Taurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and genomic-ecophysiological characterization of the first taurine-respiring mouse gut bacterium. Taurinivorans muris represents a new widespread species with protective capacity against pathogens and differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism and host distribution. Despite alternative physiologies, taurine respiration was the main in vivo lifestyle of T. muris independent of mouse diet and genotype. In gnotobiotic mice, T. muris selectively enhanced the activity of a sulfur metabolism gene-encoding prophage and provided slightly increased colonization resistance against Salmonella Typhimurium, which showed reduced expression of galactonate catabolism genes. We identified T. muris as the dominant sulfidogen of a mouse microbiota that conferred H2S-mediated protection against Klebsiella pneumoniae in a previous study. Together, we revealed the realized physiological niche of a key murine gut sulfidogen and its impact on pathogen and phage gene expression.

Our work identified and characterized a new core member of the murine gut microbiota, revealed sulfidogenic taurine respiration as its predominant in vivo lifestyle, and emphasizes its protective function in pathogen colonization. metabolic gene, virus intestinal microbiome, synthetic microbiota, sulfur metabolism, taurine, Salmonella, Klebsiella, auxiliary 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

Hydrogen sulfide (H2S) is an intestinal metabolite with pleiotropic effects, particularly on the gut mucosa 1,2. H2S can have a detrimental impact on the intestinal epithelium by chemically disrupting the mucus barrier 3, causing DNA damage 4, and impairing energy generation in colonocytes through inhibition of cytochrome c oxidase and beta-oxidation of short-chain fatty acids 5,6. In contrast, low micromolar concentrations of H2S are anti-inflammatory and contribute to mucosal homeostasis and repair 7,8. Furthermore, H2S acts as a gaseous transmitter, a mitochondrial energy source, and an antioxidant in cellular redox processes, and thus its impact on mammalian physiology and health reaches beyond the gastrointestinal tract 9. For example, colonic luminal H2S can promote somatic pain in mice 10 and contribute to regulating blood pressure 11,12. The multiple (patho)physiological functions of H2S in various organs and tissues are dependent on its concentration and the health status of the host, but possibly also on the source of H2S. Mammalian cells can produce H2S from cysteine via several known pathways 13. In contrast to these endogenous sources, H2S-releasing drugs and H2S-producing intestinal microorganisms are considered exogenous sources. Compared to the colonic epithelium, sulfidogenic bacteria, which either metabolize organic sulfur compounds (e.g. cysteine) or anaerobically respire organic (e.g. taurine) or inorganic (e.g. sulfate, sulfite, tetrathionate) sulfur compounds in the gut, have a higher H2S-producing capacity and are thus potentially harmful to their hosts 1,2,14. Indeed, the abundance and activity of sulfidogenic gut bacteria were associated with intestinal diseases such as inflammatory bowel disease and colon cancer in various studies 15317 and many gut pathogens, such as Salmonella enterica and Chlostridioides difficile, are also sulfidogenic 18,19. Excess bacterial H2S production combined with a reduced capacity of the inflamed mucosa to metabolize H2S is one of many mechanisms by which the gut microbiome can contribute to disease 20. Yet, the manifold endogenous and microbial factors and processes that regulate intestinal H2S homeostasis are insufficiently understood. 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92

A major substrate of sulfidogenic bacteria in the gut is the organosulfonate taurine (2aminoethanesulfonate) that derives directly from meat- or seafood-rich diets and is also liberated by microbial bile salt hydrolases (BSHs) from endogenously produced taurocholic bile acids 21. Bilophila wadsworthia is the most prominent taurine-utilizing bacterium in the human gut. Diets that contain high quantities of meat, dairy products or fats can be associated with the outgrowth of B. wadsworthia in the gut 22,23. Consumption of high-fat food triggers taurocholic bile acid production and increases the taurine: glycine ratio in the bile acid pool 23. In mouse models, higher abundances of B. wadsworthia can promote colitis and systemic inflammation 23,24 and aggravate metabolic dysfunctions 25.

