# Methanobrevibacter smithii strain U29 whole genome sequence delineates M. smithii intermediate cell variants

Ihab Malat, Vincent Bossi, Michel Drancourt, Ghiles Grine, Raymond Ruimy

► To cite this version:

Ihab Malat, Vincent Bossi, Michel Drancourt, Ghiles Grine, Raymond Ruimy. Methanobrevibacter smithii strain U29 whole genome sequence delineates M. smithii intermediate cell variants. BMC Research Notes, 2025, 18 (1), pp.414. 10.1186/s13104-025-07444-4. hal-05372618

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https://amu.hal.science/hal-05372618v1

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# *Methanobrevibacter smithii* strain U29 whole genome sequence delineates *M. smithii* intermediate cell variants

Ihab Malat¹,², Vincent Bossi³, Michel Drancourt²*, Ghiles Grine²,⁴ and Raymond Ruimy⁵

**Abstract**

**Background** *Methanobrevibacter smithii* (*M. smithii*), the predominant methanogen in the human digestive tract, plays a key role in methane production. Despite its importance, the genomic diversity of *M. smithii* is poorly characterised, especially in extra-digestive sites such as the urinary and respiratory tracts, and the blood. Understanding this diversity would help unravel its potential role in human health and diseases.

**Results** We report the genome of *M. smithii* strain U29, isolated from urine, expanding the known diversity of the species. The *M. smithii* U29 genome (scaffold level; 1822–124-bp, 1745 protein-coding sequences) lacks *Candidatus Nanopusillus* sequences, unlike digestive tract strains. Comparative analysis has revealed a high similarity (99.86% ANI) with the reference *M. smithii* strain ATCC 35,061, although U29 contains 71 unique coding sequences including 12/71 (13 200-bp; 69% of the total extra-material size) with demonstrated *Siphoviridae* viral ancestry. Accordingly, *M. smithii* U29 has been identified as an intermediate *M. smithii* cell variant, exhibiting genomic traits potentially conferring adaptability to the urinary tract.

**Conclusion** This study enhances our understanding of *M. smithii* genomic diversity and highlights the presence of viral sequences in the urinary tract *M. smithii* strain U29. These findings open up a hypothesis that viral integration may play a role in *M. smithii* mucosae colonisation and dynamics, underscoring the need for further investigations into the mechanisms underlying its tissue translocation and potential health implications.

**Keywords** Methanobrevibacter smithii, Archaea, Urinary tract, Genome, Cell variants

**Introduction**

*Methanobrevibacter smithii* (*M. smithii*) prevails over human digestive tract methanogenic archaea (methano- gens) detected and cultivated from the oral cavity and faecal specimens in virtually all individuals, with the exception of infants suffering from deadly acute malnu- trition [1–3]. In the digestive tract, *M. smithii* has been further associated with a nanoarchaeon, *Candidatus Nanopusillus phoceensis* (*Ca*. *Nanopusillus phoceensis*) [4]. In contrast, *M. smithii* strains detected in other sites, including the lower respiratory tract [5], newborn stom- ach [6], colostrum [7], blood of patients suffering from

<footnote>*Correspondence:
Michel Drancourt
michel.drancourt@univ-amu.fr

¹IHU Méditerranée Infection, Marseille, France

²MEPHI, Aix-Marseille-Université, 19-21 Boulevard Jean Moulin, Marseille 13005, France

³Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France

⁴MI2, IHU Méditerranée Infection, Marseille, France

⁵Department of Bacteriology, University Hospital of Nice, Nice, France</footnote>
endocarditis [8], vaginosis [9], urine [10], as well as in human sewage [11], ruminant mammals [12], and animal models including pigs and mice [13, 14], were all devoid of symbiotic *Ca*. Nanopusillus phoceensis.

Despite the prominent role of *M. smithii* in convert-
ing gaseous hydrogen from bacterial fermentations in
mucosae-related niches deprived of oxygen into mechan-
ically expulsed methane [15], the genomic diversity of
*M. smithii* has been poorly studied. As of February 2025,
GenBank contained only four complete whole genome
sequences and 167 partial sequences obtained from the
human digestive tract, mainly from the gut. Our labora-
tory generated four additional digestive tract genome
sequences along with two from human blood, one from
a human brain abscess, and one from human breast
milk. The remaining 16 genomes came from non-human
sources: 11 from pig guts, two from mice digestive tracts,
two from monkey guts, and the reference genome (ATCC
35061) from an anthropic sewage digester (https://www.
ncbi.nlm.nih.gov/datasets/genome/?taxon=2173).

By investigating the presence of *M. smithii* in left-
over urine specimens routinely submitted to the clinical
microbiology laboratory in search of urinary tract infec-
tions in line with medical ethics regulations in France,
we identified an additional *M. smithii* isolate (strain U29)
[10]. This allowed us to determine the first *M. smithii*
whole genome sequence derived from a urine sample.

**Materials and methods**

Sample collection

Urine samples collected as part as the routine medical
management of patients suspected to be suffering from
urinary tract infections, were screened for *M. smithii*
using an RT-PCR followed by Sanger sequencing, as pre-
viously described [16]. Among the positive samples, the
*M. smithii* strain U29 was co-cultured with *Escherichia
coli* in an anaerobic Hungate tube with SAB medium [10,
16]. After a seven-day incubation at 37 °C, methane pro-
duction was measured by gas chromatography as a proxy
for methanogen growth. The *M. smithii* strain U29 was
then observed using the Hitachi TM4000 Plus tabletop
SEM (Hitachi, Tokyo, Japan), as previously described
[17].

DNA extraction and whole genome sequencing

After a 20-minute sonication of *M. smithii* U29 in SAB
broth [18], total DNA was extracted using the EZ1
Advanced XL extractor with the V 1.066069118 Qiagen
DNA bacteria card and the EZ1® DNA Tissue Kit (Qia-
gen, Courtaboeuf, France), as described by the manufac-
turer. This was then quantified at 0.2 ng/µL using a Qubit
assay with the high sensitivity kit (Life technologies,
Carlsbad, CA, USA). Genomic DNA was then sequenced
using MiSeq technology (Illumina Inc, San Diego, CA,

USA) followed by the paired-end strategy and barcod-
ing, in order to be mixed with 21 other genomic projects
(engaging 1 ng of each genome) prepared with the Nex-
tera XT DNA sample prep kit (Illumina). The “tagmen-
tation” step fragmented and tagged the DNA, and 12
cycles of PCR amplification completed the tag adapters
and introduced dual-index barcodes. After purification
on CleanNGS beads (CLENA, Coenecoop 75, 2741 PH
Waddinxveen, Netherlands), libraries were normalised
on specific beads according to the Nextera XT proto-
col (Illumina) and pooled into a single library for MiSeq
sequencing. Automated cluster generation and paired-
end sequencing with dual-index reads were performed in
a single 39-hour run in 2 × 250-bp. Total information of
9.34 Gb was obtained from a 1416 K/mm² cluster den-
sity, with a cluster passing quality control filters of 75%.
Within this run, the value of the representation index for
U29 was 8.03%. The 24 904 239 paired-end reads were fil-
tered according to read qualities.

