A genetic toolkit for the human gut bacterium Mediterraneibacter gnavus identifies capsular polysaccharides as a competitive colonization factor

Domain: biology
Published: 2026-03-12
Editorial outcome: minor_revision

Abstract
Mediterraneibacter gnavus is a human symbiotic gut bacterium whose abundance often increases in patients with various diseases, such as active inflammatory bowel disease (IBD). However, the genetic factors governing its gut colonization and pathogenicity remain elusive due to the lack of genetic modification systems. In this study, we developed several genetic tools for M. gnavus, including a shuttle vector, an inducible promoter, fluorescent reporters, and systems for gene disruption and deletion. Using these genetic tools, we constructed mutants for six of the eight sortase-encoding genes in M. gnavus ATCC 29149 and identified those involved in the surface presentation of capsular polysaccharide (CPS) and superantigen-like proteins. We also identified a CPS biosynthetic gene cluster adjacent to the sortase gene and demonstrated that CPS production is crucial for competitive colonization in germ-free mouse intestines. Notably, CPS production was inversely correlated with inflammatory activity, and CPS cluster-positive strains were more prevalent in healthy individuals than in Crohn’s disease patients. These findings suggest that CPS contributes to the modulation of inflammation and pathogenesis. This study highlights the potential of precise gene-modification systems to uncover genetic determinants of intestinal colonization and pathogenesis in gut bacteria.

Introduction
The human gut microbiota plays a pivotal role in maintaining host health and modulating disease processes1. Dysbiosis—an imbalance in microbiota composition—has been implicated in various conditions, including inflammatory, metabolic, and neurodevelopmental disorders2, 3. While metagenomic and metabolomic approaches have elucidated disease-associated microbial patterns, the causal roles of specific bacterial genes in host pathophysiology remain poorly understood4. This knowledge gap stems in part from the limited availability of genetic tools for non-model gut bacteria, which constitute the majority of the gut ecosystem. Recently, the functional analysis of non-model gut bacteria, along with the development of a gene modification system for specific bacteria, has been expanding and attracting attention5.

Mediterraneibacter gnavus (formerly Ruminococcus gnavus) is a symbiont in the human gut which has been linked to various diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and allergy 6 7 8 9. M. gnavus is frequently detected in the intestines of patients with these diseases10 and its increased abundance is often positively correlated with the severity of symptoms. This implies that M. gnavus may induce inflammation by blooming in the intestines or alternatively may preferentially thrive in the inflammatory environment. Since M. gnavus has been reported to produce inflammatory polysaccharides7 and degrade mucus layers11, this bacterium is likely to induce inflammatory responses in the host. Several studies have reported strain-specific pro-inflammatory properties12, 13, suggesting that certain M. gnavus genes in specific strains may contribute to mucosal colonization and inflammation, and that not all strains may be equally pathogenic. However, the genetic determinants underlying such phenotypic diversity remain largely unknown.

Given the association between M. gnavus abundance and inflammation, understanding the molecular and genetic mechanisms of intestinal colonization in M. gnavus is crucial. Bacteriocin production 14, mucin degradation activity 15, adhesin binding to mucins16 and biofilm formation 17 have been previously identified as strategies for intestinal colonization in M. gnavus. However, the number of experimentally validated colonization factors is still limited. Moreover, the strain-specific nature of certain phenotypes, such as capsular polysaccharide (CPS) production and mucin degradation, underscores the need for comparative genomic analysis, as well as functional validation using isogenic mutant strains18, 19. Such approaches, however, require a robust genetic manipulation system, something that has been notably lacking in M. gnavus.

In this study, we develop and apply a suite of genetic tools for M. gnavus, including a shuttle vector, inducible promoters, fluorescent markers, and gene disruption systems. Using these tools, we uncover the roles of specific sortase enzymes in cell-surface protein localization and identify a previously uncharacterized gene cluster responsible for CPS biosynthesis. Functional studies using germ-free mice demonstrate that CPS production is essential for competitive colonization and is inversely correlated with inflammatory potential. Furthermore, our genomic analyses reveal that CPS-producing strains are more prevalent in healthy individuals than in patients with Crohn’s disease, suggesting that CPS plays a role in both colonization and disease modulation. In this study, we develop a genetic toolkit that enables the functional dissection of specific sortase genes and capsular polysaccharide biosynthesis pathways in M. gnavus, a human gut symbiont. This work provides insights into how strain-specific genetic variation shapes surface structure and may influence host interactions and disease relevance.

Results
Development of genetic tools in M. gnavus.
Precise gene-modification systems are necessary for understanding gene functions and the causal relationships between bacteria and diseases. To facilitate genetic modifications for M. gnavus, we began by creating an E. coli-M. gnavus shuttle vector containing an origin suitable for M. gnavus (Fig. 1a and Supplementary Fig. 1; see Methods). Furthermore, to find the inducible promoter for M. gnavus, we cloned cumate-20, lactose-21, tetracycline-22, and xylose-inducible promoters23 upstream of mScarlet-I, red fluorescent protein gene24 (Supplementary Fig. 2). The cumate-inducible promoter showed constitutive expression in pNORg11, in which the cymR gene encoding the repressor is under the control of a modest promoter, P . When the cymR gene is under the control of the strong constitutive promoter P , mScarlet-I expression in pNORg12 is inducible by a high concentration of cumate in M. gnavus (Fig. 1B and Supplementary Fig. 2). However, we observed slight, leaky mScarlet-I expression in the absence of the inducer cumate. To reduce the leaky expression, we introduced a theophylline riboswitch into the Shine-Dalgarno (SD) sequence in pNORg14, resulting in no apparent leaky expression (Fig. 1a and b). mScarlet-I expression from pNORg14 can be induced in the presence of both cumate and theophylline. A series of plasmids with different expression patterns could facilitate experiments for various target genes, including those that are toxic or difficult to clone into the plasmid.

Using the constitutive expression system in pNORg11, we generated strains expressing fluorescent proteins, which differ in excitation and emission spectra, photostability, and maturation time (Fig. 1c). Because M. gnavus are usually cultured anaerobically, but these fluorescent proteins require oxygen for fluorophore maturation, we exposed the cells to oxygen for 2 h. Exposure to oxygen did not lead to obvious cell lysis of M. gnavus. After exposure to oxygen, we can observe these cells by fluorescent imaging, indicating that these fluorescent proteins allow for the fluorescent labeling of M. gnavus cells and the construction of promoter-reporter strains.

Next, we attempted to improve the gene disruption and deletion systems in M. gnavus. The Clostron system can be applied to constructing gene disruptants in M. gnavus 15, 25. We previously built a plasmid of the Clostron system for C. difficile26 and confirmed its availability for M. gnavus (Fig. 1d). However, this system utilizes the erythromycin resistance gene to select the mutant strain, which limits the construction of multiple gene mutant strains. To achieve more precise gene modification, we developed a plasmid carrying CRISPR/Cas9 (Fig. 1e). For proof of concept, we attempted to construct the double mutant strain of superantigens, which can bind to human IgG (hIgG) and IgA (hIgA), encoded by the genes ibpA and ibpB27. Using the Clostron and CRISPR/Cas9 systems, we successfully constructed the ibpA and ibpB double mutant strain (Supplementary Fig. 3a-e) as evidenced by the absence of IgA-bound protein detected by Western blotting using hIgA (Fig. 1f). We performed a whole genome sequencing analysis of this strain, showing that there are no off-target effects from the CRISPR/Cas9 system (Supplementary Fig. 3f).

Taken together, we developed plasmid-based genetic tools to construct various gene-modified strains of the human gut symbiont R. ganvus and likely other closely related bacteria, such as other members of the Lachnospiraceae family.

M. gnavus possess multiple sortases.
To identify the genetic factors that contribute to intestinal colonization in M. gnavus using the genetic tools, we selected sortase genes, which encode the enzyme responsible for mediating protein anchoring to cell walls, a process essential for proper cell wall protein localization. Sortase has divergent roles associated with recognition by the host immune system and colonization in several bacteria 28, 29. M. gnavus ATCC 29149, a type strain, has eight putative sortase genes, which we named srtB1-6 and srtC1-2 based on a phylogenetic analysis of their amino acid sequences (Supplementary Fig. 4a). Out of 8 putative sortase genes, we successfully created mutant strains for six genes (srtB1-5 and srtC1: Supplementary Fig. 5a and b). Genome resequencing analysis revealed that the srtC2 and srtB6 genes may be duplicated in our stock of the M. gnavus strain, which may explain why we were unable to disrupt these genes (Supplementary Fig. 5c).

SrtB3 is necessary for the cell wall localization of superantigen proteins.
Superantigens, which are cell-surface proteins widely conserved in M. gnavus, can bind to human IgA and stimulate B cells, prompting us to test the localization of superantigen proteins in the sortase mutants. The C-terminal domain of Ibp proteins contains the SPXTG motif, a feature also observed in sortase-processed cell wall-anchored proteins 27, 30. To test the effect of sortase genes on superantigen localization, we detected hIgA-bound proteins by western blotting of the cytoplasmic, cell wall, and extracellular fractions of wild-type and mutant strains (Fig. 2a). We confirmed that superantigens were detected in cell wall fractions, as predicted, and also detected these proteins in extracellular fractions. In the srtB3 mutant, superantigens that were localized to the cell wall fractions were abolished and were primarily localized to extracellular fractions (Fig. 2a). To confirm the loss of superantigen localization to the cell surface in the srtB3 mutant, we labeled the cells with fluorescence-tagged IgG and analyzed the IgG-bound cells by flow cytometry. The IgG-bound cell decreased in the srtB3 mutant (Fig. 2b, c, and d). Additionally, complementation by the srtB3-expressing plasmid (pNORg12-srtB3) restored the cell wall localization of Ibp proteins (Fig. 2e). These indicate that the SrtB3 enzyme anchors superantigens to the cell wall in M. gnavus.

srtB4 is necessary for capsular polysaccharide presentation.
Next, since M. gnavus is known to produce CPS, which is associated with the induction of inflammatory cytokines12, we observed CPS production by microscopy using Indian ink staining in the sortase mutants. We confirmed CPS production as a “halo” around the cells when observed after mixing the culture with Indian ink. The srtB4 mutant did not exhibit a halo around the cells (Fig. 3a). The srtB4 mutant formed tightly packed cell pellets after centrifugation, whereas the wild-type strain and the other mutants formed a less compact pellet (Fig. 3b, bottom panel). CPS production has been reported to cause cell pellets to become loose in various bacteria12. Furthermore, we detected acid-polysaccharides, the typical main constituents of CPS, in each mutant strain by SDS-PAGE using alcian blue staining31. The smear signals indicating polysaccharides in the cell wall fractions were significantly reduced in the srtB4 mutant strain (Fig. 3b top panel). Finally, we observed fibrous structures surrounding the wild-type cells but not the srtB4 mutant by transmission electron microscopy (Fig. 3c).

