the relative abundances of the two genera were also 188 correlated (χ 2 test, p-value = 1.50x10 -57 ). Our full model Cg-Mg showed that Mg-tra was 189 significant for predicting Cg-tra while controlling for BMI and age (type I ANOVA, F-190 value(339) = 274.35, p-value < 0.0001), with the interaction term between Mg-tra and 191 age also positively correlated to Cg-tra. These results indicate that the correlations 192 between the relative abundances of the two families, explained above, hold true at the 193 level of two representative genera. The association between Methanobrevibacter and 194 Christensenella is stronger in older people regardless of BMI. 195 A similar analysis at the species level indicated that C. minuta and M. smithii 196 were the most abundant species of each of their genera, and similarly to the family and 197 genus ranks, their relative abundances across samples were significantly correlated 198 (Fig. 1d and Appendix 2). The less abundant Christensenella gut species, C. 199 massiliensis and C. timonensis, also correlated with Methanobrevibacter smithii across 200 the 1,821 metagenomes (Appendix 2). C. minuta and C. timonensis transformed relative 201 abundances were significantly negatively correlated to both BMI and age, while C. does not aggregate in mono-culture before 7-10 days of culture (data not shown). In 215 that it supports the growth of M. smithii via H 2 production (25, 26), did not flocculate 217 when grown alone, (Fig. 2c) and, when co-cultured with M. smithii, displayed very 218 limited aggregation (Fig. 2e, Fig. 3k-n and Fig. S2). 219 H 2 and CH 4 production -After 6 days in mono-culture, C. minuta had produced 221 7 times more H 2 than B. thetaiotaomicron (14.2 ± 1.6 mmol.L −1 vs. 2.0 ± 0.0 mmol.L −1 , 222 Pressure effects on gas production and aggregation -Gas consuming 237 microbes, including hydrogenotrophic methanogens, grow better in a pressurized 238 environment (32-34) due to a higher gas solubility at higher pressure, as described by 239 Henry's law. We compared CH 4 production by M. smithii in mono-culture and in co-240 culture with C. minuta under 2 different pressures (i.e., 2 bar and atmospheric 241 pressure). Similar to the flocculation at 2 bar (Fig. 2d), C. minuta and M. smithii also 242 aggregated at atmospheric pressure ( Fig. S3a-b). Accordingly, C. minuta supported Along with the reduced butyrate production, we also observed slightly but 277 significantly higher acetate production in co-cultures compared to mono-cultures (Fig. 278 6d-f, Fig. 5d; ANOVA, F-value(1) = 317.41 and adjusted p-value = 3.2x10 − 9 ). This 279 difference was also observed in three additional batches performed at 2 bars (Fig. S4).
The difference in acetate production between mono and co-culture conditions 281 significantly varied with the headspace and pressure conditions (interaction term 282 between the mono-or co-culture and the growth condition was significantly correlated to 283 acetate production; ANOVA, F-value(2) = 29.09 and adjusted p-value = 3.0x10 − 5 ). The 284 differences in final acetate production (after 6 days) ranged from +0.7 mmol.L The H 2 produced by the bacteria in mono-culture after 6 days of growth (6.9 ± 0.5 297 mmol.L −1 for C. massiliensis and 0.6 ± 0.1 mmol.L −1 for C. timonensis, Fig. 8a,d) was 298 lower than the levels produced by C. minuta (Fig. 4a). CH 4 production in the co-cultures 299 reached 4.0 ± 0.2 mmol.L −1 with C. massiliensis and 1.5 ± 0.3 mmol.L −1 with C. 300 for M. smithii with C. minuta (6.6 ± 0.8 mmol.L −1 ; ANOVA followed by a Tukey's post-302 hoc test, adjusted p-values = 6.8x10 -2 and 1.7x10 −3 for co-cultures respectively with C. 303 massiliensis and C. timonensis against C. minuta; Fig. 8c,e, and Fig. 4c). 304 We observed less butyrate production in the co-cultures compared to mono-305 cultures (Wilcoxon rank sum test, p-values = 0.33 for C. massiliensis and 0.5 for C. 306 timonensis; Fig. 8f,g), with butyrate measured barely above the detection limit in co-307 cultures. While in mono-cultures, C. massiliensis and C. timonensis produced 0.93 ± 308 0.06 mmol.L -1 and 1.10 ± 0.00 mmol.L −1 of butyrate, respectively; in co-culture with M. 309 smithii they produced 0.20 ± 0.14 mmol.L −1 and 0.13 ± 0.15 mmol.L −1 , respectively. 