Genome annotation

The *M. smithii* U29 genome sequence was annotated under standard parameters using the DFast annotation server [19] (https://dfast.ddbj.nig.ac.jp/), which provides automated functional and structural genome annotations. To detect viral sequences integrated within the genomes of *M. smithii* U29, we performed BLASTn searches (Basic Local Alignment Search Tool for nucleotides) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [20]. CDSs annotated in *M. smithii* U29 were compared against a custom library containing 39 561 *Caudoviricetes* genomic sequences (taxid: 2832643). BLASTn analysis was performed using standard parameters with query cover and identity ≥ 95% to ensure high matching with viral sequences. To verify and categorise the viral coding sequences, BLASTx searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were conducted using standard parameters against a custom viral protein database consisting of 801 452 389 *Caudoviricetes* (taxid: 2832643) proteins, with query cover and identity ≥ 50%, these criteria enabled the detection of viral proteins, even in cases where the sequences exhibited significant divergence from known viral proteins. CRISPR arrays and Cas proteins were identified and typed within *M. smithii* U29 genome using the CRISPRCasFinder tool (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) and CR ISPRCasTyper (https://crisprcastyper.crispr.dk), two we b-based platforms integrating four distinct CRISPR-Cas detection algorithms and providing comprehensive classification based on the latest available CRISPR-Cas system taxonomy [21, 22]. Only CRISPR arrays categorised as Level 4 (the highest confidence level) were considered in this study.
## Genome comparison

Average nucleotide identity (ANI) was conducted using Proksee (https://proksee.ca) [23] to compare *M. smithii* U29 genome with other *M. smithii* complete genomes available in NCBI genome database (https://www.ncbi.nlm.nih.gov/home/genomes/): *M. smithii* ATCC35061 (CP000678.1), *M. smithii* KB11 (CP017803.1), *M. smithii* CE91-St67 (AP025586.1), and *M. smithii* CE91-St68 (AP025587.1).

## Data Availability

The *M. smithii* strain U29 genome is available in Gen-Bank with ASSEMBLY_NAME: GCA_041537865.1; ASSEMBLY_ACC: PRJNA 1,150,393; BioSample: SAMN43285203; BioProject: PRJNA1150393; ACCES-SION JBGOSM000000000.

## Results

The 1 822 124-bp (111 scaffolds and 31% G+C) *M. smithii* strain U29 genome, devoid of any detectable nanoarchaeal sequence (NCBI Prokaryotic Genome Annotation Pipeline) [24] yielded 1745 protein-coding sequences (CDS), including 56 viral CDS, one complete 5'-16 S-23 S rRNA gene cluster, 36 tRNA genes, one Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) with 39 spacers, including 13 spacers matching with *Caudoviricetes* sp. sequences and one CRISPR-associated protein (Cas) cluster type I-B (https://crisprcastyper.crispr.dk) [22] (Fig. 1). Using Proksee

(https://proksee.ca/) [23], a comparison of the *M. smithii* U29 genome with other *M. smithii* complete genomes yielded an average nucleotide identity (ANI) of 99.86% with *M. smithii* ATCC35061 (CP000678.1) derived from sewage digester, and 97.99% with *M. smithii* strain KB11 (CP017803.1), 97.92% with *M. smithii* strain CE91-St67 (AP025586.1), and 97.78% with *M. smithii* strain CE91-St68 (AP025587.1), all derived from human gut samples.

The *M. smithii* U29 genome was 31 036-bp smaller than the closest sewage *M. smithii* ATCC35061 genome without any difference in coding capacity between the genomes (Fig. 1; Table 1). However, the urinary tract *M. smithii* U29 genome was longer than its digestive tract counterpart strains, i.e. strain KB11 (16 579-bp), strain CE91-St67 (30 971-bp), and strain CE91-St68 (30 969-bp); encoding 69 more CDS including 56 hypothetical proteins than both *M. smithii* CE91-St67 and CE91-St68 and 71 more CDS (including 54 hypothetical proteins) than *M. smithii* KB11 (Table 1). A general BLAST comparison indicated that the complementary genomic material present in U29 but absent in CE91-St67, CE91-St68, and KB11, corresponded to 53 CDS including 40 non-viral CDS (including 30 hypothetical proteins) and 13 viral CDS (including seven hypothetical proteins) matching with *M. smithii* tailed virus 1 sequences (query cover > 90%; identity > 95%). The *M. smithii* U29 genome specifically encodes ten annotated genes absent in the KB11, CE91-St67, and CE91-St68 genomes, including pyridoxamine 5'-phosphate oxidase family protein,