B. wadsworthia metabolizes taurine via the two intermediates sulfoacetaldehyde and isethionate (2hydroxyethanesulfonate) to sulfite 26 and the sulfite is utilized as electron acceptor for energy , conservation and reduced to H2S via the DsrAB-DsrC dissimilatory sulfite reductase system 27. The highly oxygen-sensitive isethionate sulfite-lyase system IslAB catalyzes the abstraction of sulfite (desulfonation) of isethionate 26,28. Alternative taurine degradation pathways in other bacteria involve direct desulfonation of sulfoacetaldehyde by the oxygen-insensitive, thiamine-diphosphate-dependent sulfoacetaldehyde acetyltransferase Xsc 29331. Xsc is employed in taurine utilization as carbon and energy source in a wide range of aerobic bacteria 30,32, as well as for anaerobic taurine fermentation by Desulfonispora thiosulfatigenes 31.

Here, we isolated the first taurine-respiring and H2S-producing bacterium from the murine intestinal tract and elucidated its fundamental and in vivo realized nutrient niche. Strain LT0009 represents a new Desulfovibrionaceae genus, termed Taurinivorans muris gen. nov., sp. nov., and differs from its human counterpart B. wadsworthia by using the Xsc pathway for taurine degradation and its distribution across 93 94 different animal hosts. We further provide insights into the protective role of this newly identified species of the core mouse microbiome against pathogen colonization. 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

Intestinal content of wild-type C57BL/6 mice was used as inoculum for the enrichment cultures. A modified Desulfovibrio medium was used for the isolation of strain LT0009 with taurine as electron acceptor and lactate and pyruvate as electron donors and for further growth experiments. Consumption of taurine and lactate and production of H2S and short-chain fatty acids (SCFA) were measured as previously reported 33.

Gram staining of the LT0009 isolate was performed u sing a Gram-staining kit according to the manufacturer9s instruction (Sigma Aldrich, 77730-1KT-F) and its cellular morphology was imaged with a scanning electron microscope (JSM-IT300, JOEL). A new probe was designed, tested, and applied for fluorescence in situ hybridization (FISH)-based microscopy of the genus Taurinivorans (Supplementary

The complete genome of strain LT0009 was determined by combined short- (Illumina) and long-read (Nanopore) sequencing. The automated annotation of the genome was manually curated for genes of interest, focusing on energy metabolism. Phylogenomic analyses comprised treeing with 43 concatenated marker protein sequences and calculation of average amino acid identities (AAI) and whole-genome average nucleotide identities (gANI). Additional phylogenetic trees were calculated with LT0009 using sequences of the 16S rRNA gene and selected sulfur metabolism proteins or genes. Source information of 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 16S rRNA gene reference sequences was manually compiled from the NCBI SRA entries (Supplementary as electron acceptor and lactate/pyruvate as electron donor were determined and compared.

The occurrence and prevalence of Taurinivorans muris- and Bilophila wadsworthia-related 16S rRNA gene sequences were analyzed across 123,723 amplicon datasets from gut samples, including 81,501 with host information. Further information on mouse studies with at least 20 samples that were positive for B. wadsworthia was manually compiled from the NCBI SRA entries or the corresponding publications (Supplementary Table 3).

The identity of Desulfovibrionaceae 16S rRNA gene sequences from amplicon sequencing data of wildR mice, which showed increased representation of Deltaproteobacteria and colonization resistance against the enteropathogen Klebsiella pneumoniae in a previous study 34, was re-analyzed.

The animal experiment was approved by the local authorities (Regierung von Oberbayern; ROB-55.22532.Vet_02-20-84). Twelve germ-free C57BL/6 mice w ere stably colonized with the 12-strain OligoMouse-Microbiota (OMM12) community 35. OMM12 mice (n=6) were orally (50 µl) and rectally (100 µl) inoculated with the LT0009 strain. The control OMM 12 mouse group (n=6) was treated with the same volume of sterile 1x phosphate-buffered saline. After 10 days, the mice were infected with the human enteric pathogen Salmonella enterica serovar Typhimurium (avirulent S. enterica Tm strain M2702; 5×107 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 c.f.u.) and sacrificed two days post infection (p. i.). Fecal microbiota composition was determined by strain-specific qPCR assays as previously described 35, including a newly developed assay for strain LT0009. Abundance of viable S. enterica Tm in feces and cecal content was determined by plating. Fecal samples of three mice from each group on day two p.i. were selected for metatranscriptome sequencing (JMF project JMF-2104-01) and analyses.

