A new study published in MicrobiologyOpen has now combined two cutting-edge technologies, CRISPR interference and nanopore native RNA sequencing, to systematically probe the epitranscriptome of Escherichia coli. The research, led by Miranda E. Pitt of the University of Technology Sydney and Lachlan J. M. Coin of the University of Melbourne, together with colleagues, demonstrates a workflow in which individual RNA modification enzymes are switched off in living bacteria, and the resulting loss of chemical marks is read out directly at the single-molecule level. The approach not only confirmed several well-known modification sites on ribosomal RNA but also revealed candidate modification sites scattered across bacterial messenger RNA, a frontier that has remained largely unmapped.

The technical challenge the team faced is formidable. Traditional methods for detecting RNA modifications, such as immunoprecipitation or chemical treatments like bisulfite conversion, are laborious, require highly purified and concentrated RNA, and generally capture only one modification type at a time. Bacterial RNA adds further complications: rigorous lysis procedures and complex cell membranes accelerate RNA degradation, and bacterial transcripts have notoriously short half-lives, in many cases under one minute. Enzymes called RNases can rapidly destroy extracted RNA unless inhibited immediately. Direct RNA sequencing, released by Oxford Nanopore Technologies in 2017, offers a faster alternative because it reads native RNA molecules as they pass through protein nanopores, preserving the chemical modifications that conventional methods destroy when RNA is converted to complementary DNA. However, the chemistry was originally incompatible with bacterial transcripts, which lack the polyadenylated tails found in eukaryotic messenger RNA.

The researchers overcame this barrier by artificially adding poly(A) tails to bacterial transcripts before sequencing, a technique they had refined in previous work. To create the perturbations needed to detect modifications, they turned to CRISPR interference, or CRISPRi. Unlike standard CRISPR gene editing, which cuts DNA, CRISPRi uses a catalytically inactive form of the Cas9 protein, dCas9, that binds to a target gene guided by a matching guide RNA and simply blocks its transcription. This knocks down, rather than knocks out, gene expression, avoiding the toxicity and off-target DNA disruption associated with genome cutting while allowing inducible, tunable silencing. The team used a plasmid system optimized for E. coli, in which dCas9 expression is triggered by the small molecule anhydrotetracycline.

Five genes encoding known ribosomal RNA modification enzymes were targeted: rlmF and rlmJ, both responsible for N6-methyladenosine marks on 23S ribosomal RNA; rsmF, which deposits 5-methylcytosine on 16S ribosomal RNA; rsmG, which installs 7-methylguanosine on 16S ribosomal RNA; and rluD, a pseudouridine synthase that modifies three positions in a functionally critical stem-loop of the 23S subunit. A sixth gene, add, encoding adenosine deaminase, was included to explore whether it might carry previously unrecognized RNA editing activity. The knockdown system was tested across multiple E. coli reference strains, including the type strain ATCC 11775, ATCC 8739, ATCC 25922, and a multidrug-resistant isolate, ATCC BAA-2452. Quantitative reverse transcription PCR confirmed that most target genes were repressed by more than 80 percent, although rsmG proved harder to silence, reaching only about 54 percent reduction, and the resistant isolate could not be transformed with the plasmid without a severe fitness cost.

With knockdown strains in hand, the researchers sequenced both total RNA and ribosomal RNA-depleted, messenger RNA-enriched samples on nanopore flow cells, using computational tools to compare ionic current signals between knockdown and control conditions. The software nanocompore, which does not require prior training and is agnostic to modification type, compared each perturbed sample against two controls: the unmodified original isolate and a plasmid control expressing dCas9 without a guide RNA. For rluD, all three known pseudouridine sites in the 23S ribosomal RNA were detected with strong statistical significance, and the sequence motifs surrounding those sites matched published reports. The rsmG knockdown likewise produced a clear signal at the known 7-methylguanosine position in 16S ribosomal RNA. In contrast, the sites modified by rlmF, rlmJ, and rsmF yielded weak signals that were difficult to distinguish from noise, echoing the findings of earlier benchmarking studies showing that certain modification types, particularly 5-methylcytosine and some N6-methyladenosine sites, are inherently harder to detect with current nanopore basecalling and current-difference tools.