To further confirm the responsibility of srtB4 in CPS presentation, since srtB4 is located within a gene cluster, we constructed the srtB4 mutant strain using homologous recombination with the CRISPR-Cas9 system (Fig. 3d). We confirmed the deletion of the srtB4 gene in the established mutant strain by diagnostic PCR (Fig. 3e). The resulting ΔsrtB4 did not produce a substantial amount of acid-polysaccharides, and the cell pellets formed after centrifugation were tightly packed (Fig. 3f). Furthermore, we confirmed that the complementation by srtB4-expressing plasmid restored CPS presentation (Fig. 3f and g). These results clearly show that srtB4 is necessary for the surface presentation of CPS in M. gnavus.

srtB4 mutant loses competitive fitness during murine intestinal colonization
CPS production is frequently associated with the colonization ability of bacteria in the host 32. To assess the involvement of sortase genes in competitive fitness for intestinal colonization, we measured the competitive advantage of wild-type (wt) strains against the sortase mutants during colonization into germ-free mouse intestines. We mixed each sortase mutant with the wild type at a 1:1 ratio and co-inoculated it into germ-free mice. After 1 or 2 weeks of inoculation, we measured the colony-forming units (CFU) of each strain in feces to calculate the competitive index (for detailed procedures, see Methods) (Fig. 4a). Most mutant strains displayed a slightly decreased competitive index after two weeks of colonization. Among them, the srtB4 mutant was remarkably outcompeted by the wild type at one week of colonization and was undetectable after two weeks. We also measured the in vitro growth rate of sortase mutant strains. The in vitro growth rate of the srtB4 mutant is comparable to that of the wild type (Fig. 4b). In addition, the wild type did not outcompete the srtB4 mutant strain under in vitro co-culture conditions (Fig. 4c). These results indicate that srtB4-encoding sortase is indispensable for competitive colonization of M. gnavus ATCC 29149 in the murine intestine.

Identification of a gene cluster responsible for CPS production in M. gnavus ATCC 29149
Since sortase is unlikely to catalyze the direct production of CPS, we sought to identify genes involved in CPS biosynthesis. We found that a gene cluster containing the srtB4 gene is composed of genes predicted to be involved in CPS biosynthesis, including sugar biosynthesis enzymes, glycosyltransferases, and a peptidoglycan attachment enzyme (Fig. 5a). Furthermore, we confirmed promoter activity in several intergenic regions in the gene cluster in our experimental conditions using the fluorescent reporter system (Supplementary Fig. 6). We chose several genes putatively encoding nucleotide sugar biosynthesis protein (RGna_11905), glycosyltransferase (RGna_11920), the LytR-CpsA-Psr (LCP) family enzyme (RGna_12040), and bacterial tyrosine phosphatase (RGna_12055) to test if the genes were necessary for CPS production (Fig. 5a). Nucleotide sugar biosynthesis is required to generate the substrates for the reaction of glycosyltransferase, which mediates polysaccharide repeat unit production 33. The LCP family enzyme is known to catalyze cell wall attachment of CPS34. The BY-kinase/phosphatase pair is reported to regulate CPS production35. All of the mutants exhibited defective CPS presentation phenotypes as evidenced by microscopic observations, tightly packed cell pellets after centrifugation, and reduced levels of acid-polysaccharides detected in SDS-PAGE (Fig. 5b, c, and d). These results suggest that the gene cluster around srtB4 is responsible for CPS biosynthesis in M. gnavus ATCC 29149. A previous study predicted another gene cluster as CPS biosynthesis genes in the M. gnavus ATCC 29149 genome12. We also tested CPS presentation in the mutant strain of genes located in this locus (RGna_00045 and RGna_00055, which are putatively encoding glycosyltransferase and LCP family protein, respectively). However, in our experimental conditions, these gene disruptions did not affect the phenotypes tested in our study (Fig. 5b, c, and d).

CPS biosynthesis genes are necessary for competitive fitness in the murine intestine
We reasoned that if SrtB4-mediated colonization fitness is attributed to CPS presentation to cell surfaces, the disruption of the CPS gene should also result in decreased colonization fitness during an in vivo competition assay against the wild-type strain. We co-inoculated the CPS mutant strains (RGna_11905 and RGna_12040) with the wild-type strain into germ-free mice and measured competitive fitness to test the hypothesis. While the RGna_00045 mutant did not exhibit any deficiency in colonization ability, the RGna_11905 and RGna_12040 mutants were rapidly outcompeted by the wt strain during intestinal colonization (Fig. 5e). These findings indicate that CPS production, facilitated by its biosynthesis genes, is crucial for competitive colonization in M. gnavus. We observed that the in vitro growth rate of RGna_11905 mutant was slightly decreased compared to that of the wt strain, but RGna_11905 was not outcompeted by the wild-type strain in the in vitro competition assay (Supplementary Fig. 7 and Fig. 5f). Because RGna_11905 is a putative nucleotide-sugar synthesis gene, it may affect the production of nucleotide-linked sugars necessary for other cell glycans. This is a possible explanation for the mutant strain exhibiting decreased competitive fitness. However, results including RGna_12040, which may only be involved in polysaccharide attachment, are likely to rule out a simple difference in growth rate as the cause of the CPS mutant strains being outcompeted by wild-type in the intestinal environment.

Taken together, the CPS produced by the gene products of the identified locus is necessary for competitive fitness during intestinal colonization in M. gnavus.

CPS production in M. gnavus is inversely correlated with inflammation activities both in vitro and in vivo
Because CPS is attributed to have various roles in immune recognition or modulation, CPS production in M. gnavus may influence the innate immune response of host cells. To determine the influence of CPS production, we incubated murine macrophage-like J774.1 cells with M. gnavus wild-type and CPS mutant strains and measured cytokine production from host cells. TNF-α production is stimulated by M. gnavus cells and is significantly increased in the presence of the srtB4 mutant (Fig. 6a). The RGna_11905 mutant also displayed increased TNF-α production compared to wild-type, suggesting that CPS production is inversely correlated with cytokine induction by M. gnavus cells (Fig. 6a). To further confirm whether CPS production is involved in intestinal inflammation induced by M. gnavus, we prepared M. gnavus wild-type or srtB4 mutant-mono-colonized mice and then compared inflammation during administration with dextran-sodium sulfate (DSS) (Fig. 6b). Despite no substantial difference in colonization levels between wt and srtB4 mutant in gnotobiotic mice at day 0 and a modestly increased number of srtB4 mutant strains at 8 days post DSS treatment (Fig. 6c), we observed more severe body weight loss and increased disease activity index (DAI) in srtB4 mutant-colonized mice compared to wild-type (Fig. 6d and e). These data suggest that genetic differences in M. gnavus colonizing the intestine influence inflammation, and that loss of CPSs may exacerbate its severity.

The srtB4 gene cluster is predominantly lost in M. gnavus isolates from Crohn’s disease patients
Loss of the srtB4 gene and CPS production are disadvantageous for competitive colonization but are associated with aggravated intestinal inflammation in M. gnavus mono-associated mice. These findings suggest that the presence or absence of the srtB4 gene cluster may be one of the factors contributing to M. gnavus-associated disease. Recently, complete genome sequences have become available for M. gnavus strains isolated from healthy individuals and from patients with Crohn’s disease. Using this dataset of M. gnavus, 26 and 14 strains isolated from healthy persons and Crohn’s disease patients, respectively, we investigated the distribution of the srtB4 gene cluster (RGna_12030 to RGna_12060) (Fig. 7a). We found that the genomes of healthy person isolates tend to possess the srtB4 gene cluster more frequently compared to Crohn’s disease patient isolates (srtB4 frequency: 0.69 in healthy person isolates vs. 0.21 in Crohn’s disease patient isolates), despite the limited sample size. To further confirm the association between srtB4 and CPS production, we cultured the RI136 and NBRC 114413 strains, which possess and do not possess the srtB4 gene cluster, respectively. We found that the RI1 strain displayed robust CPS production, whereas NBRC 114413 did not (Fig. 7b). We then generated a srtB4 disruptant in the RI1 strain and confirmed reduced CPS presentation (Fig. 7b and c). These findings suggest that the srtB4 gene cluster is associated with CPS production and that non-capsulated and inflammatory M. gnavus strains may be more frequently found in the intestines of Crohn’s disease patients.