Cecal and fecal metatranscriptomes from a high-glucose diet experiment in mice (HG study, Hanson et al., unpublished) (JMP project JMF-2101-5) were analyzed for LT0009 gene expression. Mouse experiments were conducted following protocols approved by Austrian law (BMWF-66.006/ 0032-WF/V/3b/2014). Additionally, mouse gut metatranscriptomes from a previous study were analyzed for LT0009 gene expression (Plin2 study) 36. Sequence data (PRJNA379425) derived from eight-we ek-old C57BL/6 wild-type and Perilipin2-null (Plin2) mice fed high-fat/low-carbohydrate or low-fat/high-carbohydrate diets.

Strain LT0009 has been deposited in the German Coll ection of Microorganisms and Cell Cultures (DSMZ) as DSM 111569 and the Japan Collection of Microorga nisms (JCM) as JCM 34262. The genome and the 16S rRNA gene sequence of T. muris LT0009 are available at NCBI GenBank under accessi on numbers CP065938 and MW258 658, respectively .

Sequencing data of the LT0009 pure culture transcri ptome (JMF-2012-1) and the mouse gut metatranscriptomes from the HG study (JMF-2101-05) and the gnotobiotic study (JMF-2104-01) were deposited to the Sequence Read Archive (SRA) under BioProject accession PRJNA867178. 165

Desulfovibrionaceae The first taurine-respiring bacterium isolated from the murine gut represents a new genus of the family Strain LT0009 was enriched from mouse gut contents (cecum and colon) using an anoxic, non-reducing, modified Desulfovibrio medium with L-lactate and pyruvate as electron donors (and carbon source) and taurine as the sulfite donor for sulfite respiration. Its isolation was achieved by several transfers in liquid medium, purification by streaking on ferric-iron supplemented agar plates, indicating sulfide production by black FeS formation and picking of black colonies, and by additional purification using dilution-toextinction in liquid medium. Strain LT0009 produced sulfide and acetate during taurine degradation. We sequenced and reconstructed the complete LT0009 gen ome, which has a size of 2.2 Mbp, a G+C content of 43.6%, and is free of contamination as assessed by CheckM. The genome comprises 2,059 proteincoding genes, 56 tRNA genes, 4 rRNA operons (with 5S, 16S, and 23S rRNA genes), 4 pseudogenes, and 6 miscellaneous RNA genes.

LT0009 formed a monophyletic, genus-level (>94.5% s imilarity) lineage with other 16S rRNA gene sequences from the gut of mice and other hosts. It has <92% 16S rRNA gene sequence identity to the closest related isolates Marseille-P3669 and Mailhella massiliensis Marseille-P3199 T, two strains isolated from human stool (Fig. 1a). Phylogenomic treeing and an AAI of <60% to other described species strongly suggested that LT0009 represents the type strain of a novel genus in the family Desulfovibrionaceae of the phylum Desulfobacterota 37 for which we propose the name Taurinivorans muris (Fig. 1b, Supplementary Fig. 2, Supplementary Information). The previously described mouse gut MAGs UBA8003 and extra-SRR4116659.59 have >98% ANI and AAI to LT 0009 and thus would also belong to the species T. muris 38340. Furthermore, the mouse gut MAGs extra-SRR4116662.45 and single-China-D-Fe10-120416.2 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 showed <80% AAI/ANI to strain LT0009 and 84% AAI an d 82% ANI to each other, which indicates that each of these two MAGs would likely represent a separate species in the novel genus Taurinivorans. Notably, the genome of T. muris LT0009 with only 2.2 Mbp in size is considerably sm aller than that of other freeliving bacteria of the Desulfovibrio-Bilophila-Lawsonia-Mailhella-lineage (Fig. 1b). Only the obligate intracellular intestinal pathogen Lawsonia intracellularis has a smaller genome at 1.5-1.7 Mbp 41,42. The Gram-staining of T. muris LT0009 was negative. FISH imaging of the LT0009 pu re culture with the newly-developed genus-specific 16S rRNA-targeted probe TAU1151 showed cells with a conspicuous spiral-shaped morphology and considerably varying lengths (1.7 to 28 µm) (Fig. 1c). SEM imaging furthe r indicated that LT0009 cells have multiple polar fla gella and are thus motile (Fig. 1c).