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Genomic map of *M. smithii* U29 created with Proksee software (https://proksee.ca). CDS, viral CDS, CRISPR-Cas system, rRNA, tRNA, and GC content were presented in this map, also *M. smithii* U29 alignment with the reference strain genome (ATCC 35061) in blue, and the three other complete genomes of *M. smithii* derived from gut samples: KB11, CE91-St67, and CE91-St68, indicated in purple, yellow, and green, respectively</figcaption></figure>
<table><caption>**Table 1** Overview of 87 CDS unique to *M. smithii* U29 from urine and absent in gut-derived strains</caption><thead><tr><th>Locus name</th><th>Contig</th><th>Start</th><th>Stop</th><th>Product</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th><th>ATCC35061</th><th>Viral sequence</th><th>Name of virus</th></tr></thead><tbody><tr><td>LOCUS_00740</td><td>sequence001</td><td>65,122</td><td>65,523</td><td>pyridoxamine 5'-phosphate oxidase family protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_00790</td><td>sequence001</td><td>69,845</td><td>70,228</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_01590</td><td>sequence003</td><td>7546</td><td>8706</td><td>ATP-binding protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02050</td><td>sequence004</td><td>106</td><td>357</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02110</td><td>sequence004</td><td>4926</td><td>6554</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02130</td><td>sequence004</td><td>7128</td><td>7475</td><td>Serine protease</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02140</td><td>sequence004</td><td>7478</td><td>8092</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02150</td><td>sequence004</td><td>8151</td><td>9161</td><td>Major capsid protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02160</td><td>sequence004</td><td>9452</td><td>9817</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02170</td><td>sequence004</td><td>9857</td><td>10,300</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02180</td><td>sequence004</td><td>10,297</td><td>10,758</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02190</td><td>sequence004</td><td>10,764</td><td>12,005</td><td>Putative tail tube protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Locus name</th><th>Contig</th><th>Start</th><th>Stop</th><th>Product</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th><th>ATCC35061</th><th>Viral sequence</th><th>Name of virus</th></tr></thead><tbody><tr><td>LOCUS_02200</td><td>sequence004</td><td>12,088</td><td>12,522</td><td>Tail assembly chaperone protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methano-brevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02210</td><td>sequence004</td><td>12,509</td><td>12,742</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methano-brevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02220</td><td>sequence004</td><td>12,742</td><td>17,223</td><td>Tail tape measure protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methano-brevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02230</td><td>sequence004</td><td>17,230</td><td>17,967</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methano-brevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02240</td><td>sequence004</td><td>17,993</td><td>20,815</td><td>Tail protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methano-brevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_02260</td><td>sequence004</td><td>23,361</td><td>25,010</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_02300</td><td>sequence004</td><td>26,894</td><td>27,046</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_02970</td><td>sequence006</td><td>5361</td><td>5822</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_02980</td><td>sequence006</td><td>5725</td><td>6030</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_02990</td><td>sequence006</td><td>6156</td><td>6638</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_04540</td><td>sequence010</td><td>21,873</td><td>22,304</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_04670</td><td>sequence011</td><td>647</td><td>775</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_05160</td><td>sequence012</td><td>2977</td><td>3306</td><td>TIGR04076 family protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_05250</td><td>sequence012</td><td>19,696</td><td>20,451</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_07270</td><td>sequence018</td><td>2709</td><td>2924</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_07950</td><td>sequence020</td><td>351</td><td>497</td><td>Hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_07970</td><td>sequence020</td><td>1503</td><td>2108</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_07990</td><td>sequence020</td><td>2953</td><td>3441</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08630</td><td>sequence022</td><td>940</td><td>1455</td><td>Hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08640</td><td>sequence022</td><td>1466</td><td>5050</td><td>BREX system P-loop protein BrxC</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08650</td><td>sequence022</td><td>5055</td><td>5633</td><td>DUF1788 domain-containing protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08660</td><td>sequence022</td><td>5633</td><td>6226</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08670</td><td>sequence022</td><td>6300</td><td>8423</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Locus name</th><th>Contig</th><th>Start</th><th>Stop</th><th>Product</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th><th>ATCC35061</th><th>Viral sequence</th><th>Name of virus</th></tr></thead><tbody><tr><td>LOCUS_08680</td><td>sequence022</td><td>8712</td><td>12,230</td><td>BREX-1 system adenine-specific DNA-methyl-transferase PglX</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08690</td><td>sequence022</td><td>12,233</td><td>13,801</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08700</td><td>sequence022</td><td>13,817</td><td>15,757</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08710</td><td>sequence022</td><td>15,858</td><td>16,292</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08720</td><td>sequence022</td><td>16,312</td><td>19,428</td><td>BREX-1 system adenine-specific DNA-methyl-transferase PglX</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08740</td><td>sequence022</td><td>20,643</td><td>21,557</td><td>DUF5655 domain-containing protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08860</td><td>sequence023</td><td>4163</td><td>4705</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08900</td><td>sequence023</td><td>7182</td><td>7355</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_08920</td><td>sequence023</td><td>8544</td><td>8819</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_09550</td><td>sequence026</td><td>1876</td><td>2622</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_09580</td><td>sequence026</td><td>4133</td><td>5605</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_11620</td><td>sequence036</td><td>4871</td><td>5560</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_11640</td><td>sequence036</td><td>5910</td><td>6131</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12070</td><td>sequence038</td><td>14,970</td><td>15,629</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12150</td><td>sequence039</td><td>8399</td><td>8761</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12620</td><td>sequence043</td><td>175</td><td>1257</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12660</td><td>sequence043</td><td>5555</td><td>5902</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12670</td><td>sequence043</td><td>5904</td><td>6632</td><td>ImmA/IrrE family metallo-endopeptidase</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12680</td><td>sequence043</td><td>6625</td><td>7161</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12890</td><td>sequence045</td><td>2189</td><td>2611</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_13020</td><td>sequence045</td><td>12,792</td><td>13,253</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_13440</td><td>sequence048</td><td>5996</td><td>6427</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_13460</td><td>sequence048</td><td>7890</td><td>8063</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_13520</td><td>sequence048</td><td>9354</td><td>9671</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>Yes</td><td>Methanobrevibacter smithii tailed virus 1</td></tr><tr><td>LOCUS_13570</td><td>sequence048</td><td>11,318</td><td>11,563</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14270</td><td>sequence053</td><td>9529</td><td>10,005</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14280</td><td>sequence053</td><td>9986</td><td>10,183</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_15410</td><td>sequence062</td><td>9125</td><td>10,072</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_16390</td><td>sequence074</td><td>6695</td><td>7735</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_16580</td><td>sequence077</td><td>2352</td><td>3251</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_16590</td><td>sequence077</td><td>3331</td><td>3645</td><td>hypothetical protein</td><td>Abscent</td><td>Present</td><td>Present</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_16630</td><td>sequence077</td><td>6184</td><td>6315</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_17310</td><td>sequence093</td><td>527</td><td>2575</td><td>protease Lon-related BREX system protein BrxL</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_17390</td><td>sequence096</td><td>275</td><td>676</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_17400</td><td>sequence096</td><td>976</td><td>1482</td><td>hypothetical protein</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_17540</td><td>sequence106</td><td>79</td><td>1017</td><td>DNA-processing protein DprA</td><td>Abscent</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_00750</td><td>sequence001</td><td>65,526</td><td>65,903</td><td>helix-turn-helix domain-containing protein</td><td>present</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_00840</td><td>sequence002</td><td>4592</td><td>5353</td><td>Hypothetical protein</td><td>present</td><td>Abscent</td><td>Abscent</td><td>Present</td><td>No</td><td></td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Locus name</th><th>Contig</th><th>Start</th><th>Stop</th><th>Product</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th><th>ATCC35061</th><th>Viral sequence</th><th>Name of virus</th></tr></thead><tbody><tr><td>LOCUS_00910</td><td>sequence002</td><td>10,519</td><td>10,719</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_03840</td><td>sequence008</td><td>14,355</td><td>15,356</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_04120</td><td>sequence009</td><td>8065</td><td>9078</td><td>iron ABC transporter permease</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_11630</td><td>sequence036</td><td>5564</td><td>5893</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_12630</td><td>sequence043</td><td>1302</td><td>2042</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14150</td><td>sequence052</td><td>6497</td><td>7216</td><td>methyltransferase domain-containing protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14580</td><td>sequence055</td><td>7076</td><td>8245</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14590</td><td>sequence055</td><td>8248</td><td>10,809</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14600</td><td>sequence055</td><td>10,964</td><td>11,428</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14610</td><td>sequence055</td><td>11,701</td><td>11,946</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14630</td><td>sequence056</td><td>1375</td><td>2076</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_14640</td><td>sequence056</td><td>2090</td><td>2254</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_16570</td><td>sequence077</td><td>669</td><td>2123</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr><tr><td>LOCUS_16640</td><td>sequence077</td><td>6310</td><td>6480</td><td>Hypothetical protein</td><td>present</td><td>Absent</td><td>Absent</td><td>Present</td><td>No</td><td></td></tr></tbody></table>