Methods
Bacterial culture conditions
Bacterial strains and plasmids used in this study are listed in Supplementary Data 1 and 2. We used M. gnavus JCM 6515 (= ATCC 29149) or NBRC 114413, obtained from JCM and NBRC, respectively. We used E. coli HB101/pRK24 or Stellar™ for conjugation or DNA cloning. M. gnavus strains were grown in GAM (Shimadzu) or YCFAg (YCFA medium44 with 0.4% glucose) media under an atmosphere of 80% N , 20% CO , and 4% H in an anaerobic chamber (COY). E. coli was routinely grown aerobically in LB medium. Antibiotics were supplemented in the media when necessary; 50 µg/ml carbenicillin, 20 µg/ml chloramphenicol, 10 µg/ml Thiamphenicol, 250 µg/ml D-cycloserine, 100 µg/ml kanamycin and 200 or 10 µg/ml erythromycin for E. coli or M. gnavus.

Plasmid construction
Oligonucleotides used in this study are listed in Supplementary Data 3. We used an indigenous M. gnavus NBRC 114413 plasmid to construct the E. coli-M. gnavus shuttle vectors. The M. gnavus NBRC114413 genome contains a small plasmid that should include a replicon of origin suitable for M. gnavus. We PCR amplified the putative M. gnavus ori region, including the repA gene, from the NBRC114413 plasmid (Supplementary Data 4). We also amplified a fragment containing an antibiotic resistance gene (catP), traJ, ColE1 ori, and MCS from pMTL83151, a shuttle vector used in Clostridium species45. These PCR fragments were combined by the NEB Hifi assembly system (New England Biolabs). The resultant plasmids were used for conjugative transfer to M. gnavus from E. coli. We confirmed that one of the plasmids, pNORg10, was suitable for replication in M. gnavus since many M. gnavus transconjugants could be obtained (Supplementary Fig. 1). We constructed pNORg50, in which the chloramphenicol resistance gene in pNORg10 was switched with the erythromycin resistance gene. We cloned a putative promoter region and CDS of the srtB4 gene into pNORg50 to construct the srtB4 complementation plasmid. To construct a suitable expression system for M. gnavus, we cloned cumate-20, lactose-21, tetracycline-22, and xylose-inducible promoter23 to the upstream of mScarlet-I, red fluorescent protein gene24, which are amplified from synthetic DNA (Invitrogen), pCPO051446, pRFP185, pXCH, respectively. The cymR and cumate operator (CuO) was artificially synthesized. We used the artificially synthesized codon-optimized fluorescent protein gene for the low GC content bacterium C. perfringens. For strict regulation of cumate-inducible expression, we cloned strong promoter P into the upstream of cymR genes and theophylline riboswitch47 into the SD sequence of mScarlet-I. We tested several fluorescent proteins, mVenus48, oxStayGold49, mNeonGreen50, mKate251, for fluorescent labelling of R. ganvus.

For the precise deletion of target genes, we applied the CRISPR-Cas9 system. We amplified the ncas9 (nickase) gene, sgRNA gene under the control of a synthetic constitutive promoter, and 1 kb homology arms for homologous recombination from pNICKclos2.052, SS9_RNA53 and M. gnavus genome, respectively. pNICKclos2.0 was a gift from Sheng Yang (Addgene plasmid # 73228). SS9_RNA was a gift from Ryan Gill (Addgene plasmid # 71656). These DNA fragments are joined by HiFi DNA Assembly.

Conjugation
The plasmids were introduced into M. gnavus via conjugation as described with some modifications 26. Briefly, E. coli HB101/pRK24 harboring the desired plasmid was cultured overnight in 2 ml LB medium at 30°C with vigorous shaking. E. coli cells were collected from 1 ml culture by centrifugation (1,500 × g, 3 min), washed with PBS, and placed into an anaerobic chamber. The E. coli cell pellet was suspended in 200 μL of M. gnavus culture grown in YCFAg medium overnight at 37°C. We dropped the mixture (10 × 20 µL) onto the GAM plate and incubated it for 6 h at 37°C. The transconjugants on the plate were collected in 1 ml PBS and spread onto GAM-TCK (Thiamphenicol-D-cycloserine-kanamycin) or -ECK (Erythromycin-D-cycloserine-kanamycin) plates. We typically observed the colonies of the transconjugants after 1-2 days of incubation at 37°C.

To construct erm-inserted mutant strains, the transconjugants were cultivated in YCFAg liquid culture containing thiamphenicol, which was then spread onto GAM plates containing erythromycin. We confirmed intron-erm gene insertion in the appropriate locus by PCR.

For the construction of deletion mutants using the CRISPR-Cas9 system, we picked the colonies of transconjugants and re-streaked them onto GAM containing thiamphenicol or erythromycin. We checked the deletion of the target locus by PCR.

Fluorescent reporter assay
M. gnavus strains harboring plasmids expressing fluorescent proteins were grown anaerobically in YCFAg media containing the appropriate antibiotics. After culture, we removed the culture from the anaerobic chamber and washed the cells with PBS. The cell suspensions were agitated by using a ThermoMixer (Eppendorf) at 1400 rpm to be exposed to oxygen for 2 h at room temperature or overnight at 4°C to prompt the maturation of fluorescent proteins. The fluorescent values were measured using a microplate reader Synergy H1 (BioTek).

Western blotting
M. gnavus cells were pelleted by centrifugation at 20,000 × g for 2 min. The supernatant was mixed with 2 volumes of ethanol. Then, the extracellular proteins were collected by centrifugation at 20,000 × g for 5 min. The cell pellets were washed with PBS and suspended in protoplast buffer (50 mM Tris-HCl pH 7, 50 mM MgCl , and 20% (w/v) sucrose) containing 25 mg/ml lysozyme (Wako 129-06723) and 40 U/ml mutanolysin (Merck M9901)54. The suspensions were incubated at 37°C and 1400 min-1 for 2 h and then centrifuged at 20,000 × g for 2 min. Microscopic observation confirmed that all cells were converted to protoplasts after the treatment. We used the supernatant as a cell wall fraction and the pellets as a cytoplasmic fraction. All samples were suspended in SDS-sample buffer (0.125 mol/L Tris-HCl, pH 6.8, 4% SDS, 20% Glycerol, 0.01% BPB, 10% 2-mercaptoethanol) and heated at 95°C for 10 min. The protein samples were separated by SDS-PAGE and electroblotted onto PVDF membranes. The membranes were blocked with Bullet Blocking One (Nacalai tesque). Superantigen proteins were probed with human IgA (Bethyl Laboratories P80-102) diluted 1:5000 with PSB containing 0.05% Tween 20. These antibodies were labeled with anti-human IgA-HRP (Invitrogen A18787) diluted 1:20000. The bound antibodies were labeled with Immunostar LD (Wako) and detected using a C-DiGit System (LI-COR Biosciences).

Flowcytometry
Cells grown for 6 h were adjusted to a concentration of 1 × 107 CFU/ml with PBS. After centrifugation, the cells were resuspended in 2% BSA/PBS and incubated at room temperature for 15 min. The cells were then probed with 5 µg/mL of mouse IgG Alexa Fluor 488 conjugates (Thermo Fisher Scientific, A-10631; lot number, 2400911) in 2% BSA/PBS and incubated for 30 min at 4°C. After staining, the cells were washed twice with PBS and analyzed by flow cytometry using a FACS Aria II (BD Biosciences). Cell-bound IgG was evaluated based on all events detected by flow cytometry. Bacterial cell populations were gated by FSC-A/SSC-A. Fluorescence was excited with a 488-nm laser, and emission was subsequently collected using a 525/50-nm filter. Data were analyzed using FlowJo V10.8. Representative examples of the gating strategies used are provided in Supplementary Fig. 10.

Gnotobiotic mouse experiment
The University of Tsukuba Animal Experiment Committee approved all germ-free mouse experiments. Mice were housed in a room maintained at 23.5 ± 2.5°C and 52.5 ± 12.5% relative humidity under a 14:10-h light: dark cycle. We cultured M. gnavus overnight in YCFAg at 37°C and collected the cell pellets by centrifugation in the anaerobic chamber. The cells were resuspended in GAM medium at a concentration of 1 × 108 CFU/ml. We orally gavaged 200 μL suspension into female BALB/cCr germ-free mice (6-12 weeks old). We collected fecal samples weekly and spread the fecal suspensions onto GAM with or without 10 μg/ml erythromycin to measure the CFU of wt and mutant strains in the fecal samples. We calculated competitive fitness (CI) by dividing the CFU of the mutant strain by that of the wild type.

To assess whether M. gnavus mutant exacerbates intestinal inflammation, we administered 2.5% dextran sodium sulfate (MP Biomedicalsas 160110) as drinking water to M. gnavus mono-associated male BALB/cCr mice (8-10 weeks old) and daily monitored body weight changes and disease activity scores (DAI). The DAI scores were evaluated on a scale of 0–4 for the following observed parameters weight loss, stool consistency, and bleeding55. Weight loss was scored as follows: 0 for no weight loss, 1 for 0-5% weight loss, 2 for 5-10% weight loss, 3 for 10-15% weight loss, and 4 for more than 15% weight loss. Stool consistency was scored as 0 for normal stool, 2 for soft stool, and 4 for diarrhea. Bleeding was scored as 0 for no bleeding, 2 for slight bleeding, and 4 for severe bleeding. Total scores were used to indicate intestinal inflammation.

Sex as a biological variable was not specifically analyzed because only one sex was used for each assay (female mice for colonization, male mice for DSS).

In vitro competition assay
The overnight cultures of wt and mutant strains were diluted 1:50 in 200 µL of YCFAg medium in a 96-well plate and anaerobically grown at 37°C. We sub-cultured the cells every 24 h. On day 7, we diluted the culture daily in fresh YCFAg and spread it on the GAM plate to count the CFUs of each strain.

CPS analysis
We mixed M. gnavus culture with Indian ink at an equal volume and then observed the cells with a phase contrast microscope.

