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PubMed2026

Alternative transcription of the mouse Gh gene identifies an immune-associated transcript with species-specific structural divergence.

Growth hormone (GH) in mice is primarily expressed in the anterior pituitary, although Gh expression has been reported in extrapituitary tissues, including immune organs. However, the structure of immune-associated Gh transcripts remains poorly characterized. To determine whether splenic Gh transcripts differ from pituitary Gh mRNA, 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses were performed. While 3' RACE showed a shared polyadenylation site, 5' RACE identified a novel exon located approximately 2 kb upstream of the conventional exon 1, generating a transcript (spl-Gh mRNA) with a distinct first exon but shared downstream exons with pituitary Gh mRNA (pit-Gh mRNA). RT-PCR analysis revealed that spl-Gh mRNA is predominantly expressed in immune tissues such as spleen and bone marrow, and its distribution did not correlate with Pit-1 mRNA expression. Quantitative RT-PCR further demonstrated that spl-Gh mRNA was expressed at levels comparable to those of pit-Gh mRNA in the mouse spleen, indicating that spl-Gh is one of the major Gh transcript forms in this tissue. Sequence analysis indicated that spl-Gh mRNA is predicted to retain coding potential for a GH protein. Comparative genomic analyses further demonstrated that genomic features associated with the spl-Gh transcriptional unit are conserved only in a subset of closely related Mus species. In contrast, although a spl-Gh-related transcript was detected in rat spleen, no properly spliced mouse-like transcript was identified under the present experimental conditions. The detected transcript exhibited intron retention and an in-frame stop codon, suggesting that it is unlikely to produce a functional GH protein. These findings identify a distinct immune-associated Gh transcript generated through alternative transcription of the mouse Gh gene and suggest that immune-associated Gh transcriptional mechanisms have undergone species-specific divergence among rodents. Together, these findings reveal previously unrecognized complexity in Gh gene regulation and highlight species-specific differences in immune-associated Gh transcripts.

Animals↗
PubMed2025

Biallelic MINAR2 variant is associated with nonsyndromic severe to profound sensorineural hearing loss.

MINAR2 is essential for normal hearing by regulating cholesterol localization in stereocilia in hair cells. MINAR2 knockout results in rapidly progressive sensorineural hearing loss (SNHL) in mice and zebrafish models. Recently, biallelic variants in MINAR2 have been reported to cause SNHL in four unrelated families with nonsyndromic severe to profound SNHL. Here we provide a second report of an additional family with SNHL. The index patient presented with nonsyndromic severe to profound SNHL. The family history was remarkable for a 20-year-old male sibling with nonsyndromic severe to profound SNHL. Both patients did not have any neurological involvement. Trio whole-exome sequencing of the index and his parents revealed a homozygous nonsense variant in MINAR2 (NM_001257308.2:c.319A>T; p.(Lys107*) in the index. Parents were heterozygous for the same variant. This variant introduces an early stop codon and probably results in a loss of function because of the predicted nonsense-mediated decay. Our study provides the first independent confirmation of the MINAR2-related SNHL.

Journal Article↗
PubMed2026

Targeting oncogenic FLT3 uncovers a ferroptosis vulnerability through selenocysteine recoding in acute myeloid leukaemia.

Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, has emerged as a potential therapeutic strategy for therapy-resistant cancers. Glutathione peroxidase 4 and the selenoprotein biosynthesis pathway essential for its translation are key regulators of ferroptosis but lack effective therapeutic targeting. In a drug screening using a selenoprotein translation reporter, here we identify FMS-like tyrosine kinase 3 (FLT3) inhibitors as suppressors of selenoprotein translation that induce ferroptosis in FLT3-mutant acute myeloid leukaemia. Mechanistically, FLT3 inhibition disrupts selenocysteine recoding, in which a UGA stop codon is recoded as selenocysteine via the SECIS element and associated binding proteins. Notably, the antileukemic efficacy of the FLT3 inhibitor gilteritinib was markedly reduced by dietary vitamin E, which attenuated ferroptosis. This study highlights ferroptosis as a vulnerability in FLT3-mutant acute myeloid leukaemia and suggests that high vitamin E intake may compromise tyrosine kinase inhibitor efficacy partly by suppressing ferroptosis.