Complete utilization of taurine as electron acceptor in modified Desulfovibrio liquid medium with electron donors lactate and pyruvate in excess, resulted in production of nearly quantitative amounts of H2S (Fig. 1d). Strain LT0009 in pure culture did not grow in absence of 1,4-naphthoquinone and yeast extract, indicating an absolute requirement of menaquinone (vitamin K2) and other essential growth factors, respectively. Both growth rate and final growth yields were increased when taurine was provided at 20 and 40 mmol/l concentration in comparison to 10 mmol/l, while growth was inhibited at concentrations g60 mmol/l taurine (Supplementary Fig. 3a). Strain LT0009 grew with a lower growth rate and final grow th yield when pyruvate was omitted as additional electron donor (Supplementary Fig. 3b). Strain LT0009 grew equally well at a pH range of 6 to 8.5 (Supple mentary Fig. 3c) and temperatures between 27-32°C, but with reduced final growth yield at 37 and 42°C (Supplementary Fig. 3d). No colony formation was observed on agar plates under aerobic conditions, suggesting a strict anaerobic lifestyle of T. muris LT0009. 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236

Based on a genome-inferred metabolism prediction of strain LT0009 (Fig. 2a, Supplementary Table 6), we tested its growth with substrates that could serve as energy and sulfur sources in the gut. Fermentative growth with only pyruvate or only taurine, i.e., as both electron donor and acceptor, was not observed. In addition to pyruvate and lactate, LT0009 also us ed formate as electron donor for growth under taurinerespiring conditions, albeit with an extended lag phase and a lower growth yield. For the alternative electron acceptors tested, LT0009 used 3-sulfolacta te and thiosulfate in combination with lactate and pyruvate, but did not grow with 2,3-dihydroxypropane-1-sulfonate (DHPS), isethionate, cysteate, and not with inorganic sulfate or sulfite (Fig. 2b).

The metabolic pathways used for growth by respiration with taurine, sulfolactate or thiosulfate and with lactate/pyruvate as electron donors were further analyzed by differential transcriptomics and proteomics. This demonstrated that taurine is degraded via the Tpa-Xsc pathway and the produced sulfite respired via the DsrAB-DsrC pathway (Fig. 2c,d). Pyruvate-dependent taurine transaminase Tpa catalyzes initial conversion of taurine to alanine and sulfoacetaldehyde 31. Oxidative deamination of alanine to pyruvate is catalyzed by alanine dehydrogenase Ald (Fig. 2a,c,d). Lack of sarD and islAB and an inability to grow with isethionate showed that LT0009 does not have the ta urine degradation pathway of B. wadsworthia 26. Instead, sulfoacetaldehyde is directly desulfonated to acetyl-phosphate and sulfite by thiaminediphosphate-dependent sulfoacetaldehyde acetyltransferase Xsc 29331. The acetyl-phosphate is then converted to acetate and ATP by acetate kinase AckA. Strain LT0009 seems to lack candidate genes for the TauABC taurine transporter 43. While homologs of tauABC are encoded in the genome, the individual genes do not form a gene cluster like in Escherichia coli 44 and were not expressed during growth on taurine (Supplementary Table 7). Instead, the LT0009 genome encodes three copies of gene sets for tripartite ATP-independent periplasmic (TRAP) transporter 45 that are co-encoded in the taurine 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 degradation gene cluster and were expressed during growth with taurine, thus are most likely involved in taurine import, including DctPQM1 (with fused DctQM1) (TAUVO_v1_1026 and 1027) and DctPQM3 (TAUVO_v1_1467-1469) (Fig. 2c,d). (2S)-3-sulfolactate is degraded by LT0009 via the S lcC-ComC-SuyAB pathway as shown by differential expression of these enzymes in cells grown with racemic sulfolactate (Fig. 2c, d, Supplementary Table 7). The dehydrogenases (S)-sulfolactate dehydrogenase SlcC and (R)-sulfolactate oxidoreductase ComC isomerize (2S)-3-sulfolactate to (2R)-3-sulfolactate via 3-sulfopyruvate. (2R)-3-sulfolactate is desulfonated to pyruvate and sulfite by sulfo-lyase SuyAB. The neighboring gene clusters slcHFG-slcC-comC and hpsNdctPQM-suyAB were both significantly upregulated in the transcriptome of sulfolactate-grown cells (Fig. 2d). The DctPQM2 (with fused DctQM2) (TAUVO_v1_1434 and 1435) TRAP transporter and the SlcGFH tripartite tricarboxylate transporters (TTT) 45,46 are putative sulfolactate importers. The gene cluster further includes a homolog to hpsN, encoding sulfopropanediol-3-dehydrogenase 47. This enzyme converts (R)-DHPS to (R)-sulfolactate during aerobic catabolism of DHPS by diverse bacteria in soils 47 and the ocean 48. However, LT0009 did not grow with racemic DHPS wh en tested (Fig. 2b). The hpsN gene was transcribed in LT0009 with taurine, sulfolactate, a nd thiosulfate treatments, but the HpsN protein was not detected (Supplementary Table 7). LT0009 did not gr ow with cysteate as electron acceptor under the conditions we used, although it encodes a homolog of L-cysteate sulfo-lyase CuyA that desulfonates Lcysteate to pyruvate, ammonium, and sulfite 49,50 (Fig. 2b). The cuyA gene was transcribed in the presence of taurine, sulfolactate, and thiosulfate. Furthermore, CuyA was significantly higher expressed in LT0009 with taurine (P<0.001) compared with sulfolactate and thiosulfate (Supplementary Table 7), yet its physiological role in LT0009 remains unclear. 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282