To detect acid polysaccharide production, we anaerobically cultured M. gnavus cells in YCFAg at 37°C for 16 h and collected cell pellets by centrifugation at 20,000 × g for 2 min. The cell pellets were washed with PBS and resuspended in H O. The suspension was heated at 95°C for 10 min to collect cell-bound CPS. We collected the suspension supernatant after centrifugation and used it for SDS-PAGE. Samples corresponding to 0.5 O.D. unit were loaded into each lane. Extracted CPS was visualized by the cationic dye alcian blue (0.125% alcian blue, 40% ethanol, and 5% acetic acid) for 2 hours and washed with 40% ethanol and 5% acetic acid for 2 hours56.

Transmission electron microscopy was outsourced to Hanaichi UltraStructure Research Institute (Aichi, Japan). M. gnavus cells were fixed in 2.5% glutaraldehyde/1.25% paraformaldehyde/ 0.03% picric acid with 10 mM lysine and 0.1% ruthenium red in 0.1 M sodium cacodylate buffer (pH 7.4).

Cytokine ELISA
J774.1, a mouse macrophage-like cell line, purchased from RIKEN BRC, was cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C under 5% CO atmosphere. M. gnavus grown in YCFAg overnight was suspended in PBS to reach O.D. = 10 (approximately 1 × 109 cells/ml). One mL of J774.1 cell suspension at a 1 × 105 cells/ml concentration was incubated with 10 µL of M. gnavus cells (MOI = 1: 100) for 24 h. The cell culture supernatant was collected to detect cytokine production by the Murine TNF-α ELISA Development Kit (PeproTech 900-K54K) according to the manufacturer's instructions.

Bacterial quantification by qPCR
Bacterial DNA was extracted from fecal samples by a bead-beating method. Freeze-dried feces (10 mg) were suspended in 400 µL of 1% SDS/TE buffer and mixed with 400 µL glass beads (Merck G8772) and an equal volume of phenol-chloroform-isoamyl alcohol (Nippon Gene). Bead beating was carried out using Shake Master Neo at 1400 rpm for 15 min. After centrifugation at 20,000 × g for 5 min, supernatants were collected for further purification of nucleic acids using Monarch® Spin gDNA Extraction Kit (New England Biolabs) according to the manufacturer’s instructions. DNA concentration was quantified using Quant-iT™ PicoGreen™ dsDNA Assay Kits (Thermo Fisher Scientific).

Bacterial quantification by qPCR was performed as described previously57. The 16S rRNA gene fragments were amplified by PCR and diluted to 1.67 ng/µL, corresponding to 109 copies/µL. The DNA solutions serially diluted to 108 to 103 copies/µL were used as the standard. The qPCR was carried out using TB Green® Premix Ex Taq™ II FAST qPCR (TAKARA bio) and StepOnePlus™ Real-Time PCR Systems (Thermo Fisher Scientific) according to the manufacturer’s instructions. The number of bacterial cells/g feces was calculated using the Ct values, the standard curve and the amount of feces used for DNA extraction.

Genome analysis
M. gnavus complete genome information was obtained from NCBI. The BLASTn analysis against srtB4 and its adjacent genes in ATCC 29149, the reference sequence, was performed using Geneious Prime 2025.0.3. (https://www.geneious.com)