Extending the analysis to messenger RNA, the team identified significant modification-site changes on several bacterial transcripts shared across strains. Genes harboring candidate modification sites included ompC, which encodes an outer membrane porin; lpp1; salPB; cspC; dbhA and dbhB; and secY, which encodes a core component of the protein secretion machinery. For the rluD and rsmG knockdowns, which had produced the strongest ribosomal RNA signals, the researchers searched messenger RNA for sequence motifs matching those seen at the known ribosomal sites. Strikingly, a significant change was found on the well-characterized pseudouridine motif in the gene encoding elongation factor Tu, the workhorse protein of translation, as well as on transcripts of lpp1, gapA, and fusA. For rsmG, significant sites matching the known 7-methylguanosine motif appeared in hupA, lpp1, rplT, rpsA, and ompC. Because these messenger RNA sites were detected in more than one strain background, they represent plausible, though not yet chemically verified, targets of bacterial modification enzymes acting beyond the ribosome.

The study also examined the downstream consequences of losing these modifications, measuring bacterial growth and profiling the proteome by quantitative mass spectrometry. Growth effects varied by strain: activation of dCas9 itself imposed a measurable burden in ATCC 11775, which carries an extra plasmid, while ATCC 8739 tolerated the system well. Statistically significant growth delays were observed for knockdowns of rluD and rsmF in one strain and rlmF, rluD, and rsmF in another, consistent with prior reports that the phenotypic impact of ribosomal RNA modification loss depends heavily on genetic background. Proteomic analysis detected roughly 1,800 to 2,400 proteins per strain and revealed modest but reproducible changes, including alterations in citrate lyase, the trehalose-specific phosphotransferase component TreB, the xylose-binding protein XylF, the sodium/proline symporter PutP, formate hydrogenlyase subunit HycB, and the oligopeptide transporter OppC. The authors note these changes require further validation and may only become pronounced under stress conditions such as antibiotic exposure or temperature shifts, which previous work has shown can reshape the bacterial epitranscriptome.

The broader significance of the work lies in its implications for antimicrobial resistance and bacterial virulence. RNA modifications are already known to help pathogens evade entire antibiotic classes, including aminoglycosides, chloramphenicol, and macrolides, and to contribute to antibiotic tolerance in organisms such as Vibrio cholerae. Loss of tRNA modification enzymes can impair a bacterium’s ability to infect and cause disease in a host. A reliable, rapid, genome-wide method for mapping modification dynamics in bacteria could therefore illuminate how pathogens respond to clinical interventions at the level of their RNA. The authors caution that fine-tuning is still needed, particularly for messenger RNA modification detection across diverse bacterial species and under varying growth conditions, and newer nanopore chemistry promises higher accuracy and greater yield. Yet as a proof of principle, pairing CRISPR interference with native RNA sequencing offers a scalable blueprint for interrogating the bacterial epitranscriptome, one silenced enzyme and one molecule of RNA at a time.

Subject of Research: Mapping RNA modifications in the Escherichia coli epitranscriptome using CRISPR interference gene knockdown and nanopore direct RNA sequencing

Article Title: Interrogating the Escherichia coli Epitranscriptome Via CRISPR Interference and Nanopore Native RNA Sequencing

Article References: Pitt, M. E., Zhang, J., Nguyen, A. N. T., Hall, M. B., Jebeli, L., Featherstone, L. A., Myers, G. S. A., Scott, N. E., & Coin, L. J. M. (2026). Interrogating the Escherichia coli Epitranscriptome Via CRISPR Interference and Nanopore Native RNA Sequencing. MicrobiologyOpen, 15(5), Article e70413. https://doi.org/10.1002/mbo3.70413

Image Credits: AI Generated

DOI: 10.1002/mbo3.70413

Keywords: epitranscriptome, E. coli, CRISPR interference, nanopore sequencing, direct RNA sequencing, RNA modifications, ribosomal RNA, messenger RNA, pseudouridine, m6A, antimicrobial resistance, MicrobiologyOpen

A new study published in MicrobiologyOpen reports the isolation and mechanistic validation of Mitsuokella jalaludinii strain PMC73, a gut commensal that its discoverers describe as a

The rationale for looking to the intestine as a therapeutic reservoir in gout rests on a long-recognized but often overlooked facet of urate physiology. While the kidneys excrete the great majority of uric acid each day, roughly one third of renal-independent elimination occurs through the gut, where commensal bacteria capable of degrading urate and its purine precursors participate in what researchers describe as an intestinal uricolytic axis. When this microbial community is intact, urate that is secreted into the intestinal lumen can be metabolized before reabsorption; when dysbiosis depletes these uricolytic populations, more purine substrate recirculates and the systemic urate burden climbs. This ecological perspective reframes hyperuricemia not merely as an enzymatic problem within host cells but as a breakdown in a metabolic partnership between host and microbiome, one that a well-chosen microbial therapeutic could theoretically restore.

