Desulfovibrio diazotrophica sp. nov., a sulphate reducing bacterium from the human gut capable of nitrogen fixation

Sulphate-reducing bacteria (SRB) are widespread in human guts, yet their expansion has been linked to colonic diseases. We report the isolation, genome sequencing, and physiological characterisation of a novel SRB species belonging to the class Deltaproteobacteria (QI0027T). Phylogenomic analysis revealed that the QI0027T strain belongs to the genus Desulfovibrio with its closest relative being Desulfovibrio legallii. Metagenomic sequencing of stool samples from 45 individuals, as well as comparison with 1690 Desulfovibrionaceae metagenome-assembled genomes, revealed the presence of QI0027T in at least 22 further individuals. QI0027T encoded nitrogen fixation genes and based on the acetylene reduction assay, actively fixed nitrogen. Transcriptomics revealed that QI0027T overexpressed 45 genes in nitrogen limiting conditions as compared to cultures supplemented with ammonia, including nitrogenases, an urea uptake system and the urease enzyme complex. To the best of our knowledge, this is the first Desulfovibrio human isolate for which nitrogen fixation has been demonstrated. This isolate was named Desulfovibrio diazotrophica sp. nov., referring to its ability to fix nitrogen (‘diazotroph’). Importance Animals are often nitrogen limited and have evolved diverse strategies to capture biologically active nitrogen. These strategies range from amino acid transporters to stable associations with beneficial microbes that can provide fixed nitrogen. Although frequently thought as a nutrient-rich environment, nitrogen fixation can occur in the human gut of some populations, but so far it has been attributed mainly to Clostridia and Klebsiella based on sequencing. We have cultivated a novel Desulfovibrio from human gut origin which encoded, expressed and actively used nitrogen fixation genes, suggesting that some sulphate reducing bacteria could also play a role in the availability of nitrogen in the gut.


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Sulphate reducing bacteria (SRB) are present in the mouth and the gut of ~50% of the human 54 population (1, 2). SRB thrive in the gut, releasing hydrogen sulphide (H2S) as a by-product of 55 sulphate reduction. H2S is a potent genotoxin and has been linked to chronic colonic 56 disorders and inflammation of the large intestine (3). Likewise, the presence of some 57 Desulfovibrio species has been implicated in chronic periodontitis, cell death and 58 inflammatory bowel diseases such as ulcerative colitis and Crohn's disease (4). The 59 detrimental role of SRB is, however, not firmly established. H2S can also act as a signalling 60 molecule or energy source for mitochondria (5, 6). Moreover, by using hydrogen, SRB help 61 in the efficient energy acquisition and complete oxidation of substrates produced by 62 fermentative bacteria (7). SRB could, therefore, have a dual role in the gut microbiome. Biological nitrogen fixation is the process by which gaseous dinitrogen (N2) is reduced to 69 biologically available ammonia (NH3) by diazotrophic microbes (10). Diazotrophy was 70 reported in some Desulfovibrio species from free-living communities and the termite gut (11, 71 motile, they will show a sharp edge where the inoculation stab is made. In contrast, if they are 122 motile, they will show a cloudy growth around the stabbing site. D. legallii showed a diffuse 123 area of growth, in agreement with the previously reported presence of a flagellum (8). In 124 contrast, QI0027 T had a sharp edge area of growth. However, there were slight signs of iron 125 sulphide outside the area of the stabbing site, which could be due to some cells being motile 126 or the diffusion of the hydrogen sulphide gas produced by QI0027 T (20)). QI0027 T also synthesized the negative regulator of flagellin 138 synthesis flgM. We thus hypothesize that QI0027 T regulates the expression of flagella 139 synthesis, which might be a key factor during the colonization and sensing of areas rich in 140 nutrients in the gastrointestinal tract (21). 