Statistics and reproducibility
Data are represented as mean values ± SD (standard deviation) or ±SEM (standard error of the mean), calculated using ggplot package in R. Significance was calculated by an unpaired Welch’s t-test with Bonferroni correction for multiple comparisons using Microsoft Excel for Microsoft 365. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study reports the development of a genetic toolkit for the gut bacterium Ruminococcus gnavus. The authors established a shuttle vector, inducible expression, fluorescent protein reporter, and two gene deletion methods for this organism. Using the genetic deletion approach, they generated mutants with individual deletions of putative sortase-encoding genes, and found that srtB3 is responsible for anchoring a bacterial superantigen to the cell wall, and srtB4 is essential for capsular polysaccharide production and bacterial in vivo fitness. This work will be impactful for the future mechanistic studies of bacterial physiology and host-microbe interactions in R. gnavus and potentially other closely related bacteria.\nMajor:\nGenetic toolkit:\nFor Line 103 and Fig. 1D-1F, please include genotyping results from PCR and/or sequencing in the supplementary figures.\nRegarding the strategy to create the ibpA/B double mutant, why did authors use a clostron-based method to delete ibpA and then use CRISPR/Cas to remove ibpB?\nFrom Fig. 1E, it appears that only the promoter region and upstream portion of the ibpB CDS were deleted rather than a full deletion. Have the authors attempted to delete the entire ibpB CDS or even the ibpA/ibpB region together?\nIs it possible to cure the CRISPR plasmid and perform iterative gene deletions in Ruminococcus gnavus?\nHow successful is the genetic manipulation strategy in other Ruminococcus strains?\nSortase deletion:\nFor the duplicated srtA2 and srtB6 genes, would it be possible to use a different antibiotic resistance marker after the first deletion to disrupt the second copy?\nHow different are these sortases in terms of protein sequences and genomic context? Any predictions regarding their substrate preferences? An illustration in the SI would aid in inferring their biological roles.\nSrtB3 function:\nControl proteins should be included in the WB to demonstrate clear separation of different bacterial fractions.\nSrtB4 function:\nThe paper does not explain how srtB4 is involved in CPS biosynthesis. Have the authors tested which proteins are the actual substrates of SrtB4?\nFig. 6: What are the colonization levels of the two strains in the mono-colonization experiments? Could the observed differences in colitis severity be explained by differences in colonization levels?\nFig. 7: The comparison of three strains is not strong evidence to link srtB4 to CPS production. Including additional strains or demonstrating gene deletion in the RI1 strain would strengthen the conclusion.\nMinor:\nLine 52: The description linking R. gnavus to IBD severity is vague. Please clarify whether it is the abundance or presence of R. gnavus that is associated with symptoms, and whether this is a positive correlation.\nLine 93: please explain the term “SD” sequence.\nFig. 1F, 2A, and others: Please indicate the expected location of the targeted proteins in the WB.\nThe methods section should describe how complementation experiments corresponding to Fig. 3F and 3G were performed. These complementation strains are not listed in Table S1.\nPlease consider depositing the plasmids to Addgene to facilitate distribution. Including the replicon sequence in the SI table would also be helpful.\nLine 288: Please provide more detail regarding strain dependence. In Supplementary Figure 6, many strains appear to lack the upstream genes involved in sugar biosynthesis and glycosylation. In those strains, which genes are present instead? Are they homologs of the genes in ATCC 29149, or are they entirely different genes?", "reviewer_2": "This study begins with the construction of a suite of genetic tools that greatly enhance the genetic systems currently available to work with M. gnavus. These include an inducible gene expression system where two inducers lead to reduced leaky expression. The team also designs constructs for gene deletions and constructs for fluorescent protein analysis in M. gnavus. The bulk of the study addresses the role of various sortases in the linking of CPS and proteins to the cell using isogenic mutants. Most of these studies are well performed and provide new important data. The study includes the use of gnotobiotic mice to show fitness defects of particular mutants during competitive colonization with WT. The studies included in Figure 6 and 7 are the weakest of the study and relate to inflammatory responses or protection due to the CPS, and genetic analyses of the CPS locus in healthy and Crohn’s disease patients. In total, this study significantly contributes to the field, but some areas need additional experimentation or analyses to better justify the conclusions.\nThe genetic tools are a significant advance and must be made available through AddGene or another not for profit plasmid distribution company.\nThe newly reclassified genus must be used in the title and throughout and initially the authors should write “formerly Ruminococcus gnavus”.\nLinese 27 and 28 of the abstract should be rewritten to state that “mutations of six of the eight sortase encoding genes allowed for the identification of which are involved in the surface localization of the CPS and proteins predicted to have superantigen properties.” As written, it sounds as if you show that sortases are involved in surface attachment of molecules in Gram positive bacteria, which is well established.\nTo say that the organism is a “commensal” and then say that it is associated with many diseases is not correct terminology. The term symbiont should be used or you can should say it if frequently a member of the human gut microbiota.\nThe caveat that these aero-intolerant bacteria must be exposed to oxygen for two hours for fluorophore maturation should be discussed. It is assumed that cultures were shaken during this exposure, which if so, should be stated in the methods.\nLine 53 “This implies that R. gnavus could induce inflammation by blooming in the intestines.” This does not make that implication. They may just be able to better survive in inflammatory states.\nLinr 60 – change “disease activities” to “inflammation”\nLine 71 “specific sortase enzymes”\nThe last sentence of your introduction is another obvious statement and could be better tailored to your findings\nLine 335 – remove the word “sequence”. Give a brief description of pMTL83151 “a shuttle plasmid used in Clostridiodes species”.\nLine 108 – what is meant by two copies of the gene, ibpA, and ibpB? Do you mean one copy of each? If so, just write encoded by genes ibpA, and ibpB.\nLine 110 – Did you do a whole genome sequence of this strain? This is now quite inexpensive and is necessary to show that there are not off-target effects from the CRISPR/Cas system.\nLine 113- better to write “other members of the Lachnospiraceae family”.\nLine 121 – give your rationale for naming two of the sortase genes srtA and 6 of them srtB.\nLine 130 – maybe better to say “in sortase-processed cell wall anchored proteins”\nFigure S3C –As you have a genome sequence, did it reveal two copies of this gene cluster? This should be performed with long read technology to overcome problems assembling repetitive regions. Is this region likely a mobile genetic element? If so, these srt genes may not be present in present in all M. gnavus strains. MGEs often insert into tRNA genes, which you could search for at the junctions of the duplicated region. What other genes are present in this duplicated region?\nLine 148 – better to say “formed a less compact pellet”\nFig 3B and 3F must show a MW range so that we can determine the size of the CPS. They are typically quite heterogeneously sized high MW molecules.\nFig 3C – are there any other phenotypes of the srtB4 mutant? In the image, it appears to have shorter chains. Do these bacteria clump at all as is a characteristic or some bacteria when their capsules are removed.\nLine 152-154 – this image is not enough proof to say that these are fimbriae. These bacteria were processed for EM which introduces all types of artifacts. This statement about fimbriae should be removed.\nThe figure legend of Fig 3 (and line 161) should be modified, srtB4 is not responsible for capsular polysaccharide production, it is likely important in linking the CPS to the bacterial surface. Did you analyze the supernatants for the presence of CPS? An antibody to this molecule would substantially help with analyses – which should be easily obtained by adsorbing antibodies to the WT bacterium with the CPS mutant.\nLine 167 –state “competitive advantage of the WT strains against the sortase mutants”, rather than the other way around.\nLines 200-201 – This statement is not supported as written. Bacteria frequently make more than one CPS/EPS. It is quite likely that the locus identified in the other study also produces CPS that is not the same as the CPS of your study. Many CPS do not extend so far from the cell and are not nearly as visible with India ink staining as the CPS you identified. This statement should be qualified to say “these gene disruptions did not affect the phenotypes tested in our study”. In fact, the cell pellets of these deletion mutants seem to be even more loosely packed than the WT strain, suggesting some role in production or presentation of surface PSs.\nLine 204 – there is no evidence that SrtB4 affects CPS production, just likely its surface attachment, or the attachment of another protein that is necessary for its surface localization.\nThere are some concerns with the CPS mutants analyzed for competitive colonization in the mouse gut. As gene 11905 encodes a nucleotide-sugar synthesis gene, it may affect the production of nucleotide linked sugars necessary for other cell glycans. However, there are also concerns with any mutants that allow the incomplete building or flipping and polymerization of the repeat unit, as these mutants sequester and prevent recycling of undecaprenol-P, which is necessary for PG synthesis and can contribute to growth defects. The best strategy is to delete all glycosylatranserase genes so that the repeat unit is not built at all. These caveats should at least be discussed for the competitive colonization assays, especially since the strains have an in vitro growth defect. However, the 12040 mutant, which based on gene proximity may only be involved in attaching the CPS (although that is unknown), also confirms the competitive defect.\nLines 215-216 – “The CPS produced by the gene products of the identified locus….”. There may be more than one CPS/EPS produced by this organism.\nLine 220 – this statement is too broad, and the opposite has also been shown. Maybe say “to have various roles in immune recognition or modulation”.\nFig 6 – there is some concern for the very different amounts of TNF in the strains comparing panels A and B. Why is there such a difference in the WT and srtB::erm strains in these panels?\nFigure 6C – it is not evident when these mice were gavaged, please put an arrow on the time line. Also, it is surprising that mice monocolonized with M. gnavus survived for 8 days on 2.5% DSS.\nFig 6D – for this experiment, you must quantify the cfu/gram of bacteria in these mice over the course of the experiment (for a few time points). It is very possible that the mutant strain was not able to colonize the DSS treated mouse to the same extent as the WT, which would be relevant data. Based on the data shown, I disagree with the statement “These results suggest that CPS production inhibits the recognition of R. gnavus cells from host immune cells and that CPS233 deficient R. gnavus has the potential to induce more severe inflammation during intestinal colonization”. The inflammation is induced by the DSS, not the M. gnavus. Without cfu/g, there cannot be a statement about effects of the CPS in this assay.\nStudies related to figure 7 are problematic\n1) are you sure that all of these strains were isolated from separate people and are not replicates (possible longitudinal samples) from the same individual? In addition, the geographic location of the isolates (from where they live) is necessary as there can be clustering of isolates in different countries which would confound the data. There is some concern for the low number of samples for this analysis, which may be lower if duplicate samples from the same person are revealed.\n2) CPS loci are often heterogeneous across strains of a species. Do the strains that do not have the same CPS locus as described in this study have a distinct CPS locus in this region? In Fig S6, please show gene maps of this region in other strains.\n3) it is quite possible that another Srt can serve the function of SrtB4 if there are diverse CPS loci in this region.\n.\nStatements in the discussion related to findings of Crohn’s disease patients are overstated based on the caveats mentioned above."}, "author_response": "Response: We sincerely appreciate the reviewer’s positive evaluation. In response to your suggestions, we have carefully revised the manuscript as the reviewer suggested and have addressed each comment point-by-point. We hope the revisions improve the manuscript and meet your expectations.\nMajor:\nGenetic toolkit:\nFor Line 103 and Fig. 1D-1F, please include genotyping results from PCR and/or sequencing in the supplementary figures.