310 Acetate production by C. massiliensis was higher in co-culture compared to mono-311 culture (7.83 ± 0.49 mmol.L − 1 of acetate produced in mono-culture by day 6 and 9.75 ± 312 0.78 mmol.L − 1 produced in co-culture with M. smithii), although this difference was not 313 significant (Wilcoxon rank sum test, p-value = 0.2). And in contrast with the co-cultures 314 of C. minuta with M. smithii, acetate production by C. timonensis was not higher in the 315 co-cultures compared to mono-cultures: C. timonensis produced 5.05 ± 0.21 mmol.L −1 316 in mono-culture and 4.33 ± 1.21 mmol.L −1 in co-culture (Wilcoxon rank sum test, p-value 317 = 0.8; Fig. 8h,i). 318 Discussion 320 The link between the relative abundance of the Christensenellaceae and host 321 BMI now stands as one of the most reproducible associations described between the 322 gut microbiome and obesity (4-15). Here, we confirm in a meta-analysis of 323 metagenomes across 10 populations, the previously observed association between 324 leanness and the Christensenellaceae family (4, 20-22). We could also show that 325 provided. For the latter, we restricted our analyses to individuals for which the following 423 information was available: gender, age, country of origin, and BMI. Individuals with 424 Schistosoma (n = 4), or Wilson's disease (n = 2), were excluded from the analysis, as 425 were samples from two pregnant women. In all, 1,534 samples from 9 studies were 426 downloaded from the sequence read archive (SRA) and further processed (Table S3)  Christensenellaceae is Cf-tra). Covariates in null models were selected using a 448 backward feature selection approach based on a type II ANOVA (i.e., by including all 449 covariates and removing the non-significant ones step-by-step until all remaining 450 variables were significant, Appendix 2). We made 4 null models predicting the 451 transformed relative abundance of the family Christensenellaceae (Cf-null), the genus 452 Christensenella (Cg-null), the family Methanobacteriaceae (Mf-null) and the genus 453 Methanobrevibacter (Mg-null). To evaluate the correlation between taxa, we made 454 Reciprocally, we made model Cg-Mg by adding Mg-tra and its interaction with age to 456 the covariates of Cg-null. The same approach was performed at the species level and it 457 is described in Appendix 2. 458 We used the likelihood ratio test to compare the nested models via the χ 2 467 distribution (i.e. Cf-null vs. Cf-Mf and Cg-null vs. Cg-Mg). To characterize the correlation 468 of Cf-tra with Mf-tra, and Cg-tra with Mg-tra, after correcting for BMI and age, we used a 469 type I ANOVA to evaluate the importance of the variables in the order they appear in Cf-470 Mf and Cg-Mg. The F-value, degree of freedom and p-value are reported for each 471 variable. All analyses were performed using R (60). 472  (Table  493   S1). Each batch of experiments was carried out once with 3 biological replicates per 494 culture conditions (Table S1). Statistical analyses -We used Wilcoxon rank sum tests to compare gas 524 production between cultures after 6 days of growth. We performed ANOVA tests when 525 more than one culture condition (i.e., headspace composition and pressure, Table S1) 526 was included in the comparison. The conditions in the ANOVA tests (i.e., headspace 527 composition and pressure, in mono-or co-culture) were evaluated to explain the 528 variance of CH 4 production after 6 days of growth. A Tukey's post-hoc test was then 529 performed to discriminate between the effects of the different conditions. SCFA 530 concentrations were compared using a two-way ANOVA where the culture conditions 531 (i.e., headspace composition and pressure, Table S1) and the sample (mono-and co-532 culture) were evaluated to explain the variance of butyrate and acetate concentrations 533 after 6 days of growth. p-values were adjusted using the Benjamini-Hochberg method. A 534

Culturing of methanogens and bacteria -