Strain LT0009 respired thiosulfate, such as B. wadsworthia strain RZATAU 51, but lacks genes for PhsABC thiosulfate reductase 52 and thiosulfate reductase from Desulfovibrio (EC 1.8.2.5) 53, which both (i) disproportionate thiosulfate to sulfide and sulfite and (ii) are present in the human B. wadsworthia strains ATCC 49260, 4_1_30, and 3_1_6. LT0009 has a gene fo r a homolog of rhodanese-like, sulfur-trafficking protein SbdP (TAUVO_v1_1430) (Supplementary Fig. 4d) 54. Rhodaneses (EC 2.8.1.1.) can function as thiosulfate sulfurtransferase and produce sulfite 55. Homologs of SbdP are broadly distributed in members of the

We next performed metatranscriptome analysis and re-analyzed published metatranscriptome datasets of gut samples from different mouse models to reveal the metabolic pathways that are most expressed by T. muris in its murine host.

In our gnotobiotic model, strain LT0009 or a mock c ontrol were added to germ-free mice stably colonized with the synthetic OMM12 community (Fig. 4a). Strain-specific qPCR assays showed that ten OMM12 strains and strain LT0009 colonized the mice (Fig. 4b). Con sistent with previous studies, strains A. muris KB18 and B. longum subsp. animalis YL2 were not detected 35,70. Colonization of LT0009 did not affect the abundan ce of other strains, which indicated that LT0009 occup ied a free niche in the intestinal tract of this gnotobiotic mouse model. The taurine metabolism (tpa, ald, xsc) and sulfite reduction (dsrAB, dsrC) genes were in the top 5% expression rank of all LT0009 genes (Fig. 4c ). In contrast, gene expression of the putative thiosulfate transferase (sbdP) ranked at 17% and of sulfolactate degradation (suyAB, slcC, comC) ranked from 62% to 88% of all LT0009 genes (Fig. 4c). T. muris LT0009 thus largely occupied the vacant taurinenutrient niche in the intestinal tract of OMM12 mice. 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377