The evolutionary context makes this microbial capacity especially interesting to gout researchers. Most mammals possess functional uricase, a hepatic enzyme that converts uric acid into the far more soluble compound allantoin, which is excreted readily in urine. Humans and certain other primates lost uricase activity through a series of mutations during the Miocene epoch, a change frequently attributed to the antioxidant properties of urate or to proposed survival advantages under purine-rich diets. The consequence, however, is that humans depend almost entirely on renal and intestinal excretion to maintain urate homeostasis, and any impairment of either route predisposes to hyperuricemia. Microorganisms retained their uricolytic enzymes, and the urate transporter and uricase machinery of gut bacteria have therefore become attractive targets for understanding how uric acid is naturally degraded outside the liver.

Purine metabolism provides a second, upstream point of microbial intervention. Dietary and endogenous purines are broken down through a cascade in which hypoxanthine is oxidized to xanthine and then to uric acid by xanthine oxidoreductase, the very enzyme targeted by allopurinol and febuxostat. Bacteria in the intestinal lumen can intercept this pathway at multiple levels: some taxa assimilate purine bases directly as nutrients, incorporating them into nucleic acid biosynthesis rather than allowing their conversion to urate; others express enzymes that funnel hypoxanthine and xanthine toward degradation or interconversion routes that bypass uric acid formation. A candidate strain capable of consuming hypoxanthine, as the Mitsuokella isolate was evaluated for, would in principle reduce the substrate available to host xanthine oxidase, offering an indirect complement to xanthine oxidase inhibitor drugs without sharing their hepatic mechanism of action.

The inflammatory arm of gout pathophysiology is equally central to the study’s logic. Monosodium urate crystals do not cause tissue damage simply through mechanical abrasion; they are recognized as danger signals by macrophage pattern recognition receptors, leading to assembly of the NLRP3 inflammasome, a multiprotein complex that activates caspase-1 and enables proteolytic maturation of interleukin-1 beta and interleukin-18. Mature interleukin-1 beta drives the intense neutrophil recruitment, pain, warmth, and swelling characteristic of an acute flare. Because the macrophage sits at the initiating step of this cascade, the RAW 264.7 cell line triggered by synthetic urate crystals provides a reductionist but informative system for asking whether a candidate treatment dampens inflammasome activation, suppresses cytokine release, or limits the oxidative burst that accompanies crystal phagocytosis. Reactive oxygen species generated during this response feed back positively on inflammasome activity, making antioxidant effects and anti-inflammatory effects mechanistically intertwined.

Preclinical models of this kind carry inherent limitations that temper expectations. A murine macrophage line, even one authenticated and mycoplasma-free, cannot reproduce the complexities of human joint biology: the contribution of synovial lining cells, resident mast cells, neutrophil extracellular traps, and the adaptive immune system are all absent. Species differences between murine and human inflammasome regulation are well documented, and doses of crystal or bacterial conditioning media that inhibit cytokine release in vitro may behave differently in vivo, where pharmacokinetics, gut colonization dynamics, and host immune status shape outcomes. For this reason, the macrophage experiments are best understood as mechanistic screening that establishes plausibility and identifies signaling pathways worth interrogating, rather than as proof of clinical efficacy, which will require animal models of hyperuricemia and, ultimately, controlled human studies.

The safety evaluation framework applied to new microbial candidates deserves attention because it differs substantially from conventional drug development. Genomic analysis serves as the first gate: the complete chromosome sequence allows identification of virulence factor genes, toxin-encoding elements, and acquired antibiotic resistance determinants that could compromise clinical use. Average nucleotide identity calculations against reference genomes confirm the taxonomic identity of the strain at species-level resolution, which matters because probiotic safety and function can be strain-specific rather than species-wide. Physical characterization, including assessment of bile tolerance, acid survival, adhesion properties, and hemolytic behavior, then addresses whether the organism can survive gastrointestinal transit and whether it behaves as a commensal rather than an opportunistic pathogen. This layered vetting reflects lessons from rare infections involving lactobacilli and other typically benign bacteria in profoundly immunocompromised patients.