141 Fatty acid analysis from QI0027 T and D. legallii showed that the main fatty acid was iso 142 C17:1 (29% for QI0027 T and 24.2% for D. legallii), which is consistent with most 143 Desulfovibrio species (22). Other predominant fatty acids had also a similar composition and 144 abundance for both strains: iso fatty acid methyl ester (FAME) C17:0 (24.9% for QI0027 T and 145 26.12% for D. legallii ), FAME C15:0 (19.3% for QI0027 T and 20.6% for D. legallii)), and iso 146 C17:0 (17.2-17.5% for QI0027 T and 19.9-20.2% for D. legallii) (Table 2). However, 10 fatty 147 acids were detected at low abundance (< 2%) only in QI0027 T , which likely reflects small 148 differences in the metabolism of QI0027 T compared to D. legallii (Table 2). 149 We used a microplate system, which allows for the high-throughput monitoring of 96 carbon 150 substrates under anaerobic conditions, to identify the carbon substrates used by QI0027 T and 151 D. legallii (Biolog, Technopath, Ireland). Substrates that could be used by both, QI0027 T and 152 D. legallii strains, included L-rhamnose, D-fructose, L-fucose, D-galactose, D-galacturonic 153 acid, palatinose, D,L-lactic acid, L-lactic acid, D-lactic acid methyl ester, L-malic acid, 154 pyruvic acid, methyl pyruvate, and L-glutamic acid, glucose and D-mannose (Table 3). Only 155 QI0027 T used as carbon substrate B-gentiobiose, D-glucose-6-phosphate, and D-melibiose. 156 Likewise, only D. legallii used arbutin, α-keto-butyric acid, α -ketovaleric acid, L-157 methionine, L-valine, and uridine-5'-monophosphate. However, replicates of each species did 158 not show consistent use of these carbon substrates, suggesting that their utilization might not 159 be a reliable method to distinguish between the two bacterial species. 160

Distribution of QI0027-related species in human microbiome metagenomes 173
We detected the presence of the same species as QI0027 T on at least two further individuals 174 based on sequencing of 45 stool metagenomes from Chinese donors. We used three methods 175 to detect QI0027 T in these Chinese metagenomes. First, we used mOTUS2, a taxonomic 176 classifier that uses clade-specific marker genes (23). The whole-genome sequencing (WGS) 177 reads from the pure culture QI0027 T were used as a control to identify the assigned species 178 by the taxonomic classifier. mOTUS2 classified the WGS reads only at genus level as 179 Desulfovibrio. By including in the mOTUS2 reference database our QI0027 T genome 180 assembly, mOTUS2 was able to detect QI0027 T in one individual. Second, we used 181 MetaPhlAn2 (24), a taxonomic classifier that uses a different database of clade-specific 182 marker genes. MetaPhlAn2 is widely used in studies that combine metagenomic sequencing 183 efforts from diverse human microbiome studies (e.g. 25, 26). MetaPhlAn2 classified the reads 184 of QI0027 T as 'Desulfovibrio desulfuricans', even though the closest relative is the type 185 strain D. legallii. Reads classified as 'D. desulfuricans' by MetaPhlAn2 will therefore include 186 other species such as QI0027 T . The lack of specificity or mis classification of QI0027 T likely 187 reflects the lack of representative genomes in the MetaPhlAn2 database. Third, we used read 188 mapping against our QI0027 T genome assembly using a high similarity threshold to 189 overcome the identification limitation by these taxonomic profilers.  We demonstrated that strain QI0027 T was able to fix nitrogen based on an acetylene 229 reduction assay performed in similar growth conditions used for the RNA-seq transcription 230 analysis ( Figure 3C). The specific nitrogenase activity rate for strain QI0027 T was estimated 231 as 7.6±5.2 nmol ethylene · mg protein -1 · h -1 . As expected, no acetylene reduction was 232 observed when the growth media was supplemented with yeast extract or with 18.54 mM 233 NH4Cl (referred as nitrogen excess). To our knowledge, this is the first Desulfovibrio isolate 234 from the human gut for which diazotrophy has been physiologically demonstrated. 