Ferroptosis↗
PubMed2026

O'nyong-nyong virus adaptive mutations in non-structural protein 1 and 3 enhance RNA replication and overcome FHL1 requirement.

Arthritogenic alphaviruses, like o'nyong-nyong virus (ONNV), cause debilitating musculoskeletal diseases and are geographically expanding. To predict their emergence, we seek to better understand evolutionary mechanisms that enable changes in virus tropism. Here, we identify adaptive mutations in the ONNV non-structural proteins (nsPs) that arose during cellular serial passaging and enabled ONNV to infect non-permissive Lunet cells. Using shotgun proteomics, we show that this human hepatoma cell line lacks the four-and-a-half-LIM domain protein 1 (FHL1), an essential host factor in ONNV RNA replication. Individual single nucleotide mutations in the nsP1 ring-aperture membrane-binding and oligomerization domain, the nsP3 macrodomain, and the nsP3 opal stop codon overcome FHL1 deficiency in Lunet cells by enhanced RNA replication. These findings demonstrate how subtle genomic changes in nsPs can profoundly influence alphavirus replication and tropism.

LIM Domain Proteins↗
PubMed2021

Drug resistance mutations in HIV provirus are associated with defective proviral genomes with hypermutation.

BACKGROUND: HIV proviral sequencing overcomes the limit of plasma viral load requirement by detecting all the 'archived mutations', but the clinical relevance remains to be evaluated. METHODS: We included 25 participants with available proviral sequences (both intact and defective sequences available) and utilized the genotypic sensitivity score (GSS) to evaluate the level of resistance in their provirus and plasma virus. Defective sequences were further categorized as sequences with and without hypermutations. Personalized GSS score and total GSS score were calculated to evaluate the level of resistance to a whole panel of antiretroviral therapies and to certain antiretroviral therapy that a participant was using. The rate of sequences with drug resistance mutations (DRMs) within each sequence compartment (intact, defective and plasma viral sequences) was calculated for each participant. RESULTS: Defective proviral sequences harbored more DRMs than other sequence compartments, with a median DRM rate of 0.25 compared with intact sequences (0.0, P&#x200a;=&#x200a;0.014) and plasma sequences (0.095, P&#x200a;=&#x200a;0.30). Defective sequences with hypermutations were the major source of DRMs, with a median DRM rate of 1.0 compared with defective sequences without hypermutations (0.042, P&#x200a;<&#x200a;0.001). Certain Apolipoprotein B Editing Complex 3-related DRMs including reverse transcriptase gene mutations M184I, E138K, M230I, G190E and protease gene mutations M46I, D30N were enriched in hypermutated sequences but not in intact sequences or plasma sequences. All the hypermutated sequences had premature stop codons due to Apolipoprotein B Editing Complex 3. CONCLUSION: Proviral sequencing may overestimate DRMs as a result of hypermutations. Removing hypermutated sequences is essential in the interpretation of proviral drug resistance testing.

Anti-HIV Agents↗
PubMed2025

Translational reading frame determines the pathogenicity of C-terminal frameshift deletions in MeCP2: an alternative therapeutic approach.

Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) lead to loss of ~100 amino acids at the C-terminus of the MeCP2 protein, and account for approximately 10% of RTT-causing mutations. The pathogenicity of C-terminal deletions (CTDs) is unexpected, as this C-terminal domain is non-essential in mice. Utilising databases of pathogenic and benign human MECP2 mutations, we find that some individuals with apparently typical CTDs do not exhibit Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human DNA sequence data and mouse models, we demonstrate that pathogenicity results from a drastic reduction in MeCP2 levels and is determined by the presence of the short amino acid motif proline-proline-stop (-PPX) at the C-terminus, which results from a shift to the +2 reading frame. Individuals with CTDs that shift to the +1 frame avoid this motif and do not develop Rett syndrome. Mutating the stop codon of the PPX motif to tryptophan rescues MeCP2 expression and RTT-like phenotypes in a CTD mouse model. Finally, we demonstrate that an adenine base editor can efficiently introduce this tryptophan substitution in cultured cells. Overall, our findings uncover a simple and reliable prognostic distinction between benign and pathogenic CTDs and provide proof-of-concept for an editing strategy that potentially corrects all disease-causing CTD mutations.

Journal Article↗
PubMed2025

High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation.

Noncanonical amino acids (ncAAs) enable diversification of protein functions, but the efficiency of genetic code expansion (GCE) in eukaryotes is hindered by competition between suppressor tRNAs and release factors. Prior work has identified eukaryotic release factor 1 (eRF1) mutants that improve ncAA incorporation, suggesting that screens for improved variants may lead to further enhancements. Here, we developed a high-throughput system to screen eRF1 mutants in Saccharomyces cerevisiae where eRF1 mutants are coexpressed on a plasmid alongside genomically encoded, wild-type eRF1. This strategy enabled recovery of live cells expressing eRF1 variants that enhance ncAA incorporation, even with mutants known to severely affect cell viability in the absence of WT eRF1 expression. We prepared and screened a million-member library of randomly mutated eRF1 variants for clones exhibiting improved ncAA integration phenotypes. Deep sequencing revealed a diverse set of enriched mutations across all three major domains of eRF1. Interestingly, several enriched mutations identified here are also found in naturally occurring eRF1 homologs from species that recode canonical stop codons. When eRF1 variants were combined with yeast knockout strains also known to enhance ncAA incorporation, this resulted in further improvements to efficiency, highlighting the complementarity of release factor engineering to other GCE enhancement strategies. This work demonstrates that high-throughput engineering of the eukaryotic translational apparatus is a powerful approach to identify previously unknown solutions for enhancing ncAA incorporation, with implications for elucidating and precisely manipulating the molecular functions of essential translational machinery.

Noncanonical amino acids↗
PubMed2026

An antisense antidote to oncogenic poison exons.

Splicing factors are frequently mutated in myeloid cancers, causing splicing aberrations that derail the expression of tumor suppressor genes. In SRSF2 mutated cancers, a key oncogenic splicing event is the inclusion of a "poison" exon that introduces an early stop codon in EZH2 mRNA, causing its destabilization. In this issue of Genes & Development, Islam et al. (doi:10.1101/gad.353628.126) define how mutant SRSF2 binding to the poison exon mediates its inclusion and identify an antisense oligonucleotide that represses the exon to restore EZH2 function and rescues hematopoietic defects. Thus, targeting of poison exons, many of which show protumorigenic and antitumorigenic properties, is a promising new avenue to treat cancer.

Oligonucleotides, Antisense↗
PubMed2026

Endogenous CRISPR-Based Removal of Tetracycline Resistance in Bifidobacterium animalis subsp. lactis Through a Safe-by-Design Approach.