\nResponse: In the revised manuscript, we have added PCR-based genotyping results to Supplementary Fig. S3. The insertion of intron-ermB into the ibpA locus resulted in a larger amplicon when PCR was performed with ibpA-specific primers, confirming the successful insertion of the intron. As for ibpB, deletion of the promoter region resulted in a smaller amplicon in PCR with ibpB-specific primers. In addition, when using primers targeting the internal region of the deleted sequence, we did not obtain any PCR amplicon, consistent with the expected deletion. Despite the PCR amplicon was detected in the ΔibpB single mutant, primers annealing to the ibpA region, which possesses a sequence similar to ibpB, likely amplify the PCR product. These results confirm the successful disruption of both ibpA and ibpB genes.\nRegarding the strategy to create the ibpA/B double mutant, why did authors use a clostron-based method to delete ibpA and then use CRISPR/Cas to remove ibpB?\nResponse: We initially attempted to construct each ibpA and ibpB mutant using the Clostron system. However, the ibpB disruptant retained a truncated version of the IbpB protein, likely due to the position of the intron insertion site, which was located near the C-terminal region of the ibpB CDS.\nWe confirmed the truncated IbpB expression by Western blotting with human IgA, indicating that the truncated IbpB retained functionality. Therefore, we employed the CRISPR/Cas system to generate a loss-of-function mutation in ibpB.\nFrom Fig. 1E, it appears that only the promoter region and upstream portion of the ibpB CDS were deleted rather than a full deletion. Have the authors attempted to delete the entire ibpB CDS or even the ibpA/ibpB region together?\nResponse: At first, we attempted to generate a complete deletion of the ibpB gene by homologous recombination, but this approach was not successful despite several attempts. We have not attempted to delete the entire ibpA/ibpB region.\nGiven that shorter deletions are generally more feasible with homologous recombination, we instead attempted to delete the promoter region and the upstream portion of the ibpB CDS.\nIs it possible to cure the CRISPR plasmid and perform iterative gene deletions in Ruminococcus gnavus?\nResponse: Yes, we have confirmed that the CRISPR plasmid (TmR) can be cured by conjugative transfer of another plasmid carrying the same origin of replication and a different antibiotic resistance marker (ErmR). The resulting strain lost resistance to thiamphenicol (TmR), indicating that the original plasmid is displaced by the newly introduced one. Theoretically, this approach could enable the iterative introduction of CRISPR plasmids targeting different genes. However, we have not yet tested this strategy for multiple deletions in practice. We appreciate the reviewer’s suggestion and agree that iterative deletions would be a valuable application for future studies. We added the above description to the revised manuscript.\nL285. “We employed the Clostron and CRISPR-Cas systems simultaneously to construct a multiple-deletion strain. We also confirmed that the CRISPR plasmid (TmR) can be cured by conjugative transfer of another plasmid carrying the same origin of replication and a different antibiotic resistance marker (ErmR). The resulting strain lost resistance to thiamphenicol (TmR), indicating that the original plasmid is displaced by the newly introduced one. Theoretically, this approach could enable iterative introduction of CRISPR plasmids targeting different genes. However, we have not yet tested this strategy for multiple deletions in practice. ”\nHow successful is the genetic manipulation strategy in other Ruminococcus strains?\nResponse: We have attempted to use our shuttle vectors for fluorescent tagging in other gut bacterial species closely related to R. gnavus. So far, we have successfully introduced plasmids into several Lachnospiraceae strains, such as R. gnavus isolates other than the type strain, Enterocloster clostridioformis, and E. bolteae. These results suggest that our genetic manipulation strategy may be broadly applicable to other members of the Lachnospiraceae family. We added the above description to the revised manuscript.\nL291. “We have attempted to use our shuttle vectors for fluorescent tagging in other gut bacterial species closely related to M. gnavus. So far, we have successfully introduced plasmids into several Lachnospiraceae strains, such as M. gnavus isolates, Enteroclosterclostridioformis, and E. bolteae. These results suggest that our genetic manipulation strategy may be broadly applicable to other members of the Lachnospiraceae family.”\nSortase deletion:\nFor the duplicated srtA2 and srtB6 genes, would it be possible to use a different antibiotic resistance marker after the first deletion to disrupt the second copy?\nResponse: In the Clostron system, the ermB gene is initially disrupted by the insertion of the group II intron and becomes functional after the intron is spliced out upon successful retrohoming into the genome. To our knowledge, only ermB and kanR have been practically adopted as selective markers for the Clostron system. However, R. gnavus exhibits intrinsic resistance to kanamycin, limiting the availability of selection markers. Therefore, we did not attempt to use the intron for multiple gene disruption in this study.\nHow different are these sortases in terms of protein sequences and genomic context? Any predictions regarding their substrate preferences? An illustration in the SI would aid in inferring their biological roles.\nResponse: We appreciate the reviewer’s insightful suggestion. To address this point, we have added a phylogenetic analysis of sortase proteins derived from R. gnavus and various other Gram-positive bacteria (supplementary Fig. 4A). This analysis revealed that most R. gnavus sortase B homologs (RGna_01825, RGna_09760, RGna_10250, RGna_12035, RGna_13420) form a distinct clade, separate from known sortase B proteins in other species. This suggests that they may recognize unique substrate motifs. Additionally, we found that RGna_09235 and RGna_13410, which were annotated initially as SrtA, were clustered with the sortase C proteins in the phylogenetic tree. Based on this observation, we have renamed these genes as srtC1 and srtC2, respectively.\nIn addition, we checked genes encoded near these sortase and found candidate targets, which have putative cell wall sorting signals (supplementary Fig. 4B). This suggest that each sortase recognizes specific cell wall sorting signals. We agree that these results suggest potential functional divergence among sortase paralogs and have incorporated this interpretation into the revised manuscript.\nL348. “Additionally, we performed a phylogenetic analysis of sortase proteins derived from M. gnavus and various other Gram-positive bacteria (supplementary Fig. S4A). This analysis revealed that most M. gnavus sortase B homologs (RGna_01825, RGna_09760, RGna_10250, RGna_12035, RGna_13420) form a distinct clade, separate from known sortase B proteins in other species. Furthermore, we found candidate target proteins encoded near these sortase genes, whose C-terminal sequences contain putative cell wall sorting signals (supplementary Fig. S4B). This suggests that they may recognize unique substrate motifs. Identifying their target substrates may facilitate understanding the functional repertoire of M. gnavus surface proteins that may contribute to environmental adaptation in this bacterium as a pathobiont28.”\nSrtB3 function:\nControl proteins should be included in the WB to demonstrate clear separation of different bacterial fractions.\nResponse: We appreciate the reviewer’s suggestion. However, due to the limited functional and mechanistic characterization of R. gnavus to date, there are very few proteins with well-established subcellular localization in this organism. Furthermore, commercially available antibodies specific to R. gnavus proteins are not currently accessible. This makes it challenging to include canonical controls for protein fractionation (e.g., cell wall fraction) in our Western blotting. We also note that enzymatic reactions with lysozyme and mutanolysin were used to extract cell wall fractions. Microscopic observation confirmed the formation of protoplast-like cells (see below). And we added the description in the Materials and Methods section of the revised manuscript as follows:\nL438. “Microscopic observation confirmed that all cells were converted to protoplasts after the treatment.”\nSupporting information 1. Phase contrast microscopy of R. gnavus cells after the treatment of lysozyme and mutanolysin.\nFurthermore, to address this limitation, we complemented our analysis with flow cytometry using human IgG, which binds to Ibp proteins on the bacterial surface. Because flow cytometry detects surface-bound proteins on intact cells without cell lysis, it serves as an independent validation of surface localization. The flow cytometry results are consistent with our Western blot results, demonstrating that srtB3 is required for surface localization of Ibp proteins.\nSrtB4 function:\nThe paper does not explain how srtB4 is involved in CPS biosynthesis. Have the authors tested which proteins are the actual substrates of SrtB4?\nResponse: We appreciate the reviewer’s suggestion. We identified the RGna_12050 gene, located downstream of the srtB4 gene, as a candidate substrate because it is highly conserved and contains a putative sortase recognition motif (SPQTG) at the C-terminal end (Supplementary Fig. 4B). To investigate whether this protein is a substrate of SrtB4, we constructed plasmids expressing FLAG- or fluorescent protein-tagged RGna_12050 and introduced them into both wt and srtB4 mutant strains. However, we did not observe cell wall localization of the fusion proteins or any apparent differences in the protein localization between the strains.\nThese results suggest that RGna_12050 protein is not a target of SrtB4 or that our detection system was not suitable for assessing its localization. At present, the mechanism by which srtB4 contributes to CPS biogenesis remains unclear. We agree that this is an important issue that should be explored in future investigations.\nFig. 6: What are the colonization levels of the two strains in the mono-colonization experiments? Could the observed differences in colitis severity be explained by differences in colonization levels?\nResponse: We appreciate the reviewer’s insightful suggestion. We measured CFUs of both wt and srtB4::erm in the feces of mono-associated mice at day 0. Additionally, the number of M. gnavus cells was quantified by qPCR at days 0 and 8 post-DSS treatment. These values were not substantially different, as shown in revised Fig. 6C. These results suggest that the observed differences in colitis severity are likely due to the genetic characteristics of the mutant strain rather than differences in colonization levels. In the revised manuscript, we have added this description and the relevant data.\nL243. “To further confirm whether CPS production is involved in intestinal inflammation induced by M. gnavus, we prepared M. gnavus wild-type or srtB4 mutant-mono-colonized mice and then compared inflammation during administration with dextran-sodium sulfate (DSS) (Fig. 6B). Despite no substantial difference in colonization levels between wt and srtB4 mutant in gnotobiotic mice at day 0 and a modestly increased number of srtB4 mutant strains at 8 days post DSS treatment (Fig. 6C), we observed more severe body weight loss and increased disease activity index (DAI) in srtB4 mutant-colonized mice compared to wild-type (Fig. 6D and E). These data suggest that genetic differences in M. gnavus colonizing the intestine influence inflammation, and that loss of CPSs may exacerbate its severity.”\nFig. 7: The comparison of three strains is not strong evidence to link srtB4 to CPS production. Including additional strains or demonstrating gene deletion in the RI1 strain would strengthen the conclusion.\nResponse: Thank you for this valuable suggestion. In response, we constructed a srtB4 homologue disruptant in the RI1 strain. This mutant strain exhibited a marked reduction in CPS presentation, as observed under a microscope with Indian-ink staining. This additional result demonstrates that the srtB4 gene plays an important role in CPS presentation, not only in the type strain but also in other M. gnavus strains. We have incorporated this new data into the revised manuscript (Fig. 7B and C, L269).\nMinor:\nLine 52: The description linking R. gnavus to IBD severity is vague. Please clarify whether it is the abundance or presence of R. gnavus that is associated with symptoms, and whether this is a positive correlation.\nResponse: We replaced the sentence with “M. gnavus is frequently detected in the intestines of patients with these diseases, and its increased abundance is often positively correlated with the severity of symptoms. (L53)” We believe the revised sentence clarifies the correlation between M. gnavus abundance and symptom severity.\nLine 93: please explain the term “SD” sequence.\nResponse: We added the abbreviation “the Shine-Dalgarno (SD) sequence (L98)“.\nFig. 1F, 2A, and others: Please indicate the expected location of the targeted proteins in the WB.\nResponse: We have revised the Western blotting images, including Fig. 1F, 2A, and others, to add arrows indicating the expected locations of the target proteins. We hope these revisions improve the clarity of the data.\nThe methods section should describe how complementation experiments corresponding to Fig. 3F and 3G were performed. These complementation strains are not listed in Table S1.\nResponse: We have now added a detailed description of the construction of the srtB4 complementation plasmid to the Methods section (L387). Additionally, this plasmid has been listed in Supplementary Table S2, which provides an overview of all plasmids used in the study.