235 Further evaluation of our whole transcriptome analysis revealed that the strain QI0027 T 236 overexpressed 43 genes under nitrogen limiting conditions, while an additional 19 genes were 237 overexpressed under nitrogen excess conditions ( Figure 3B). Under nitrogen excess 238 conditions, QI0027 T had a higher expression of genes related to cell cycle and cell division, 239 RNA processing, glycolysis, respiration, as well as cobalamin, ATP, and tryptophan synthesis 240 ( Figure 3A-B). Moreover, QI0027 T overexpressed the antitoxin HigA, a system that has been 241 linked to survival response during environmental and chemical stresses, including amino acid 242 starvation, growth, and programmed cell death (36). 243 Besides the nitrogen fixation genes overexpressed under nitrogen limiting conditions 244 discussed above, we observed overexpression of the urease enzyme complex ureABCEFGD 245 and the reversible urea uptake system urtABCDE. ureABCEFGD is used to reversibly 246 transform urea into ammonia and CO2. Since QI0027 T cultures with limiting nitrogen did not 247 have urea in the media, ammonia resulting from N2 fixation was likely converted to urea and 248 the excess excreted. Urea is less toxic than ammonia, more soluble, and can be used as an is not present. To overcome the limitations from binning, we used a targeted assembly 285 approach using the metagenomic reads from the four samples for which the four QI0027 T 286 related MAGs did not encode the nitrogenase genes and as reference the QI0027 T genome. 287 With this method, we could recover the nitrogenase genes for the four QI0027 T MAGs, and 288 based on phylogenetic analysis, the nifH gene grouped closer to nifH encoded by QI0027 T . In 289 agreement with our genome searches on the closed genome of D. piger, as well as on draft 290 genomes of isolates from D. fairfieldensis, Bilophila and Lawsonia, none of the human-291 derived MAGs from these Desulfovibrionaceae species encoded the nitrogenase genes 292 ( Figure 1). From the isolates and MAGs that have been sequenced thus far from 'sensu-293 stricto' Desulfovibrios that encode the genomic repertoire for nitrogen fixation, only D. 294 legallii, which was isolated from a shoulder joint infection, D. desulfuricans and QI0027 T 295 species have been isolated from mammals ( Figure 1). The ability to fix nitrogen is therefore 296 not present in the Desulfovibrionaceae that are the most abundant in the gut, but rather 297 prevails in Desulfovibrio species that tend to be in lower abundance. 298 nifH was likely acquired through horizontal gene transfer 299 The genes required for nitrogen fixation had a patchy distribution among 300 Desulfovibrionaceae bacteria. This can be explained by two scenarios that are not mutually 301 exclusive: 1) multiple events of horizontal gene transfer (HGT) have occurred among the 302 Desulfovibrionaceae or 2) the gene has been lost multiple times in evolutionary history. To 303 disentangle which of these scenarios was more likely to occur for 'sensu-stricto' 304 Desulfovibrio, we used phylogenetic analysis of the key gene for nitrogen fixation nifH. nifH 305 has an ancient history of horizontal gene transfer (46). Consistent with previous phylogenetic 306 analyses based on the nifH gene product, nifH formed four major clades, with clades 1-3 307 being functional nitrogenases and clade 4 grouping paralogs that are non-functional (46). 308 Most nifH sequences from Desulfovibrionaceae grouped with nifH from cluster 3 ( Figure 3). 309 This cluster is known to be present mainly in obligate anaerobes (46). Desulfovibrionaceae 310 nifH were not monophyletic and instead formed three major subclades (D1, D2 and D3) 311 Desulfovibrio grouped within subclades D1 and D2, while 'sensu-stricto' Desulfovibrio 315 bacteria grouped within subclade D3, suggesting at least three independent acquisitions of 316 this gene with multiple gene losses ( Figure 1). Subclade D3 is also nested within a group 317 comprising nifH from Clostridia and Methanomicrobia (Archaea), further supporting an 318 independent acquisition of nifH by "sensu stricto" Desulfovibrio by HGT from Clostridia, 319 which was likely to have been more recent compared to D1 and D2 ( Figure 1). 320