Metatranscriptome analysis of intestinal samples from conventional laboratory mice on various diets (e.g. high-glucose; high-fat/low-carbohydrate; low-fat/high-carbohydrate) and with different genetic backgrounds (wildtype; plin2) also showed that taurine degradation and sulfite respiration were within the top 5% of expressed LT0009 genes (Supplementary Fig.5). Collectively, this demonstrated that taurine is the predominant electron acceptor for energy conservation of T. muris in the murine intestinal tract. Free taurine in the murine intestine largely derives from microbial deconjugation of host-derived taurocholic bile acids 71,72. LT0009 does not encode genes for bile salt hydrol ase (BSH) and is thus likely dependent on other gut microbiota members for liberation of taurine from taurocholic bile acids. BSH genes are encoded across diverse bacterial taxa in the human and mouse gut 71,73,74. In agreement with previous studies of bile acid transformations in the OMM12 model 75,76, we identified BSH genes in seven OMM12 strains (Supplementary Table 5). The expression of BSH genes in these strains did not change significantly with the presence of LT0009. Yet, BSH gene transcription increased in E. clostridioformis YL32, E. faecalis KB1, B. caecimuris I48, and M. intestinale YL27, and decreased in L. reuteri I49 (Supplementary Table 5). Bile acid deconjugation by some of these OMM12 strains has been confirmed in vitro 76. Specifically, B. caecimuris I48, B. animalis YL2, E. faecalis KB1, and M. intestinale YL27 were tested positive for deconjugation of taurine-conjugated deoxycholic acid, while E. clostridioformis YL32 was either tested negative or inhibited by addition of the bile acids. The down-regulation of the BSH gene in L. reuteri I49 is consistent with the in vitro deconjugation capacity of this strain for glycine-conjugated deoxycholic acid but not for taurine-conjugated deoxycholic acid 76 and the generally lower abundance of glycineconjugated bile acids in rodents 72. We hypothesize that taurine degradation by LT0009 could provide a selective feedback mechanism on expression of BSHs for taurocholic bile acid deconjugation in the OMM12 model. 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401

Thiosulfate is presumably a constantly present electron acceptor for microbial respiration in the mammalian gut as it is generated by mitochondrial H2S oxidation in the gut epithelium 1. The sulfide oxidation pathway is mainly located apically in the crypts of human colonic tissue at the interface to the gut microbiota 77. The amount of thiosulfate supplied into the gut lumen will depend on epithelial H2S metabolism 1,77. The expression level of the putative SbdP thiosulfate transferase of T. muris ranked relatively high with 15-24% of all LT0009 genes acr oss all mouse gut samples (Fig. 4c, Supplementary Fig. 5). However, the function of this protein remains unconfirmed as its expression was not differentially upregulated in the thiosulfate-metabolizing T. muris pure culture (Fig. 2a,d, Supplementary Table 7). In vivo taurine respiration, and potentially thiosulfate respiration, are likely fueled by pyruvate, H2, and lactate as electron donors, as expression of genes for their oxidation ranked at 5-7% (por), 1.5-9.1% (hybAC), and 2.5-31% (lutABC, lutP) of all LT0009 genes across all mouse gut metatran scriptomes, respectively (Fig. 4c, Supplementary Fig. 5).

Taurinivorans muris LT0009 slightly increased colonization resistance against S. enterica and activated a sulfur metabolism gene-encoding prophage in a gnotobiotic mouse model The human enteropathogen S. enterica Tm can invade and colonize the intestinal tract by utilizing various substrates for respiratory growth that are available at different infection stages 78. The gnotobiotic OMM12 mouse model provides intermediate colonization resistance against S. enterica Tm 35 and is widely used as a model system of modifiable strain composition for investigating causal involvement of cultivated mouse microbiota members in diverse host diseases and phenotypes 79. Yet, a bacterial isolate from the mouse gut with proven dissimilatory sulfidogenic capacity was not available until now 80. T. muris has fundamental physiological features that could on the one hand contribute to colonization resistance against S. enterica Tm by direct competition for pyruvate 81, lactate 82, H2 83, formate 84, and host-derived 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 thiosulfate 84,85. On the other hand, T. muris could also promote S. enterica Tm expansion during inflammation by fueling tetrathionate production through enhanced intestinal sulfur metabolism 18,86. Furthermore, expansion of sulfidogenic Deltaproteobacteria commensals and the tpa-xsc-dsr pathway in the metagenome fueled by host-derived taurine was shown to increase protection against the enteropathogen Klebsiella pneumoniae in mice; with sulfide-mediated inhibition of aerobic respiration by pathogens being proposed as a generic protective mechanism 34. Notably, our re-analysis of the 16S rRNA gene amplicon data from the wildR mouse model of this study identified T. muris as the dominant deltaproteobacterium (Desulfobacterota) of the protective community (Supplementary Fig. 6). Given that taurine respiration via the sulfidogenic tpa-xsc-dsr pathway is the main energy niche of T. muris in the mouse gut (Fig. 4c, Supplementary Fig. 5), enhanced resistance in the wildR mouse model was thus likely primarily due to the activity of T. muris.