\nPlease consider depositing the plasmids to Addgene to facilitate distribution. Including the replicon sequence in the SI table would also be helpful.\nResponse: We have included the replicon sequence in Supplementary Table S4, as suggested. We are currently depositing the plasmids used in this study with Addgene (Addgene Deposit number: 86940).\nLine 288: Please provide more detail regarding strain dependence. In Supplementary Figure 6, many strains appear to lack the upstream genes involved in sugar biosynthesis and glycosylation. In those strains, which genes are present instead? Are they homologs of the genes in ATCC 29149, or are they entirely different genes?\nResponse: We appreciate the reviewer's important comment. In the revised Supplementary Fig. 8A, we reanalyzed the genomes of the srtB4-positive isolates using tBLASTx with more lenient criteria to identify distant homologs of the CPS gene cluster from the type strain (ATCC 29149). This analysis revealed that all srtB4-positive isolates possess at least one homologous gene related to sugar biosynthesis and glycosyltransferase. In addition, we added a comparison of the genetic contexts between ATCC 29149 and RI1 strains, indicating that the overall gene composition and organization vary among strains (Supplementary Fig. 8B). These data support the idea that R. gnavus strains harbor diverse CPS gene clusters, potentially resulting in capsular polysaccharides with different sugar compositions and structures.\nReviewer #2 (Remarks to the Author):\nThis study begins with the construction of a suite of genetic tools that greatly enhance the genetic systems currently available to work with M. gnavus. These include an inducible gene expression system where two inducers lead to reduced leaky expression. The team also designs constructs for gene deletions and constructs for fluorescent protein analysis in M. gnavus. The bulk of the study addresses the role of various sortases in the linking of CPS and proteins to the cell using isogenic mutants. Most of these studies are well performed and provide new important data. The study includes the use of gnotobiotic mice to show fitness defects of particular mutants during competitive colonization with WT. The studies included in Figure 6 and 7 are the weakest of the study and relate to inflammatory responses or protection due to the CPS, and genetic analyses of the CPS locus in healthy and Crohn’s disease patients. In total, this study significantly contributes to the field, but some areas need additional experimentation or analyses to better justify the conclusions.\nResponse: Thank you for your insightful comments. We have carefully revised the manuscript as the reviewer suggested, especially in Fig. 6 and 7, and have addressed each comment point-by-point. We hope the revisions improve the manuscript and meet your expectations.\nThe genetic tools are a significant advance and must be made available through AddGene or another not for profit plasmid distribution company.\nResponse: We are currently depositing the plasmids used in this study with Addgene (Addgene Deposit number: 86940).\nThe newly reclassified genus must be used in the title and throughout and initially the authors should write “formerly Ruminococcus gnavus”.\nResponse: We have thoroughly revised the manuscript and have used “Mediterraneibacter gnavus” in the revised manuscript.\nLinese 27 and 28 of the abstract should be rewritten to state that “mutations of six of the eight sortase encoding genes allowed for the identification of which are involved in the surface localization of the CPS and proteins predicted to have superantigen properties.” As written, it sounds as if you show that sortases are involved in surface attachment of molecules in Gram positive bacteria, which is well established.\nResponse: Thank you for your careful and constructive comment. We have revised the abstract as suggested to clarify that our study identifies which sortase genes are involved in the surface localization of CPS and predicted superantigen proteins, rather than reiterating the general role of sortases in Gram-positive bacteria.\nTo say that the organism is a “commensal” and then say that it is associated with many diseases is not correct terminology. The term symbiont should be used or you can should say it if frequently a member of the human gut microbiota.\nResponse: Thank you for your valuable comment. To address this point, we have replaced the term “commensal” with “symbiont” or “symbiotic bacterium” throughout the manuscript to more accurately reflect the role of M. gnavus in the context of its association with various diseases.\nThe caveat that these aero-intolerant bacteria must be exposed to oxygen for two hours for fluorophore maturation should be discussed. It is assumed that cultures were shaken during this exposure, which if so, should be stated in the methods.\nResponse: As you mentioned, the maturation of fluorescent proteins requires oxygen, and we briefly described the necessity of oxygen exposure before fluorescence imaging in the main text (L105). We agitated the cell suspension using a ThermoMixer at 1400 rpm to ensure efficient oxygen exposure, and the procedure has been described in the Materials and Methods section (L427). We did not observe substantial cell lysis after 2 h exposure, supporting the applicability of this approach for labeling M. gnavus.\nLine 53 “This implies that R. gnavus could induce inflammation by blooming in the intestines.” This does not make that implication. They may just be able to better survive in inflammatory states.\nResponse: We appreciate your constructive comments. As you pointed out, we do not have direct evidence that M. gnavus induces inflammation. It is also possible that M. gnavus is simply better adapted to survive and proliferate under the inflammatory conditions in the gut. We have revised the sentence accordingly to\n(L55) “This implies that M. gnavus may induce inflammation by blooming in the intestines, or alternatively, may preferentially thrive in the inflammatory environment.”\nLinr 60 – change “disease activities” to “inflammation”\nResponse: We have revised the phrase as you suggested.\nLine 71 “specific sortase enzymes”\nResponse: We have revised the phrase as you suggested.\nThe last sentence of your introduction is another obvious statement and could be better tailored to your findings\nResponse: We appreciate the reviewer’s constructive comments. We have revised the sentence to clarify our findings, accordingly to\n(L79) “In this study, we developed a genetic toolkit that enables the functional dissection of specific sortase genes and capsular polysaccharide biosynthesis pathways in M. gnavus, a human gut symbiont. This work provided novel insights into how strain-specific genetic variation shapes surface structure and may influence host interactions and disease relevance.\nLine 335 – remove the word “sequence”. Give a brief description of pMTL83151 “a shuttle plasmid used in Clostridiodes species”.\nResponse: We have removed the word “sequence” from the original text. In addition, we revised the sentence, including the description of pMTL83151, accordingly to “We PCR amplified the putative M. gnavus ori region, including the repA gene, from the NBRC114413 plasmid (Supplementary Table 4). We also amplified a fragment containing an antibiotic resistance gene (catP), traJ, ColE1 ori, and MCS from pMTL83151, a shuttle vector used in Clostridium species. (L381)”\nLine 108 – what is meant by two copies of the gene, ibpA, and ibpB? Do you mean one copy of each? If so, just write encoded by genes ibpA, and ibpB.\nResponse: Thank you for pointing this out. We agree that the original phrasing was misleading. We have revised the sentence, as follows: “encoded by the genes ibpA and ibpB, (L117)” as we intended to indicate one copy of each gene.\nLine 110 – Did you do a whole genome sequence of this strain? This is now quite inexpensive and is necessary to show that there are not off-target effects from the CRISPR/Cas system.\nResponse: Thank you for your suggestion. We performed whole-genome sequencing of these strains as you suggested and added a description stating that there are no off-target effects from the CRISPR/Cas system (L120).\nLine 113- better to write “other members of the Lachnospiraceae family”.\nResponse: We revised the phrase as the reviewer suggested.\nLine 121 – give your rationale for naming two of the sortase genes srtA and 6 of them srtB.\nResponse: In the revised manuscript, we have added a phylogenetic analysis of sortase proteins derived from R. gnavus and various other Gram-positive bacteria (supplementary Fig. S4A). This analysis revealed that most M. gnavus sortase B homologs (RGna_01825, RGna_09760, RGna_10250, RGna_12035, RGna_13420) are phylogenetically close to known sortase B proteins in other species. Additionally, we found that RGna_09235 and RGna_13410, which were initially annotated as SrtA, clustered with the sortase C proteins in the phylogenetic tree. Based on this observation, we have renamed these genes as srtC1 and srtC2, respectively. We have revised the original sentence in the revised manuscript, as follows: “M. gnavus ATCC 29149, a type strain, has eight putative sortase genes, which we named srtB1-6 and srtC1-2 based on a phylogenetic analysis of their amino acid sequences (Supplementary Fig. S4). (L131)”\nLine 130 – maybe better to say “in sortase-processed cell wall anchored proteins”\nResponse: We have revised the phrase as you suggested.\nFigure S3C –As you have a genome sequence, did it reveal two copies of this gene cluster? This should be performed with long read technology to overcome problems assembling repetitive regions. Is this region likely a mobile genetic element? If so, these srt genes may not be present in present in all M. gnavus strains. MGEs often insert into tRNA genes, which you could search for at the junctions of the duplicated region. What other genes are present in this duplicated region?\nResponse: We have observed a ~2-fold increase in read depth across the locus spanning RGna_13305 to RGna_13530, strongly suggesting that this region is duplicated in the genome. However, as you pointed out, because we used short-read sequencing for genome resequencing, we cannot determine the precise location and orientation of this duplicated region.\nIn addition, we identified a putative transposase gene in this duplicated region, immediately upstream of tRNA genes, suggesting that this region may represent an MGE. We have revised Supplementary Fig. S5 to show the location of a putative transposase gene and the tRNA genes. These findings imply that the presence of srtC2 and srtB6 could be strain-dependent.\nLine 148 – better to say “formed a less compact pellet”\nResponse: We have revised the phrase as you suggested.\nFig 3B and 3F must show a MW range so that we can determine the size of the CPS. They are typically quite heterogeneously sized high MW molecules.\nResponse: We have revised Fig. 3B and 3F to include molecular weight markers, as you suggested. We used a pre-stained protein ladder and enlarged the cropped regions to show a broader molecular weight range of the SDS-PAGE images. We detected CPS bands above the largest molecular weight marker, suggesting they are high-molecular-weight polysaccharides, consistent with the typical characteristics of CPS.\nFig 3C – are there any other phenotypes of the srtB4 mutant? In the image, it appears to have shorter chains. Do these bacteria clump at all as is a characteristic or some bacteria when their capsules are removed.\nResponse: Thank you for your comment. Based on microscopic observation, the chain length of the srtB4 mutant appears comparable to that of the wt strain in our experimental conditions. Representative phase contrast images of both strains at the exponential phase are shown below.\nSupporting information 2. Phase contrast microscopy of wt and ΔsrtB4. Cells were grown at the exponential phase (O.D.600 1.0). Bars = 10 µm.\n≒\nLine 152-154 – this image is not enough proof to say that these are fimbriae. These bacteria were processed for EM which introduces all types of artifacts. This statement about fimbriae should be removed.\nResponse: To avoid overinterpretation, we have replaced “fimbriate-like” with “fibrous” to state only the morphology observed in the TEM images, as you suggested.\nThe figure legend of Fig 3 (and line 161) should be modified, srtB4 is not responsible for capsular polysaccharide production, it is likely important in linking the CPS to the bacterial surface. Did you analyze the supernatants for the presence of CPS? An antibody to this molecule would substantially help with analyses – which should be easily obtained by adsorbing antibodies to the WT bacterium with the CPS mutant.\nResponse: Thank you for this important and thoughtful comment. We agree that our original statement may have overinterpreted the role of SrtB4. Based on our data, we observed a substantial reduction of CPS structures on the cell surface in the srtB4 mutant, as assessed by microscopy and SDS-PAGE. We analyzed the CPS in the supernatants and found that the amount of the CPS is not greatly changed between the wt and srtB4 mutant strains (see below), suggesting that reduction of the CPS in the mutant is not simply explained by the release into the medium.\nSupporting information 3. Detection of CPS in the supernatants. Capsular polysaccharide fractions (C) and supernatants (S) collected from cells grown overnight were separated by SDS-PAGE. Polysaccharides were detected by acid polysaccharide staining dye, alcian blue. Each lane contains 0.5 O.D. 600 units.