Bifidobacterium animalis subsp. lactis is widely used as a probiotic; however, the presence of the tetracycline resistance gene tetW raises safety and regulatory concerns due to its potential mobility within the gut microbiome. Here, we applied a Safe-by-Design strategy using the endogenous CRISPR-Cas system of B.&#x2009;animalis subsp. lactis BLC01 to inactivate tetW through the introduction of premature stop codons. Whole-genome sequencing confirmed the intended editing and excluded relevant off-target effects. tetW inactivation markedly reduced the tetracycline minimum inhibitory concentration, restoring susceptibility below the tetracycline cut-off value for bifidobacteria (8&#x2009;&#x3bc;g/mL). Comparative phenotypic analyses demonstrated that the edited strain (BLC01-2F3G10) retained key probiotic traits, including tolerance to acid, bile, and osmotic stress, exopolysaccharide production, aggregation capacity, survival during simulated gastrointestinal digestion and adhesion to intestinal epithelial cells. Importantly, no reversion to tetracycline resistance was observed after prolonged exposure to sub-inhibitory minimal selective antimicrobial concentration, indicating genetic stability of the edited phenotype. Collectively, these findings demonstrate that endogenous CRISPR-based genome editing can be leveraged to selectively remove antimicrobial resistance determinants from probiotic strains while preserving functionality, supporting the development of next-generation probiotics with an improved safety profile and reduced potential for antimicrobial resistance dissemination in the human gut.

Tetracycline Resistance↗
PubMed2026

A novel deep intronic EIF2AK3 variant disrupts splicing and causes Wolcott-Rallison syndrome.

AIM: Deep intronic variants can disrupt splicing and cause monogenic disease but are missed by routine genetic testing. This study assessed the contribution of deep intronic variants to Wolcott-Rallison syndrome (WRS), a recessive disorder characterized by early-onset diabetes and progressive multisystem disease caused by loss-of-function EIF2AK3 variants. METHODS: We investigated a cohort of 116 individuals referred to the Exeter Genomics Laboratory for genetic testing who had diabetes diagnosed at &#x2264;2&#x2009;years and at least one additional feature consistent with WRS: hepatic dysfunction, skeletal abnormalities or developmental delay. No genetic cause had been identified after testing all known early-onset diabetes genes. We screened genome-sequencing data for rare homozygous intronic EIF2AK3 variants. Candidate variants predicted to affect splicing by SpliceAI were assessed using a minigene exon-trapping assay. RESULTS: We identified two rare homozygous intronic EIF2AK3 variants in two siblings. Only one variant, c.1651-180G>T, was predicted to disrupt splicing in silico. The two children, born to consanguineous parents, were diagnosed with early-onset diabetes (diagnosed at 1&#x2009;year and 21&#x2009;weeks), hepatic dysfunction, skeletal abnormalities, developmental delay, thyroid dysfunction, hip dysplasia and gait abnormalities. The minigene assay showed that c.1651-180G>T creates a cryptic donor splice site within intron 9, resulting in inclusion of a 79-nucleotide pseudoexon, causing a frameshift and premature stop codon. Using this evidence, the variant was reclassified as likely pathogenic according to ACMG/ACGS guidelines. CONCLUSIONS: We report the first deep intronic EIF2AK3 variant causing WRS, highlighting the need to consider systematic intronic analysis in unresolved cases.

EIF2AK3↗
PubMed2023

Spontaneous allelic variant in deafness-blindness gene Ush1g resulting in an expanded phenotype.

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw /+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.

Mice↗
PubMed1978

Complete nucleotide sequence of the simian-virus 40 Hind-G fragment and localisation of the carboxyl terminus of the VP1 protein.

The restriction fragment Hind-G represents 7.0% of the simian virus 40 (SV40) genome. The information present in fragment Hind-G is expressed as part of the major, late 16-S messenger RNA. The complete nucleotide sequence of the fragment Hind-G has now been determined by application of the procedure of Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. (1977) 74, 560-564]. It contains 369 nucleotide base pairs. On the basis of the termination code words in the strand with the same polarity as the late mRNA, two illegitimate reading frames can be defined. Therefore the third, open frame must code for the carboxyl terminal part of the VP1 protein. It terminates within fragment Hind-G with a TGA signal. This stop codon is followed by a non-translated region of the mRNA of about 83 nucleotides. The latter contains the sequence A-A-U-A-A-A, common to all other eukaryotic mRNA molecules so far studied. The Hind-G fragment also contains sequences which presumably play a role in the synthesis, processing and/or expression of early mRNA; these aspects are discussed in the following paper.