This table presents a detailed comparison of 87 CDS that are exclusively found in *M. smithii* U29 isolated from urine and *M. smithii* ATCC35061 isolated from a sewage digester but are absent in the gut-derived strains *M. smithii* KB11, *M. smithii* CE91-St67, and *M. smithii* CE91-St68. It provides the exact genomic positions of each CDS within the U29 genome, along with the corresponding protein products encoded. Additionally, the table specifies the origin of each CDS, indicating whether it is of viral origin or non-viral origin. This comprehensive comparison helps to elucidate the genetic variations that may be associated with the adaptation of *M. smithii* U29 to the urinary environment

ATP-binding protein, and TIGR04076 family protein. In addition, it encodes proteins associated with the BREX system, including the BREX system P-loop protein BrxC, BREX-1 system adenine-specific DNA-methyltransferase PglX (appearing twice), and the protease Lon-related BREX system protein BrxL. Additionally, it encodes domain-containing proteins such as DUF1788 and DUF5655, along with the DNA-processing protein DprA (Table 1). Furthermore, six known viral CDS exclusively present in the *M. smithii* U29 genome include a serine protease, a major capsid protein, a putative tail tube protein, a tail assembly chaperone protein, a tail tape measure protein, and a tail protein (Tables 1 and 2) (Fig. 2).

In addition, 18 CDS including 17 hypothetical proteins exclusively lack in KB11, while 16 CDS, including 13 hypothetical proteins, were absent in CE91-St67 and CE91-St68 (Table 1). The only annotated protein missing in KB11 but present in the other genomes was an ImmA/ IrrE family metallo-endopeptidase. Meanwhile, the three known proteins absent exclusively in CE91-St67 and CE91-St68 were a helix-turn-helix domain-containing protein, an iron ABC transporter permease, and a methyltransferase domain-containing protein (Table 1). Notably, out of 53 CDS found exclusively in *M. smithii* U29 and *M. smithii* ATCC 35,061 but absent in *M. smithii* KB11, *M. smithii* CE91-St67, and *M. smithii* CE91-St68, only one CDS, encoding the TIGR04076 family protein, was present in the genomes of *M. smithii* strains derived

from the blood and brain, while the remaining 52 CDS were absent.

## Discussion

This study suggests that the *M. smithii* U29 strain, recovered from a urine sample, nevertheless originated from the digestive tract, expanding the knowledge of *M. smithii* methanogen diversity in this particular ecosystem. Three *M. smithii* variants are now recognised, distinguished by their genome size and relationship with nanoarchaea [25]. *M. smithii* small cell variants exhibiting a small (<1 805 545-bp) genome size have the potential to translocate (i.e., cross intestinal barriers) in extra-urinary tract tissues. *M. smithii* intermediate cell variants exhibiting an intermediate (1 822 124 to 1 853 160-bp) genome size have the potential to translocate to urinary tract tissues. Finally, *M. smithii* associated with nanoarchaea forming large cell variants exhibiting a large (> 2 413 006-bp) genome size (previously named *M. smithii* nanogens) have no potential to translocate [25]. The specific translocation pathways, i.e. the routes by which *M. smithii* U29 may have disseminated beyond the gut, remain to be elucidated. Theoretical routes include haematogenous dissemination (via the bloodstream), lymphatic dissemination (through the lymphatic system), and neural dissemination (along nerve-associated structures) (Fig. 3).