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We have presented evidence that QI0027 T is a novel species with nitrogen-fixing ability 322 distributed in humans from America, Europe and Asia, but with higher representation in 323 Chinese individuals. This novel species was misclassified by commonly used taxonomic 324 classifiers that rely on clade-specific marker genes from metagenomes and could only be 325 identified using whole-genome sequencing and assembly (of MAGs or pure cultures). 326 Although taxonomic classifiers based on clade-specific marker genes claim to be able to 327 distinguish bacteria at species level, these methods rely on proper genus/species classification 328 and characterization, which is problematic among the Desulfovibrionaceae as shown by our 329 phylogenomic analyses. Our study combines cultivation, genome sequencing, physiological 330 characterization, differential expression analysis and metagenome sequencing to overcome 331 the bias inherent to each technique. 332 Based on our acetylene reduction assay and differential expression analyses, strain QI0027 T 333 fixed nitrogen only when we did not provide a source of fixed nitrogen in the media, such as 334 yeast extract or NH4Cl, which are known to inhibit the metabolically expensive process of 335 nitrogen fixation (47). Our results show therefore that the genes for nitrogen fixation are fully 336 functional in strain QI0027 T . However, the rate of nitrogen fixation by QI0027 T was lower 337 compared to the environmental isolates D. desulfuricans, D. vulgaris, D. gigas, D. salexigens, 338 D. africanus, and D. thermophilus (7.6±5.2 vs. 42-918 nmol ethylene · mg protein -1 · h -1 ) 339 (11). Postgate and Kent (11) showed with the acetylene reduction assay that 3 out of 15 340 environmental strains belonging to five Desulfovibrio species could not fix nitrogen (11) and 341 hypothesized that the right conditions for nitrogen fixation might have yet to be found. 342 However, this pattern can be explained now as a true lack of the ability to fix nitrogen, since 343 the key gene for nitrogen fixation, nifH, has been gained and lost several times in the 344 Desulfovibrio clade (Figure 1). 345 The nifH gene present in 'sensu stricto' Desulfovibrio was likely acquired through HGT from 346 a Clostridia ancestor. The gut is an environment where Clostridia and Desulfovibrio 347 frequently encounter each other at high numbers. In this environment, microorganisms are 348 known to rapidly exchange genetic material (48). We hypothesize that nifH exchange could 349 have occurred between Desulfovibrio and Clostridia in an environment where they often 350 encounter each other, such as the gut. 351 Only a few members of the 'sensu-stricto' Desulfovibrio encoded nifH. D. desulfuricans a 352 member of the 'sensu-stricto' Desulfovibrio, is often recovered from human stool samples 353 (3). Some of these D. desulfuricans strains might also fix nitrogen, such is the case of our 13 354 D. desulfuricans Chinese gut isolates that encoded the nitrogenase operon. However, not all 355 strains of D. desulfuricans encode the genes to fix nitrogen ( Figure 1). Indeed, D. 356 desulfuricans formed two major clusters with an 80-85% ANI between the two clusters, 357 which shows that these species will need to be reclassified in future studies. The contribution 358 to nitrogen fixation in the human gut by Desulfovibrionaceae is therefore likely not limited to 359 QI0027 T . 360

Nitrogen fixation in a nutrient-rich environment 361