\nHowever, at this point, we cannot determine whether srtB4 affects CPS production, surface anchoring, or both. To avoid overinterpretation, we have revised the description to state srtB4 is responsible for the surface presentation of CPS.\nRegarding your suggestion to generate anti-CPS antibodies, we appreciate this valuable idea and agree that such a tool would facilitate more detailed analysis. However, due to time constraints and resource limitations, we were unable to generate such antibodies in the current study. We hope to pursue this approach in future work.\nLine 167 –state “competitive advantage of the WT strains against the sortase mutants”, rather than the other way around.\nResponse: We have revised the phrase as you suggested.\nLines 200-201 – This statement is not supported as written. Bacteria frequently make more than one CPS/EPS. It is quite likely that the locus identified in the other study also produces CPS that is not the same as the CPS of your study. Many CPS do not extend so far from the cell and are not nearly as visible with India ink staining as the CPS you identified. This statement should be qualified to say “these gene disruptions did not affect the phenotypes tested in our study”. In fact, the cell pellets of these deletion mutants seem to be even more loosely packed than the WT strain, suggesting some role in production or presentation of surface PSs.\nResponse: Thank you for your thoughtful comment. We agree that our original statement was overinterpreted, and we did not intend to exclude the possibility that the genes identified in the previous study may be involved in the biosynthesis of polysaccharides other than those we tested in this study. Accordingly, we have revised the sentence as you suggested.\nIn addition, we appreciate your insightful observation that these mutants showed the looser cell pellets. Although we did not quantify this phenotype under our experimental conditions, we agree that this implies the involvement of these genes in surface polysaccharide production or presentation. We consider exploring it as an intriguing direction of future studies.\nLine 204 – there is no evidence that SrtB4 affects CPS production, just likely its surface attachment, or the attachment of another protein that is necessary for its surface localization.\nResponse: As mentioned earlier, we agree that our original statement may have overinterpreted the role of SrtB4. Based on our data, we cannot determine whether srtB4 affects CPS production, surface anchoring, or both. To avoid overinterpretation, we have revised the description to state srtB4 is responsible for CPS presentation to cell surfaces.\nThere are some concerns with the CPS mutants analyzed for competitive colonization in the mouse gut. As gene 11905 encodes a nucleotide-sugar synthesis gene, it may affect the production of nucleotide linked sugars necessary for other cell glycans. However, there are also concerns with any mutants that allow the incomplete building or flipping and polymerization of the repeat unit, as these mutants sequester and prevent recycling of undecaprenol-P, which is necessary for PG synthesis and can contribute to growth defects. The best strategy is to delete all glycosylatranserase genes so that the repeat unit is not built at all. These caveats should at least be discussed for the competitive colonization assays, especially since the strains have an in vitro growth defect. However, the 12040 mutant, which based on gene proximity may only be involved in attaching the CPS (although that is unknown), also confirms the competitive defect.\nResponse: Thank you for your thoughtful comment. As you suggested, we cannot exclude the possibility that the inability to produce nucleotide sugars influences a growth defect other than polysaccharide surface presentation and causes decreased competitive fitness in the intestine. We have revised the paragraph that describes the competitive colonization assay of RGna_11905 and RGna_12040 mutants, as follows:\n(L226) “Because RGna_11905 is a putative nucleotide-sugar synthesis gene, its disruption may affect the production of nucleotide-linked sugars necessary for other cell glycans beyond CPS. This possibly contributes to the observed decrease in competitive fitness. However, the competitive defect was also observed in the RGna_12040 mutant, which is likely involved in CPS attachment rather than synthesis. These results are likely to rule out a simple difference in growth rate as the cause of the CPS mutant strains being outcompeted by wild-type in the intestinal environment. Taken together, CPS production and surface localization are important for competitive fitness during intestinal colonization in M. gnavus.”\nWe believe these revised sentences clarify and address the caveats raised by the reviewer.\nLines 215-216 – “The CPS produced by the gene products of the identified locus….”. There may be more than one CPS/EPS produced by this organism.\nResponse: We agree with the reviewer’s suggestion. We have revised the phrase as you suggested.\nLine 220 – this statement is too broad, and the opposite has also been shown. Maybe say “to have various roles in immune recognition or modulation”.\nResponse: Thank you for your suggestion. We have revised the sentence as you suggested.\nFig 6 – there is some concern for the very different amounts of TNF in the strains comparing panels A and B. Why is there such a difference in the WT and srtB::erm strains in these panels?\nResponse: We acknowledge the difference in TNF concentrations between Fig. 6A and B in the original figure. In our experimental setting, these variations were occasionally observed even within the wt strain group across three independent experiments. Nevertheless, we consistently observed the same tendency across the experiments: srtB4 or CPS mutants induced higher TNF production than the wt strains. To avoid potential confusion, we have replaced the original Fig. 6A and B with a new Fig. 6A that shows representative data from a single experiment, including all relevant strains.\nFigure 6C – it is not evident when these mice were gavaged, please put an arrow on the time line. Also, it is surprising that mice monocolonized with M. gnavus survived for 8 days on 2.5% DSS.\nResponse: We have revised the original Fig. 6C to include arrows indicating the time point of oral gavage, as you suggested. Regarding the DSS concentration, BALB/c mice are reported to be more tolerant to DSS treatment than C57BL/6 mice [1], which likely explains the survival of M. gnavus-mono-associated mice for 8 days in our experiment.\n[1] Chassaing, Benoit, et al. \"Dextran sulfate sodium (DSS)‐induced colitis in mice.\" Current protocols in immunology 104.1 (2014): 15-25.\nFig 6D – for this experiment, you must quantify the cfu/gram of bacteria in these mice over the course of the experiment (for a few time points). It is very possible that the mutant strain was not able to colonize the DSS treated mouse to the same extent as the WT, which would be relevant data. Based on the data shown, I disagree with the statement “These results suggest that CPS production inhibits the recognition of R. gnavus cells from host immune cells and that CPS233 deficient R. gnavus has the potential to induce more severe inflammation during intestinal colonization”. The inflammation is induced by the DSS, not the M. gnavus. Without cfu/g, there cannot be a statement about effects of the CPS in this assay.\nResponse: We have revised Fig. 6 to include CFUs/g feces and the number of cells calculated by qPCR for each strain, as you suggested. These data indicate that colonization levels were not substantially different between the wt and mutant strains. Thus, the observed difference in DSS-induced inflammation is not due to differences in colonization efficiency but rather to genetic differences between these strains. We also replaced the last sentence of this paragraph as follows:\n(L243) “To further confirm whether CPS production is involved in intestinal inflammation induced by M. gnavus, we prepared M. gnavus wild-type or srtB4 mutant-mono-colonized mice and then compared inflammation during administration with dextran-sodium sulfate (DSS) (Fig. 6B). Despite no substantial difference in colonization levels between wt and srtB4 mutant in gnotobiotic mice at day 0 and a modestly increased number of srtB4 mutant strains at 8 days post DSS treatment (Fig. 6C), we observed more severe body weight loss and increased disease activity index (DAI) in srtB4 mutant-colonized mice compared to wild-type (Fig. 6D and E). These data suggest that genetic differences in M. gnavus colonizing the intestine influence inflammation, and that loss of CPSs may exacerbate its severity.”\nStudies related to figure 7 are problematic\n1) are you sure that all of these strains were isolated from separate people and are not replicates (possible longitudinal samples) from the same individual? In addition, the geographic location of the isolates (from where they live) is necessary as there can be clustering of isolates in different countries which would confound the data. There is some concern for the low number of samples for this analysis, which may be lower if duplicate samples from the same person are revealed.\nResponse: We carefully reviewed the literature [2] describing the isolated strains used in this analysis. While there was no clear statement whether all strains were isolated from different individuals, available clinical metadata, such as age, sex, and nationality, suggest that most strains were likely derived from different individuals.\nTo address your concern, we have revised Fig. 7A to include the geographic locations of each isolate. These data suggest that there is no strong geographic bias in srtB4 possession across countries. Despite the small sample size, we believe the trends of association between disease and the srtB4 gene remain informative. In addition, we have added a description of the limitations of this analysis, due to sample size, in the Discussion section.\n(L303) “Additionally, the srtB4 gene, which is required for CPS presentation, appears to be less prevalent in isolates from Crohn’s disease patients than in those from healthy individuals in the available complete genome dataset, although the sample size in this study is limited. This highlights a key limitation of comparative genomics alone and underscores the importance of functional validation using a bacterium-specific genetic modification system. Nevertheless, due to the limited number of isolates with complete genome sequences, the association between srtB4 and diseases should be interpreted with caution and warrants further investigation.”\n[2] Nooij, Sam, et al. \"Metagenomic global survey and in-depth genomic analyses of Ruminococcus gnavus reveal differences across host lifestyle and health status.\" Nature Communications 16.1 (2025): 1182.\n2) CPS loci are often heterogeneous across strains of a species. Do the strains that do not have the same CPS locus as described in this study have a distinct CPS locus in this region? In Fig S6, please show gene maps of this region in other strains.\nResponse: In the revised Supplementary Fig. 8A, we reanalyzed the genomes of the srtB4-positive isolates using tBLASTx with more lenient criteria to identify distant homologs of the CPS gene cluster from the type strain (ATCC 29149). This analysis revealed that all srtB4-positive isolates possess at least one homologous gene related to sugar biosynthesis and glycosyltransferase activity. In addition, we added a comparison of the genetic contexts of ATCC 29149, RI1, and NBRC 114413 strains, indicating that the overall gene composition and organization vary among strains (Supplementary Fig. 8B). These data support the idea that R. gnavus strains harbor diverse CPS gene clusters, potentially resulting in capsular polysaccharides with different sugar compositions and structures.\n3) it is quite possible that another Srt can serve the function of SrtB4 if there are diverse CPS loci in this region.\nResponse: In the revised Supplementary Fig. 8B, we include a genetic map of the corresponding CPS locus in NBRC 114413, a srtB4-negative strain. This region contains several putative CPS-related genes (e.g., glycosyltransferases, sugar-nucleoside biosynthesis, and flippases) but lacks sortase genes. Similarly, none of the srtB4-negative strains used in this study possess sortase genes at the corresponding locus. These suggest that, at least within this CPS locus, it is unlikely that another sortase gene is substituted for srtB4 function.\nStatements in the discussion related to findings of Crohn’s disease patients are overstated based on the caveats mentioned above.\nResponse: As noted earlier, we acknowledge the potential overstatement regarding the association between srtB4 and diseases. Therefore, we have revised the Discussion section to tone down the statements as previously described."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript shows improved quality, and I now have only a minor comment.\nAt Lines 120 and 285–295, the associated data (genomic analysis, plasmid loss, and transformation of other species) should be included in the supplementary materials.", "reviewer_2": "The authors did a good job of addressing my comments with addition of new data and text alterations. I have no further comments."}, "author_response": "Response: We thank the reviewer for the positive evaluation. We have included data from genomic analysis in Supplementary Fig. 3f, which shows no off-target effects. Additionally, we included images showing the loss of the CRISPR plasmid and the fluorescent strains of E. bolteae and E. clostridioformis in the new Supplementary Fig. 8."}]

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