Accordingly, the unique genetic composition of *M. smithii* U29 suggests potential adaptations for persistence in the urinary tract. The pyridoxamine 5'-phosphate
<table><caption>**Table 2** Overview of 56 viral CDS identified in the genome of *M. smithii* U29</caption><thead><tr><th colspan="10">Positions inside M. smithii U29</th></tr><tr><th colspan="10">Blastn</th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91 St68</th></tr></thead><tbody><tr><td>sequence001</td><td>22,600</td><td>23,388</td><td>Caudo-viricetes sp. isolate ct8Fg25</td><td>100</td><td>100</td><td>495</td><td>BK051088.1</td><td>Putative transcriptional regulator</td><td>63</td><td>98.8</td><td>176</td><td>DAU39878.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence001</td><td>23,394</td><td>24,203</td><td>Caudo-viricetes sp. isolate ct8Fg25</td><td>100</td><td>99.51</td><td>810</td><td>BK051088.1</td><td>Putative transcriptional regulator</td><td>100</td><td>100</td><td>269</td><td>DAU39867.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence001</td><td>67,333</td><td>68,703</td><td>Caudo-viricetes sp. isolate ct8Fg25</td><td>30</td><td>99.28</td><td>417</td><td>BK057997.1</td><td>Putative Zn-dependent protease</td><td>30</td><td>100</td><td>138</td><td>DAQ56314.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence001</td><td>68,722</td><td>69,279</td><td>Caudo-viricetes sp. isolate ct8Fg01</td><td>100</td><td>99.28</td><td>558</td><td>BK025467.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>185</td><td>DAL47471.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence001</td><td>70,479</td><td>71,489</td><td>Caudo-viricetes sp. isolate ctNHp2</td><td>48</td><td>99.18</td><td>490</td><td>BK032123.1</td><td>Putative GTPase</td><td>48</td><td>100</td><td>166</td><td>DAL47444.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence004</td><td>951</td><td>1124</td><td>Methanobacter smithii tailed virus 1</td><td>100</td><td>100</td><td>174</td><td>PP537965.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>57</td><td>XCD08680.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>1114</td><td>1413</td><td>Methanobacter smithii tailed virus 1</td><td>100</td><td>100</td><td>300</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>99</td><td>XCD08681.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="3">Positions inside M. smithii U29 genome</th><th colspan="3">Blastn</th><th colspan="3">Blastx</th><th colspan="3"></th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th></tr></thead><tbody><tr><td>sequence004</td><td>1572</td><td>2165</td><td>Metha-nobre-vibacter smithii-tailed</td><td>100</td><td>100</td><td>594</td><td>PP537965.1</td><td>putative terminase small subunit</td><td>99</td><td>100</td><td>197</td><td>XCD08682.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>2162</td><td>3520</td><td>Metha-nobre-vibacter smithii-tailed</td><td>100</td><td>100</td><td>1359</td><td>PP537965.1</td><td>Putative large sub-unit terminase</td><td>98</td><td>100</td><td>452</td><td>XCD08683.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>3523</td><td>4929</td><td>Metha-nobre-vibacter smithii-tailed</td><td>100</td><td>100</td><td>1407</td><td>PP537965.1</td><td>Putative portal protein</td><td>100</td><td>100</td><td>468</td><td>XCD08684.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>7128</td><td>7475</td><td>Metha-nobre-vibacter smithii-tailed</td><td>100</td><td>100</td><td>348</td><td>PP537965.1</td><td>Serine protease</td><td>99</td><td>99.13</td><td>115</td><td>DAF52396.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>7478</td><td>8092</td><td>Metha-nobre-vibacter smithii-tailed</td><td>100</td><td>100</td><td>615</td><td>PP537965.1</td><td>Hypothetical protein</td><td>100</td><td>100</td><td>204</td><td>XCD08688.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>8151</td><td>9161</td><td>Metha-nobre-vibacter smithii-tailed</td><td>100</td><td>100</td><td>1011</td><td>PP537965.1</td><td>Major capsid protein</td><td>100</td><td>100</td><td>336</td><td>XCD08689.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="3">Positions inside M. smithii U29 genome</th><th colspan="3">Blastn</th><th colspan="3">Blastx</th><th colspan="3"></th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th></tr></thead><tbody><tr><td>sequence004</td><td>9452</td><td>9817</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>366</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>121</td><td>XCD08690.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>9857</td><td>10,300</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>444</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>147</td><td>XCD08691.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>10,297</td><td>10,758</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>462</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>153</td><td>XCD08692.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>10,764</td><td>12,005</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>1242</td><td>PP537965.1</td><td>Putative tail tube protein</td><td>100</td><td>100</td><td>413</td><td>XCD08693.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>12,088</td><td>12,522</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>435</td><td>PP537965.1</td><td>Tail assembly chap-erne protein</td><td>99</td><td>98.62</td><td>145</td><td>DAF52422.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>12,509</td><td>12,742</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>234</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>98.7</td><td>77</td><td>XCD08695.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="3">Positions inside M. smithii U29 genome</th><th colspan="3">Blastn</th><th colspan="3">Blastx</th><th colspan="3"></th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th></tr></thead><tbody><tr><td>sequence004</td><td>12,742</td><td>17,223</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>4482</td><td>PP537965.1</td><td>Tail tape measure protein</td><td>100</td><td>100</td><td>1493</td><td>XCD08696.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>17,230</td><td>17,967</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>738</td><td>PP537965.1</td><td>Hypothetical protein</td><td>100</td><td>100</td><td>245</td><td>XCD08697.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>17,993</td><td>20,815</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>2823</td><td>PP537965.1</td><td>Tail protein</td><td>100</td><td>99.62</td><td>956</td><td>DAF52425.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>25,037</td><td>25,630</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>594</td><td>PP537965.1</td><td>Collagen triple helix repeat protein</td><td>99</td><td>97.97</td><td>197</td><td>DAF52389.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>25,656</td><td>25,808</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>153</td><td>PP537965.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>50</td><td>XCD08702.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence004</td><td>25,801</td><td>26,619</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>819</td><td>PP537965.1</td><td>Pseudomurein endo-isopeptidase</td><td>100</td><td>100</td><td>272</td><td>XCD08703.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence014</td><td>92</td><td>490</td><td>Caudo-viricetes sp. isolate ctejj21</td><td>100</td><td>99.25</td><td>399</td><td>BK030749.1</td><td>Hypothetical protein</td><td>99</td><td>98.48</td><td>132</td><td>DAI44027.1</td><td>1</td><td>1</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="3">Positions inside M. smithii U29 genome</th><th colspan="3">Blastn</th><th colspan="3">Blastx</th><th colspan="3"></th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th></tr></thead><tbody><tr><td>sequence033</td><td>15,544</td><td>16,761</td><td>Caudo-viricetes</td><td>100</td><td>99.59</td><td>1218</td><td>BK025422.1</td><td>Zinc finger domain-containing protein</td><td>100</td><td>99.75</td><td>405</td><td>DAY53111.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence034</td><td>5442</td><td>6269</td><td>sp. isolate ct13N6</td><td>100</td><td>99.76</td><td>828</td><td>BK026636.1</td><td>Hypothetical protein</td><td>100</td><td>99.27</td><td>275</td><td>DAZ36128.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence034</td><td>6607</td><td>7617</td><td>sp. isolate ctJX913</td><td>100</td><td>99.9</td><td>1011</td><td>BK026636.1</td><td>50 S ribosomal protein L3p</td><td>100</td><td>100</td><td>336</td><td>DAZ36127.