The human gut is a nutrient-rich environment in which most bioavailable nitrogen comes 362 from amino acids present in food. If nitrogen fixation is a high-energy demanding process 363 (49), and the gut is nutrient-rich, which conditions could favour the persistence of 364 Desulfovibrio species that can fix nitrogen? 365 It is commonly assumed that N2 fixation can occur only when bioavailable nitrogen is 366 limiting (<1 µM) (50). However, nitrogen fixation can occur in environments with relatively 367 high input of fixed nitrogen such as oceanic waters with high nitrate or ammonia 368 concentrations (30 μM NO3 -; 200 µM NH4 + ), symbiotic associations between a diazotrophic 369 cyanobacteria and a single-cell eukaryote living in waters with an excess of nitrogen (UCYN-370 A/haptophyte symbiosis), as well as the human gut (12-30 mM ammonia in stool, which can 371 be increased with higher protein consumption) (14, 50-53). A human population from Papua 372 New Guinea that had low protein intake (e.g. fixed nitrogen) was shown to have a gut 373 microbiome that was actively fixing nitrogen at higher rates as compared to populations with 374 higher protein intake (49-74 vs. 105 mg/kg body weight/day) based on acetylene reduction 375 assays using stool samples (14, 54). In this human population, nitrogen fixation by the 376 microbiome corresponded to at least 0.01% of the standard nitrogen requirement for humans. 377 Nitrogen fixation was attributed to Klebsiella and Clostridia bacteria based on nifH sequences 378 recovered from cloning and metagenomic analysis (14). However, the microbial contribution 379 to the nitrogen intake from humans could be population specific. Although the gut may have on average an excess of nitrogen because of the host diet, there 396 may be microniches where bioavailable nitrogen is locally limiting. Therefore, having 397 nitrogen fixation ability would still be a selective advantage. SRB must compete for resources 398 and space to thrive in the gut. Hydrogen, the most commonly used energy source by SRB, is 399 also used by acetogens and methanogens. Niche partitioning could be a mechanism to cope 400 with competitors, as has been observed for even members of the same species (59-61). More 401 than 90% of nitrogen is absorbed in the small intestine and studies using germ-free animals 402 have shown that most gut microbes increase the protein requirement of the host (13, 15). In  England BioLabs). Approximately 10 Gb were sequenced with a HiSeq X Ten instrument as 505 paired-end 150 bp reads. Library preparation and sequencing was done by Novogene (China). 506 Metagenomic reads were quality trimmed with a minimum quality of 2 and human host reads 507 were removed using as reference the human genome GCA_000001405 with bbduk. These 508 clean reads were used to search for QI0027 T using three methods: (i) Taxonomic profiling 509 using mOTUs2 (v.2.5.1) with the default database, as well as a modified database that 510 included the genome of QI0027 T (23). (ii) Taxonomic profiling using MetaPhlAn2 (v.2.7.7) 511 with the default database because all publicly available human metagenomes have been 512 scanned for the microbial diversity using this tool with default settings (1, 24). (iii) Finally, 513 we mapped the reads against QI0027 T genome assembly (only scaffolds >1000 bp) with 514 bbmap (v.38.43) using >95% mapping identity. We considered that QI0027 T species was 515 present when > 50% of the reference genome was covered by the reads. 516

Recovery of QI0027 T from Chinese metagenomes and MAGs 517
Clean reads of the 45 stool metagenomes were combined and mapped against all publicly 518 available and our own Desulfovibrionaceae genomes. The resulting assembly from the 519 combined reads was separated into genomes using an unsupervised binning method based on 520 nucleotide composition, differential coverage and linkage data from paired-end reads (83). 521 Resulting reads were then assembled with metaSPAdes (v. Metadata for these genomes was retrieved using the curatedMetagenomicData 535 R/Bioconductor package (26). To find genomes from the same species as QI0027T, we used 536 ANI similarity of all the Desulfovibrionaceae MAGs against the QI0027 T genome with 537 fastANI (82). 538

Detection of genes related to nitrogen-fixation and phylogenetic analysis 539