Amino Acids↗
PubMed2025

Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.

Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis &#xd7; Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.

Flavanones↗
PubMed2025

Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3' end processing in polycistronic mRNA.

"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5' galE-galT-galK-galM 3', via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3' end of the full-length galETKM mRNA is acquired by exonucleolytic processing of the 3'-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA termed galE whose 3' end forms approximately 120 nucleotides downstream of the galE stop codon, within the subsequent gene, galT, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3' end of galE mRNA. We discovered that the 3' ends of pre-galE mRNA are produced in the middle of galT as a result of the combination of two separate molecular processes-one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3' ends of pre-galE mRNA undergo exonucleolytic processing to the 3' end of galE mRNA observed in vivo. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5-10 nucleotides upstream of the 3' ends of galE mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E-contrary to its general role in mRNA degradation-produces RNA 3' ends that regulate polarity in gene expression.IMPORTANCEThis study reports the findings of two molecular mechanisms that generate the 3' ends of pre-galE mRNA in the gal operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3' ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3' end of galE mRNA within galT in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.

RNA, Messenger↗
PubMed2023

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Humans↗
PubMed2026

Genomic characterization of the attenuated human cytomegalovirus strain TR-VAC developed for subviral particle vaccine production.

We report the complete genome sequence of the attenuated human cytomegalovirus strain TR-VAC, developed for subviral particle vaccine production. Oxford Nanopore duplex sequencing confirmed all engineered modifications, including UL130 repair, UL25 stop codons, ddFKBP insertion, GFP deletion, and retention of the bacterial artificial chromosome backbone, without large-scale structural rearrangements.

Human cytomegalovirus↗
PubMed2026

EndoV does not measurably affect TadA-dependent A-to-I RNA editing in Escherichia coli under exponential-growth conditions in rich medium.

Adenosine-to-inosine (A-to-I) mRNA editing changes the genetic information post-transcriptionally and was only recently reported to occur in bacteria. Here, we examined whether endonuclease V (EndoV; encoded by nfi) cleaves inosine-containing RNAs in vivo and thereby influences the abundance and fate of A-to-I-edited mRNAs in Escherichia coli. We generated an nfi loss-of-function mutant carrying a premature stop codon and performed RNA sequencing alongside the isogenic wild-type strain. We observed that global and site-specific editing occurrence or levels in both mRNAs and tRNAs were indistinguishable between strains. Moreover, overexpression of EndoV did not affect the number of edited sites, motif enrichment, or editing levels compared with a control strain overexpressing mCherry. Our findings suggest that, in contrast to human EndoV, bacterial EndoV does not regulate the steady-state pool of edited mRNAs in E. coli under nutrient-rich, exponential-growth conditions in vivo.IMPORTANCEAdenosine-to-inosine (A-to-I) mRNA editing is an emerging regulatory layer in bacteria, but the factors that act on edited transcripts are largely unknown. Endonuclease V (EndoV) was a prime candidate because it cleaves inosine-containing nucleic acids and can act on inosine-containing RNA in vitro. By combining loss-of-function and overexpression of EndoV with genome-wide RNA editing measurements, we show that EndoV does not measurably influence TadA-dependent A-to-I mRNA editing in Escherichia coli under standard laboratory conditions. This negative result is important because it rules out a natural effector candidate and redirects attention to other bacterial pathways that may process edited RNAs. Our work, therefore, sharpens mechanistic models for bacterial RNA editing and helps focus future searches for its regulators and physiological roles.

Escherichia coli↗
PubMed2024

Development of SacB-based counterselection for efficient allelic exchange in Fusobacterium nucleatum.

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare, defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knock-in of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possibly related to the natural competence of F. nucleatum. Post-counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research.IMPORTANCEInvestigations into Fusobacterium nucleatum's role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk-based allelic exchange method, although effective, faced an inherent limitation-the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum. Our application of the sacB-based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knock-in at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum.

Fusobacterium nucleatum↗