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence048</td><td>240</td><td>929</td><td>sp. isolate ctJX913</td><td>100</td><td>100</td><td>690</td><td>BK056828.1</td><td>hisA/hisF family protein</td><td>100</td><td>100</td><td>229</td><td>DAR12815.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence048</td><td>948</td><td>1343</td><td>sp. isolate ct7Qc8</td><td>100</td><td>100</td><td>396</td><td>BK056828.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>131</td><td>DAR12859.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence048</td><td>1397</td><td>1579</td><td>sp. isolate ct7Qc8</td><td>100</td><td>100</td><td>183</td><td>BK056828.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>60</td><td>DAR12858.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence048</td><td>1677</td><td>2576</td><td>sp. isolate ct7Qc8</td><td>100</td><td>100</td><td>900</td><td>BK056828.1</td><td>Co/Zn/Cd efflux system component</td><td>100</td><td>100</td><td>299</td><td>DAR12814.1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence048</td><td>2588</td><td>3742</td><td>Metha-nobacter vibacter smithii tailed virus 1</td><td>100</td><td>100</td><td>1155</td><td>PP537965.1</td><td>site-specific recombinase XerC</td><td>95</td><td>100</td><td>384</td><td>XCD08652.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>Table 2 (continued)</caption><thead><tr><th colspan="2">Positions inside M. smithii U29 genome</th><th colspan="2">Blastn</th><th colspan="2">Blastx</th><th rowspan="2">Query cover</th><th rowspan="2">Identity Percentage</th><th rowspan="2">Length</th><th rowspan="2">Accession</th><th rowspan="2">Protein Name</th><th rowspan="2">Hypothetical protein</th><th rowspan="2">99</th><th rowspan="2">100</th><th rowspan="2">Length</th><th rowspan="2">Accession</th><th rowspan="2">ATCC 35,061</th><th rowspan="2">KB11</th><th rowspan="2">CE91-St67</th><th rowspan="2">CE91-St68</th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th></tr></thead><tbody><tr><td>sequence048</td><td>3812</td><td>4087</td><td>Caudo-viricetes sp. isolate ct13N6</td><td>100</td><td>100</td><td>276</td><td>BK025422.1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>sequence048</td><td>4107</td><td>4466</td><td>Metha-nobrevibacter smithii tailed</td><td>100</td><td>100</td><td>360</td><td>PP537965.1</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>sequence048</td><td>4471</td><td>4896</td><td>Metha-nobrevibacter smithii tailed</td><td>100</td><td>100</td><td>426</td><td>PP537965.1</td><td>putative HEPN domain containing protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>sequence048</td><td>4938</td><td>5270</td><td>Metha-nobrevibacter smithii tailed</td><td>100</td><td>100</td><td>333</td><td>PP537965.1</td><td>putative DNA-binding protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>sequence048</td><td>6488</td><td>7711</td><td>Metha-nobrevibacter smithii tailed</td><td>100</td><td>100</td><td>1224</td><td>PP537965.1</td><td>putative ORC1/Cdc6 replication initiation protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>sequence048</td><td>8242</td><td>8385</td><td>Metha-nobrevibacter smithii tailed</td><td>100</td><td>100</td><td>144</td><td>PP537965.1</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>sequence048</td><td>8382</td><td>8606</td><td>Metha-nobrevibacter smithii tailed</td><td>100</td><td>100</td><td>225</td><td>PP537965.1</td><td>Hypothetical protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="3">Positions inside M. smithii U29 genome</th><th colspan="3">Blastn</th><th colspan="3">Blastx</th><th colspan="3"></th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th></tr></thead><tbody><tr><td>sequence048</td><td>8607</td><td>8882</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>276</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>91</td><td>XCD08662.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>8879</td><td>9058</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>180</td><td>PP537965.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>59</td><td>XCD08663.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>9055</td><td>9354</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>300</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>99</td><td>XCD08664.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>9691</td><td>10,302</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>612</td><td>PP537965.1</td><td>ERF superfamily protein</td><td>100</td><td>95.07</td><td>203</td><td>DAI44006.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>10,305</td><td>10,499</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>195</td><td>PP537965.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>64</td><td>XCD08667.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>10,496</td><td>10,750</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>255</td><td>PP537965.1</td><td>putative DNA-binding protein</td><td>99</td><td>100</td><td>84</td><td>XCD08668.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Positions inside M. smithii U29 genome</th><th>Contig</th><th>Start</th><th>Stop</th><th>Phage Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>Protein Name</th><th>Query cover</th><th>Identity Percentage</th><th>Length</th><th>Accession</th><th>ATCC 35,061</th><th>KB11</th><th>CE91-St67</th><th>CE91-St68</th></tr></thead><tbody><tr><td>sequence048</td><td>10,747</td><td>11,166</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>420</td><td>PP537965.1</td><td>putative DNA-binding protein</td><td>99</td><td>100</td><td>139</td><td>XCD08669.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>11,560</td><td>11,733</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>174</td><td>PP537965.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>57</td><td>XCD08671.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>11,730</td><td>11,915</td><td>Caudo-viricetes sp. Isolate cte1D3</td><td>100</td><td>100</td><td>186</td><td>BK024908.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>61</td><td>XCD08672.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>11,921</td><td>12,262</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>342</td><td>PP537965.1</td><td>ASCH domain-containing protein</td><td>82</td><td>98.94</td><td>94</td><td>DAY47871.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>12,276</td><td>12,422</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>147</td><td>PP537965.1</td><td>Hypothetical protein</td><td>98</td><td>100</td><td>48</td><td>XCD08674.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>12,594</td><td>12,857</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>264</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>100</td><td>87</td><td>XCD08675.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>sequence048</td><td>12,844</td><td>13,074</td><td>Metha-nobre-vibacter smithii tailed</td><td>100</td><td>100</td><td>231</td><td>PP537965.1</td><td>Hypothetical protein</td><td>99</td><td>98.68</td><td>76</td><td>XCD08676.1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="2">Positions inside M. smithii U29<br>genome</th><th colspan="2">Blastn</th><th colspan="2">Blastx</th><th rowspan="2">Query<br>cover</th><th rowspan="2">Identity<br>Percentage</th><th rowspan="2">Length</th><th rowspan="2">Accession</th><th rowspan="2">Accession</th><th rowspan="2">ATCC<br>35,061</th><th rowspan="2">KB11<br>CE91-S167</th><th rowspan="2">CE91-S168</th></tr><tr><th>Contig</th><th>Start</th><th>Stop</th><th>Phage<br>Name</th><th>Query<br>cover</th><th>Identity<br>Percentage</th><th>Protein Name</th><th>Query<br>cover</th><th>Identity<br>Percentage</th><th>Length</th><th>Accession</th><th>Accession</th><th>ATCC<br>35,061</th><th>KB11<br>CE91-S167</th><th>CE91-S168</th></tr></thead><tbody><tr><td>sequence048</td><td>13,079</td><td>13,393</td><td>Metha-<br>nobre-<br>vibacter<br>smithii<br>tailed<br>virus 1</td><td>100</td><td>100</td><td>C2H2 type<br>zinc-finger-protein</td><td>98</td><td>98.06</td><td>315</td><td>PP537965.1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sequence048</td><td>13,390</td><td>13,734</td><td>Metha-<br>nobre-<br>vibacter<br>smithii<br>tailed<br>virus 1</td><td>100</td><td>100</td><td>Hypothetical<br>protein</td><td>99</td><td>100</td><td>345</td><td>PP537965.1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