We searched for the nitrogen fixation related genes among the Desulfovibrionaceae genomes 540 included in our phylogenomic tree, as well as the Desulfovibrionaceae MAGs from the 541 collection by Pasolli et al.,(1). We used as query the amino acid sequences encoded by nifH, 542 nifD, nifK, nifE and nifB from QI0027 T against the isolate genomes and MAGs with tblastn 543 (v.2.2.31+) to be able to detect partial genes at the end of scaffolds (>40% similarity, >40% 544 query coverage, minimum alignment length >50). To improve the genome assembly of the 545 four MAGs that did not encode the nitrogenase cluster, we used read mapping against the 546 QI0027 T reference genome using bbmap (v.38.43) with a similarity ≥95% against the reads 547 from projects YSZC12003_3554, SRR3736997, H1M313811and YSZC12003_36012 (Short 548 Read Archive). Results were integrated with the phylogenomic tree with iTol (87). 549 For phylogenetic analysis, we annotated all genomes with Prokka (v.1.14.0) and retrieved 550 nifH amino acid sequences. Representative nifH amino acid sequences from Clusters 1-4 551 were retrieved from Gaby and Buckley (46). Sequences were aligned using Mafft v. 1.3.7 and 552 realigned with ClustalW v2.1. The alignment was masked to remove alignment positions with 553 >75% gaps. A maximum-likelihood tree reconstruction was obtained using FastTree (v. 554

2.1.5) with SH-like branching support values (88). 555
Physiology of QI0027 T 556 pH, salinity, temperature and motility. Physiological testing was done by adjusting Postgate 557 C media without agarose in Hungate tubes (SciQuip, UK) and diluting cells 100-fold. To 558 identify the pH range at which QI0027 T could grow, pH was adjusted by injecting 559 deoxygenated 1.34 M HCl for acidic pHs, or 0.16 M NaOH with 10% NaHCO3 (w/v) for 560 alkaline pHs before autoclaving. Final pH was measured before inoculation (range from 3.98 561 to 8.83) and after the growth of the cells. QI0027 T grew in media with a pH range between 562 4.5 to 6.5, and the pH of the media became more basic after cell growth ranging from 6.8 to 563 7.2. To determine the tolerance of QI0027 T to different salinity concentrations, growth at 0-564 0.513 M NaCl was investigated in liquid Postgate C medium as described previously (89). pH 565 and salinity tolerance were investigated at 37°C for 5 days. To test for the effect of 566 temperature on growth, duplicate cultures of strain QI0012 were incubated in 10 ml Hungate 567 tubes at temperature range 10-59°C for up to 7 days. Growth was monitored with a 568 turbidimeter CO 8000 (Biochrom, UK). Motility of QI0027 T was tested using semisolid 569 Postgate C agar (5g/L of agar), using as reference for diffusion the motile D. legallii. The 570 semisolid media was stabbed with disposable inoculating loops and the diffusion was 571 observed after 3 to 5 days. 572

Determination of nitrogenase activity 631
To determine the nitrogenase activity of QI0027 T to fix nitrogen, we used the acetylene 632 reduction assay (ARA) (96, 97). The cells were first grown in media free of added fixed 633 nitrogen (ammonia, NH3), modified from Postgate B media (98), which contained per L of 634 distilled water: 3.5 g sodium lactate, 2 g MgSO4.7H2O, 0.25 g CaSO4, 0.5 g K2HPO4, 0.004 g 635 FeSO4.7H2O, 9 g NaCl, 4 ml resazurin (0.02% W/V), 0.1 g ascorbic acid, and 0.1g 636 thioglycolic acid. pH was adjusted to 7.5±0.1 using 5 M HCl. The media was dispensed into 637 Hungate tubes (6.2 mL gas phase) and sterilized by autoclaving. Media was supplemented 638 with 1 ml/L of trace element solution SL-10 (DSMZ). Growth could be observed after 5 to 7 639 days. These cells were used to inoculate 12 tubes with a 1:100 dilution in 10 ml of media. As 640 nitrogenase activity is inhibited when fixed nitrogen is available, we included as negative 641 control duplicate tubes supplemented with either 1g/L of yeast extract or 1g/L NH4Cl (47, 98, 642 99). Duplicate blank tubes without inoculum were also included as negative controls. All 643 tubes were injected 10% v/v of the gas phase with acetylene and incubated at 37°C. We thank Gemma Langridge, Claire Hill and Barry Bochner for technical support using the 716 Omnilog machine. We thank the JIC Bioimaging facility for access to electron microscopes. 717