Here, we investigated the impact of T. muris LT0009 during the initial niche invasion of S. enterica Tm using an avirulent, non-colitogenic strain 87. Compared to OMM12 mice without LT0009, mice colonized with the OMM12 and LT0009 had a slightly reduced load of S. enterica Tm at 48 p.i. that was significant in feces but not in cecum (Fig. 4d). Comparative metatranscriptome analysis did not provide evidence for a mechanism of direct interaction as only six S. enterica Tm genes were differentially expressed, i.e. significantly downregulated, in the presence of strain LT0009 (Fig. 4e, Supplementary Table 9). Three of the six genes are involved in transport and metabolism of galactonate (D-galactonate transporter DgoT, D-galactonate dehydratase DgoD, 2-dehydro-3-deoxy-6-phosphogalactonate aldolase DgoA), which is produced by some bacteria as an intermediate in D-galactose metabolism and is also present in mammalian tissue and body secretions 88. D-galactonate catabolism capability was suggested as a distinguishing genetic feature of intestinal Salmonella strains compared to extraintestinal serovars, with serovars Typhi, Paratyphi A, Agona, and Infantis lacking genes for utilizing D-galactonate as a sole carbon 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 source 89. The putative D-galactonate transporter DgoT in Salmonella enterica serovar Choleraesuis was identified as a virulence determinant in pigs 90. The OMM12 strains and LT0009 do not encode the DgoTDAKR galactonate pathway. The significance of galactonate for S. enterica Tm gut colonization and competition remains to be elucidated.