This table summarises 56 viral CDS found within the genome of *M. smithii* U29. It provides the precise genomic locations of each CDS and presents the results of BLASTn analysis, including the associated phage name, query coverage, identity percentage, alignment length, and phage accession number. Additionally, the table displays the results of BLASTx analysis, detailing the corresponding viral protein name, query coverage, identity percentage, alignment length, and protein accession number. The table also indicates the presence (score 1) or absence (score 0) of each viral CDS in other *M. smithii* strains, including ATCC35061, KB11, CE91-S167, and CE91-S168, enabling a comparative genomic analysis to identify strain-specific viral elements

oxidase family protein, involved in vitamin B6 metabolism, may confer oxidative stress resistance [26], while the ATP-binding protein could play a role in transport functions or stress adaptation in a nutrient-variable environment such as urine [27]. Additionally, the putative fimbriae-associated TIGR04076 family protein may facilitate the adhesion of *M. smithii* U29 to uroepithelial cells, while the DNA-processing protein DprA confers capacity for DNA repair and transformation, enhancing genome stability under stressful conditions [28]. BREX system-associated proteins, such as the P-loop protein BrxC, BREX-1 system adenine-specific DNA-methyl-transferase PglX (appearing twice), and BrxL protease signal a defence system against foreign DNA, including bacteriophage infections [29]. This system may enhance the genome integrity of *M. smithii* U29 and its immune evasion, allowing it thriving within the urinary tract where microbial competition and host immune pressures differ from those in the gut. Additionally, DUF1788 and DUF5655 domain-containing proteins may play roles in stress response, biofilm formation, or signalling processes that could further support the survival of *M. smithii* U29 outside its primary gut environment [30].

The detection of six phage-related sequences in the *M. smithii* U29 strain raises intriguing questions regarding their potential role in host-microbe interactions. A serine protease may assist in protein degradation and stress adaptation [31]. Additionally, the identification of structural and functional phage-related proteins, such as the major capsid protein, putative tail tube protein, tail assembly chaperone protein, tail tape measure protein, and tail protein, suggests that *M. smithii* U29 may have acquired phage-derived functions that enhance its survival and persistence in the urinary tract, potentially providing resistance to bacteriophages, stress tolerance, or metabolic advantages. More intriguingly, the presence of these phage-related components raises the possibility of a lysogenic state that could switch to a lytic cycle under specific environmental conditions. This suggests that *M. smithii* U29 might opportunistically produce virions in response to certain stressors or host environmental changes. Such a scenario could influence its adaptability, persistence, and potential role in the urinary tract microbiome. To explore this hypothesis, routine electron microscopy observations of urine samples infected with *M. smithii* could be particularly insightful. Detecting virions directly within these samples would provide compelling evidence for the occurrence of a lytic cycle and offer new perspectives on the ecological and clinical significance of *M. smithii* in extra-intestinal sites. However, without functional assays, it remains unclear whether these elements actively contribute to the strain's adaptability or translocation ability. Additionally, while *M. smithii* has been associated with various extra-intestinal
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Micrographs of *M. smithii* strain U29. (A) Diplococcus shape of *M. smithii* U29. (B) *M. smithii* U29 size with 1.28 µm length and 461 nm width. Micrographs observed using the Hitachi TM4000 Plus tabletop scanning electron microscope (Hitachi, Tokyo, Japan)</figcaption></figure>

sites, its clinical significance remains uncertain. Further investigations are required to clarify whether it plays a role in urinary tract colonization or merely represents a transient environmental contaminant.

Our findings also highlight limitations in genomic sampling. The available *M. smithii* genomes are largely derived from gut-associated strains, which clearly biases and limits inferring translocation potential. Further metagenomic, transcriptomic, and proteomic studies, as well as in vitro and in vivo experimental models, are needed to validate the hypotheses proposed here. Experimental approaches should aim to assess whether *M. smithii* can persist in the urinary tract, whether its genetic adaptations confer specific survival advantages, and whether its viral elements contribute to horizontal gene transfer or pathogenicity.

While the idea that *M. smithii* intermediate cell variants may serve as carriers of viral sequences, akin to a “Trojan Horse” mechanism, the biological relevance of this hypothesis remains to be determined by further observations and experiments, investigating whether these viral elements influence host interactions, microbial ecology, or niche adaptation, or whether their presence is incidental.

## Conclusion

Our study expands our understanding of *M. smithii* diversity and its potential for translocation beyond the gut, including adaptation of the *M. smithii* U29 strain to the urinary tract. Unique genomic features, such as stress response and adhesion-related genes, suggest possible mechanisms for its persistence, although functional validation is needed. The presence of phage-related sequences raised questions about their role in genome plasticity and microbial interactions. Future studies using metagenomic, transcriptomic, and experimental approaches are essential to confirm these hypotheses and assess the clinical significance of *M. smithii* beyond the digestive system.
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Illustration showing different potential routes of *M. smithii* translocation from the gastrointestinal tract. *M. smithii* small cell variants exhibiting a small (< 1 805 545-bp) genome size have the potential to translocate (crossing the intestinal barriers) in extra-urinary tract tissues via the bloodstream (a), lymphatic system (b), or nerve-associated structures (c). *M. smithii* U29 illustrating intermediate cell variants exhibiting an intermediate (1 822 124 to 1 853 160-bp) genome size have the potential to translocate in urinary tract tissues (d). Finally, *M. smithii* associated to nanoarchaea forming large cell variants exhibiting a large (> 2 413 006-bp) genome size have no potential to translocate (e)</figcaption></figure>

**Author contributions**

IM: Experimental work, drafting; VB: Sequencing; MD: Study design, drafting and final writing; GG: Experimental work data interpretation, final writing and RR: Study design, final writing.

**Funding**

Ihab Malat was awarded a thesis scholarship from the Fondation Méditerranée Infection, Marseille, FRANCE.