The post-NGP framing articulated by the authors responds to a genuine bottleneck in microbiome therapeutics. Cultivation-independent sequencing surveys over the past fifteen years revealed that a large fraction of gut species had never been cultured in the laboratory, and many of the most immunologically and metabolically active commensals are strict anaerobes that die rapidly on exposure to oxygen. Next-generation probiotics such as Akkermansia muciniphila and Faecalibacterium prausnitzii demonstrated that these organisms can be tamed and formulated, but also highlighted practical obstacles: manufacturing live obligate anaerobes at industrial scale, protecting them through stomach acid and bile, and maintaining viability on the shelf remain unsolved engineering problems for many candidates. Concepts such as pasteurized bacterial preparations and defined bioactive fractions have emerged as workarounds, suggesting that a live organism may not even be necessary if the responsible molecular mediators can be identified.

This is where the boundary between probiotics, postbiotics, and the proposed post-NGP framework becomes conceptually significant. Postbiotics, as defined in a 2021 expert consensus, encompass inanimate microorganisms or their components and metabolites that confer health benefits, effectively decoupling the therapeutic effect from organism viability. The post-NGP idea, as described here, is less a product category than a discovery pipeline: it emphasizes systematic isolation through culturomics, genomic validation, and disease-targeted functional screening before any candidate advances. In gout specifically, such screening can be structured around measurable functional readouts, including the capacity of a strain to consume uric acid or hypoxanthine in defined medium, to modulate inflammasome signaling in immune cells, and to survive physiological stresses encountered during oral administration. This funnel-like design contrasts with older serendipitous approaches in which commercially available strains were repurposed and tested for whatever benefits happened to emerge.

The epidemiological backdrop amplifies the value of any new mechanistic option. Gout burden correlates strongly with metabolic syndrome, nonalcoholic fatty liver disease, and chronic kidney disease, creating a therapeutic dilemma in which patients who most need urate lowering are often those least able to tolerate or benefit from existing drugs, since several require dose adjustment or carry heightened risks in renal impairment. Allopurinol dosing in particular must be reduced in kidney disease, and failure to attain target serum urate levels remains the most common reason for treatment failure in routine practice. Adherence is another persistent obstacle: because urate-lowering therapy is prophylactic rather than symptomatic, and because initiating therapy can transiently provoke flares, many patients discontinue treatment within the first year. An intervention perceived as a natural commensal with a favorable safety profile could, at least hypothetically, improve acceptability, although this presumption awaits evidence from real-world adherence studies.

Regulatory pathways will ultimately shape whether microbiome-derived candidates reach patients. In many jurisdictions, live biotherapeutic products are now treated as a distinct class requiring manufacture under pharmaceutical-grade controls, strain identity confirmation, and demonstration of absence of transferable resistance genes, in addition to conventional efficacy trials. The characterization work reported for PMC73, including complete genome sequencing on a long-read platform, phylogenomic placement among Mitsuokella reference strains, and PCR-based screening for virulence determinants, aligns with the early stages of such a pathway. What remains ahead is the harder translational sequence: demonstration of urate-lowering in animal models of hyperuricemia, evaluation of anti-inflammatory efficacy in crystal-induced arthritis models, pharmacological assessment of colonization and persistence, and finally randomized clinical testing in hyperuricemic and gouty populations. The present study supplies the mechanistic foundation and safety profile that such a program requires, and it illustrates how systematically mining human gut commensals can surface candidates that conventional probiotic development, constrained to a handful of familiar genera, would never have encountered.

Subject of Research: A human gut-derived bacterial strain, Mitsuokella jalaludinii PMC73, evaluated as a next-generation probiotic candidate for lowering uric acid and treating gout.

Article Title: A Post–NGP Mitsuokella jalaludinii as a Therapeutic Candidate for Gout

Article References: Hossain, M. S., Kim, S., Aziz, M. T., Ahmed, I., Shuvo, M. S. H., Yang, H., Jang, Y., Kim, M., Jang, S., Kim, Y., Oh, S., Nam, Y., Seo, H., & Song, H.-Y. (2026). A Post–NGP Mitsuokella jalaludinii as a Therapeutic Candidate for Gout. MicrobiologyOpen, 15(5), Article e70410. https://doi.org/10.1002/mbo3.70410

Image Credits: AI Generated

DOI: 10.1002/mbo3.70410

Keywords: gout, hyperuricemia, Mitsuokella jalaludinii, next-generation probiotics, gut microbiome, uric acid, NLRP3 inflammasome, xanthine oxidase, URAT1, postbiotics, RAW 264.7 macrophages, MicrobiologyOpen