Colonization of T. muris LT0009 in the gnotobiotic mice had variable impact on the differential gene expression pattern of the OMM12 members (Fig. 4e, Supplementary Fig. 7 and Table 9 ). While gene expression was not significantly affected in L. reuteri I49, E. clostridioformis YL32 was most affected with 84 differentially expressed genes (55 up-regulated and 29 down-regulated) (Fig. 4e). Most of the significantly up-regulated genes (n=41) in E. clostridioformis YL32 are clustered in a large genomic region (I5Q83_10075-10390) that encoded various phage gene homologs and was identified as a prophage using PHASTER (Fig. 4f). This prophage, named YL32-pp-2.059, Saumur, is among a set of thirteen previously identified prophages of the OMM12 consortium that represent novel viruses, were induced under various in vitro and/or in vivo conditions, and constitute the temporally stable viral community of OMM12 mice 91. We show that colonization of LT0009 in the OMM 12 mouse model selectively enhanced the transcriptional activity of the E. clostridioformis YL32 prophage Saumur, which carries a gene (I5Q83_1 0375) for phosphoadenosine-phosphosulfate reductase (CysH) that functions in the assimilatory sulfate reduction pathway of many bacteria (Fig. 4f). Various organosulfur auxiliary metabolic genes, particularly cysH, are widespread in environmental and human-associated viromes, which suggests viruses augment sulfur metabolic processes in these environments, including the gut 92. Addition of physiologically relevant concentrations of sulfide to a Lactococcus lactis strain culture resulted in increased production of viable particles of its phage P087 92. We thus hypothesize that T. muris LT0009 not only impacts intestinal sulfur homeostasis via its sulfur metabolism but also by H2S-mediated activation of the sulfur metabolism geneexpressing phage Saumur in E. clostridioformis YL32. If activation of prophage Saumur contributes to protection from S. enterica Tm remains subject of further study. 495 represents the new genus/species Taurinivorans muris in the family Desulfovibrionaceae. a. 16S rRNA gene tree and FISH probe coverage. Maximum likelihood branch supports (1000 resamplings) equal to or greater than 95% and 80% are indicated by black and grey circles, respectively. The scale bar indicates 0.1 estimated substitutions per residue. Accession numbers are shown in parentheses. Strain LT0009 is show n in bold and the type strains are marked with a superscript 8T9. The sequence sources are indicated with different colors (Supplementary Table 2). Sequences were assigned to Taurinivorans and Taurinivorans muris based on the genus-level similarity cutoff of 94.5 % and species-level similarity cutoff of 98.7% 96, respectively. The perfect-match coverage of probes TAU1151 for Taurinivorans and MAIL1151 for Mailhella is indicated. b. Phylogenomic tree. Ultrafast bootstrap support values equal to or greater than 95% and 80% for the maximum likelihood tree are ind icated with black and grey circles, respectively. Accession numbers are shown in parentheses. Strain LT0009 is shown in bold. Strains with complete genomes (genome size is indicated) are marked with a star. Genomes were assigned to Taurinivorans based on the genus-level AAI cut-off value of 63.4% 97. The scale bar indicates 0.1 estimated substitutions per residue. c. Morphology of LT0009 cells in pure culture. SEM: S canning electron microscopy images of cells of varying lengths. White arrows indicate the flagella. FISH: Cells hybridized with Cy3-labeled probe TAU1151 and Fluos-labeled probe EUB338mix and counterstained by DAPI. d. Growth of strain LT0009 in modified Desulfovibrio medium confirmed complete utilization of taurine as electron acceptor concomitant with nearly stoichiometric production of sulfide. Electron donors L-lactate and pyruvate were provided in excess, and their utilization also contributed to acetate formation. Pyruvate in the medium and ammonia released from deamination of taurine were not quantified in this 518 519 experiment. Lines represent averages of measures in triplicate cultures. Error bars represent one standard deviation. a. Cell cartoon of the central sulfur and energy metabolism of LT0009 as determined by genome, transcriptome, and proteome analyses. Genes/proteins detected in the transcriptome and proteome of LT0009 grown with taurine, sulfolactate or thiosulf ate as electron acceptor are shown by colored circles and squares, respectively. Circle size indicates gene transcription level normalized as TPM. Proteins of all transcribed genes that are shown were also detected in the proteome, with the exception of AprAB, TauA (TAU_v1_0027, TAU_v1_1344), TauB, TauC, TauE, DctMQ2, SlcG, DsrEFH, AscA, two [FeFe] hydrogenases (TAU_v1_1126, TAU_v1_1901), cytochrome c and Sdh. Protein complexes (e.g. Rnf, SlcFGH, DctPMQ2, Atp) are not shown with transcriptome and proteome data because one or more of the complex units were not expressed. The gene annotations are listed in Supplementary Table 6. b. Anaerobic growth tests of strain LT0009 with various substrates. Upper pan el: All substrates were added at 10 mmol/l concentration, except acetate (20 mmol/l), which was added as carbon source together with H2. Lower panel: The different sulfur compounds were added at 10 mmol/l concentration together with pyruvate, lactate, and 1,4-naphthoquinone. OD600: optical density at 600 nm. c. Organization of sulfur metabolism genes in the LT0009 genome. Numbers show the RefSeq locus tag with the prefix TAUVO_v1. d. Comparative transcriptome and proteome analysis of LT0009 grown with lactate and taurine, sulfolactate or thiosulfate as electron acceptor; each in triplicate culture. Numbers following protein names refer to RefSeq locus tag numbers (prefix TAUVO_v1). Protein expression was normalized to DsrC for each growth condition. Bars represent averages of triplicate measures with error bars representing one standard deviation. Asterisk indicates significant (p<0.05) differences in gene transcription/protein expression 542 543 compared to growth with taurine. TCA, tricarboxylic acid cycle; WL, Wood-Ljungdahl pathway; PEP, phosphoenolpyruvate; DHPS, 2,3-dihydroxypropane-1-sulfonate; TPM, transcripts per million. a. Occurence and prevalence of Taurinivorans muris- and Bilophila wadsworthia-related sequences in 16S rRNA gene amplicon datasets of human and animal guts. T. muris- and B. wadsworthia-like sequences at 97% similarity cut-off are expressed as percentages of positive samples in each host (the numbers of samples used for the analysis are shown in parenthesis) and different colors indicate percentages of samples positive for T. muris and B. wadsworthia at different relative abundance ranges. Hosts with less than 20 amplicon samples are not shown. T. muris- and B. wadsworthia-related sequences co-occur in only 28 mouse gut samples as shown by the Venn diag ram. b. Visualization of Taurinivorans in a colon tissue section of a mouse fed a polysaccharide- and fiber-deficient diet 98 by FISH. TAU1151-Cy3-labeled Taurinivorans cells appear in pink and the remaining bacterial cells and tissue in blue due to DAPI-staining.