**Data availability**

M. smithii strain U29 genome is available in GenBank with ASSEMBLY_
NAME: GCA_041537865.1; ASSEMBLY_ACC: PRJNA 1150393; BioSample:
SAMN43285203; SCAFFOLD_ACC: JBGOSM010000001-JBGOSM010000111, https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=2173.

## Declarations

### Competing interests

The authors declare no competing interests.

Received: 22 October 2024 / Accepted: 11 August 2025

Published online: 01 October 2025

## References

1. Grine G, Terrer E, Boualam MA, Aboudharam G, Chaudet H, Ruimy R, et al. Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota. Sci Rep. 2018;8:9197.

2. Miller TL, Wolin MJ. Enumeration of methanobrevibacter smithii in human feces. Arch Microbiol. 1982;131:14–8.

3. Camara A, Konate S, Tidjani Alou M, Kodio A, Togo AH, Cortaredona S, et al. Clinical evidence of the role of methanobrevibacter smithii in severe acute malnutrition. Sci Rep. 2021;11:5426.

4. Hassani Y, Aboudharam G, Drancourt M, Grine G. The discovery of candidatus Nanopusillus phoecensis sheds light on the diversity of the microbiota Nanoarchaea. *IScience*. 2024;27:109488.

5. Hassani Y, Brégeon F, Aboudharam G, Drancourt M, Grine G. Detection of methanobrevibacter smithii and methanobrevibacter oralis in lower respiratory tract microbiota. Microorganisms. 2020;8:1866.

6. Grine G, Boualam MA, Drancourt M. Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol. 2017;36:2449–55.

7. Togo AH, Grine G, Khelaifa S, des Robert C, Brevaut V, Caputo A, et al. Culture of methanogenic archaea from human colostrum and milk. Sci Rep. 2019;9:18653.

8. Drancourt M, Djemai K, Gouriet F, Grine G, Loukil A, Bedotto M, et al. Methanobrevibacter smithii archaeamia in febrile patients with bacteremia, including those with endocarditis. Clin Infect Dis. 2021;73:e2571–9.

9. Grine G, Drouet H, Fenollar F, Bretelle F, Raoult D, Drancourt M. Detection of methanobrevibacter smithii in vaginal samples collected from women diagnosed with bacterial vaginosis. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol. 2019;38:1643–9.

10. Grine G, Lotte R, Chirio D, Chevalier A, Raoult D, Drancourt M, et al. Co-culture of methanobrevibacter smithii with Enterobacteria during urinary infection. EBioMedicine. 2019;43:333–7.

11. Ufnar JA, Wang SY, Christiansen JM, Yampara-Iquise H, Carson CA, Ellender RD. Detection of the NifH gene of methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters. J Appl Microbiol. 2006;101:44–52.

12. Nicholson MJ, Evans PN, Joblin KN. Analysis of methanogen diversity in the rumen using Temporal temperature gradient gel electrophoresis: identification of uncultured methanogens. Microb Ecol. 2007;54:141–50.

13. Walters EM, Prather RS. Advancing swine models for human health and diseases. Mo Med. 2013;110:212–5.

14. Shoae S, Karlsson F, Mardinoglu A, Nookaew I, Bordel S, Nielsen J. Understanding the interactions between bacteria in the human gut through metabolic modeling. Sci Rep. 2013;3:2532.

15. Samuel BS, Hansen EE, Manchester JK, Coutinho PM, Henrissat B, Fulton R, et al. Genomic and metabolic adaptations of methanobrevibacter smithii to the human gut. Proc Natl Acad Sci U S A. 2007;104:10643–8.

16. Drancourt M, Nkamga VD, Lakhe NA, Régis J-M, Dufour H, Fournier P-E, et al. Evidence of archaeal methanogens in brain abscess. Clin Infect Dis Off Publ Infect Dis Soc Am. 2017;65:1–5.

17. Zmerli O, Bellali S, Haddad G, Hisada A, Ominami Y, Raoult D, et al. Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using phosphotungstic acid staining. Comput Struct Biotechnol J. 2023;21:3627–38.

18. Khelaifa S, Raoult D, Drancourt M. A versatile medium for cultivating methanogenic archaea. PLoS ONE. 2013;8:e61563.

19. Tanizawa Y, Fujisawa T, Nakamura Y. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinforma Oxf Engl. 2018;34:1037–9.

20. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: a better web interface. Nucleic Acids Res. 2008;36 Web Server issue:W5–9.

21. Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Néron B, et al. CRISPRCasFinder, an update of crisfinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res. 2018;46:W246–51.

22. Russel J, Pinilla-Redondo R, Mayo-Muñoz D, Shah SA, Sørensen SJ, CRISPRCas-Typer. Automated identification, annotation, and classification of CRISPR-Cas loci. CRISPR J. 2020;3:462–9.

23. Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C, et al. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023;51:W484–92.

24. Tatusova T, DiCuccio M, Badretdin A, Chetverinin V, Nawrocki EP, Zaslavsky L, et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016;44:6614–24.

25. Malat I, Drancourt M, Grine G. Methanobrevibacter smithii cell variants in human physiology and pathology: A review. Heliyon. 2024;10:e36742.

26. Ankisettypalli K, Cheng JJ-Y, Baker EN, Bashiri G. PdxH proteins of mycobacteria are typical members of the classical pyridoxine/pyridoxamine 5'-phosphate oxidase family. FEBS Lett. 2016;590:453–60.

27. Kou X, Zhao Z, Xu X, Li C, Wu J, Zhang S. Identification and expression analysis of ATP-binding cassette (ABC) transporters revealed its role in regulating stress response in Pear (Pyrus bretchneideri). BMC Genomics. 2024;25:169.

28. Sharma DK, Misra HS, Bihani SC, Rajpurohit YS. Biochemical properties and roles of DprA protein in bacterial natural transformation, virulence, and Pilin variation. J Bacteriol 205:e00465–22.

29. Goldfarb T, Sberro H, Weinstock E, Cohen O, Doron S, Charpak-Amikam Y, et al. BREX is a novel phage resistance system widespread in microbial genomes. EMBO J. 2015;34:169–83.

30. Lv P, Wan J, Zhang C, Hina A, Al Amin GM, Begum N, et al. Unraveling the diverse roles of neglected genes containing domains of unknown function (DUFs): progress and perspective. Int J Mol Sci. 2023;24:4187.

31. Baquero DP, Medvedeva S, Martin-Gallausiaux C, Pende N, Sartori-Rupp A, Tachon S, et al. Stable coexistence between an archaeal virus and the dominant methanogen of the human gut. Nat Commun. 2024;15:7702.

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