Tukey's post-hoc test was performed to discriminate between the effects of the different 535 conditions. All statistical analyses were done in R using the stats R package. and de-duplicated with the "clumpify" command of bbtools v37.78 559 (https://jgi.doe.gov/data-and-tools/bbtools/). We trimmed adapters and performed read 560 quality control using skewer v0.2.2 (65) and the "bbduk" command of bbtools. We used 561 the "bbmap" command of bbtools to filter human genome reads that mapped to the 562 hg19 assembly. Finally, we generated QC reports for all reads with fastqc v0.11.7 563 (https://github.com/s-andrews/FastQC) and multiQC v1.5a (66). 564

Confocal imaging, equipment, and settings -For confocal microscopy, SYBR ®️ 565
Green I staining was performed as previously described (61) with the following 566 modifications: 0.5 mL of culture were sampled and pelleted by centrifugation for 6 min at injection and detector temperatures were 250 and 275 ˚C, respectively. Samples were prepared as for HPLC (Methods in the main text) with the addition of an internal 614 standard (Ethyl-butyric acid) and acidification (to pH 2) with 50% formic acid. Data were 615 acquired and analysed with the Agilent OpenLAB CDS software. 616 Only acetate and butyrate were detected in the mono-and co-cultures, and none of 617 the other short and medium chain fatty acids used as standards were detected. As 618 formate was used to acidify samples for the GC measurements, to assess if it was a 619 main product in the cultures, its concentration was measured by HPLC. We also looked 620 for ethanol using HPLC but similar to formate, it was not detected in any of the Variable selection for the null model -To construct the null model, we tested the 628 effect of the following covariates with a marginal ANOVA: sequencing depth, gender, 629 country, BMI, and age. The sequencing depth was not significant (p-value = 0.73) and 630 was subsequently removed. BMI and age were correlated with the Chistensenellaceae 631 abundance (p-value = 0.0002 for the correlation with BMI and 0.02 for the correlation 632 with age). 633 As Methanobrevibacter smithii has been associated with age (23) and BMI (4, 43, The relative abundance of Christensenella massiliensis correlated with BMI (type I 670 ANOVA, p-value = 0.0028 and F-value = 9.0804) but not with age (p-value > 0.5) and 671 so, we did not correct for age in the null model nor in the model including 672 Methanobrevibacter smithii's transformed relative abundance. C. massiliensis and M. 673 smithii were also correlated (χ 2 test, p-value = 1.31x10 -61 ; type I ANOVA, p-value < 674 0.0001, F-value = 310.51), but the interaction term between M. smithii and age was not 675 significantly correlated to the bacterium's abundance. C. massiliensis is thus the only 676 Christensenella spp. for which the correlation with the methanogen abundance is not a 677 function of the age of the carrier. 678 We used the stoichiometry of hydrogenotrophic methanogenesis (CO 2 + 4 H 2 = CH 4 682 + 2 H 2 O) to calculate the amount of CH 4 that could be produced from the estimated 683 amount of H 2 consumed in each sample. For this, we used the mono-cultures of 684 bacteria as references and assumed equal H 2 production in co-culture as in mono-685 culture. We estimated the H 2 consumed after 6 days for each replicate as the difference 686 between the averaged H 2 concentrations in mono-cultures and the concentration 687 measured in co-culture (i.e., unconsumed H 2 ). The estimated H 2 consumed was then 688 divided by 4 in order to obtain the theoretical amount of CH 4 that could be produced via 689 hydrogenotrophic methanogenesis. 690 691   Table A3. Analysis of the origin of the high methane produced in co-culture 692 based on the changes in metabolism of C. minuta. CH 4 produced in co-culture was 693 higher than the theoretical amount of CH 4 that could be generated from H 2 assuming 694 that C. minuta produced the same amount of H 2 in both mono-and co-cultures. The 695 additional CH 4 observed could originate from the shift in metabolism from butyrate to 696 acetate production along with H 2 by C. minuta in co-culture. The average concentration 697 among the triplicates after 6 days of growth is given with the standard deviation (SD    Table S1). Points represent the average of the 3 biological replicates   Table S1); (f-g) butyrate 989 and ( Tables  1027   Table S1. Total pressure, headspace composition, and culture inocula for 1028 each batch of experiments described in the main text. 1029