Biology subjects

G C, B.

Publications and source records attributed to G C, B..

5 recordsLinked to original sources

bioRxiv2025

The Gene Encoding Ornithine Decarboxylase for Putrescine Biosynthesis Is Essential for the Viability of Fusobacterium nucleatum

Fusobacterium nucleatum is a Gram-negative anaerobe associated with periodontitis and colorectal cancer. It secretes putrescine, a polyamine that promotes biofilm formation by oral co-colonizers and enhances the proliferation of cancer cells. However, the physiological importance of putrescine for F. nucleatum itself remains unexplored. Here, we show that putrescine biosynthesis, mediated by the ornithine decarboxylase gene oda, is essential for F. nucleatum viability. Deletion of oda was only possible when a functional copy was provided in trans, and CRISPR interference of oda expression resulted in complete growth arrest. The essentiality of oda was conserved across multiple subspecies. Supplementation with exogenous putrescine enabled the isolation of a conditional oda mutant whose growth was strictly putrescine-dependent. Putrescine depletion caused filamentation, membrane disruption, detergent hypersensitivity, and lysis in hypoosmotic conditions, indicating a critical role in maintaining cell envelope integrity. RNA sequencing revealed broad transcriptional remodeling under putrescine-limited conditions, including upregulation of genes involved in lipid metabolism, osmoprotection, and cell wall remodeling. Notably, oda transcript levels increased when putrescine was depleted, suggesting a negative feedback mechanism. These findings demonstrate that putrescine is not only an extracellular communal metabolite but is also vital for the cellular integrity and survival of F. nucleatum under anaerobic conditions. IMPORTANCEFusobacterium nucleatum is a prominent member of the oral microbiota and has been linked to various human diseases, including periodontitis, preterm birth, and colorectal cancer. Despite its clinical significance, the metabolic requirements that support its growth and viability remain poorly understood. In this study, we identify the oda gene, which encodes ornithine decarboxylase, as essential for F. nucleatum survival due to its role in putrescine biosynthesis. We demonstrate that depletion of putrescine leads to severe growth and morphological defects, accompanied by widespread transcriptional changes. These findings reveal an underappreciated metabolic vulnerability and highlight the critical role of polyamine homeostasis in maintaining cellular integrity in this notorious anaerobe.

microbiology↗

bioRxiv2024

The CarSR Two-Component System Directly Controls radD Expression as a Global Regulator that Senses Bacterial Coaggregation in Fusobacterium nucleatum

Two-component systems (TCS) enable bacteria to sense and respond to environmental signals, facilitating rapid adaptation. Fusobacterium nucleatum, a key oral pathobiont, employs the CarSR TCS to modulate coaggregation with various Gram-positive partners by regulating the expression of radD, encoding a surface adhesion protein, as revealed by RNA-Seq analysis. However, the direct regulation of the radD-containing operon (radABCD) by the response regulator CarR, the broader CarR regulon, and the signals sensed by this system remain unclear. In this study, chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) identified approximately 161 CarR-enriched loci across the genome and a 17-bp consensus motif that likely serves as the CarR binding site. Notably, one such binding motif was found in the promoter region of the radABCD operon. The interaction of CarR with this binding motif was further validated using electrophoretic mobility shift assays (EMSA), mutagenesis, and DNase I footprinting analyses. Beyond regulating radABCD, CarR directly controls genes involved in fructose and amino acid (cysteine, glutamate, lysine) utilization, underscoring its role as a global regulator in F. nucleatum. Lastly, we discovered that RadD-mediated coaggregation enhances radD expression, and deletion of carS abolished this enhancement, suggesting that coaggregation itself serves as a signal sensed by this TCS. These findings provide new insights into the CarR regulon and the regulation of RadD, elucidating the ecological and pathogenic roles of F. nucleatum in dental plaque formation and disease processes. IMPORTANCEFusobacterium nucleatum is an essential member of oral biofilms, acting as a bridging organism that connects early and late colonizers, thus driving dental plaque formation. Its remarkable ability to aggregate with diverse bacterial partners is central to its ecological success, yet the mechanisms it senses and responds to these interactions remain poorly understood. This study identifies the CarSR two-component system as a direct regulator of RadD, the primary adhesin mediating coaggregation, and reveals its role in sensing coaggregation as a signal. These findings uncover a novel mechanism by which F. nucleatum dynamically adapts to polymicrobial environments, offering new perspectives on biofilm formation and bacterial communication in complex oral microbial ecosystems.

microbiology↗

bioRxiv2024

Development of a Conditional Plasmid for Gene Deletion in Non-Model Fusobacterium nucleatum strains

Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen. IMPORTANCEFusobacterium nucleatum is implicated in various human diseases, including periodontal disease, preterm birth, and colorectal cancer, often linked to specific strains and reflecting the species genetic and phenotypic diversity. Despite this diversity, most genetic research has centered on the model strain ATCC 23726, potentially missing key aspects of other strains pathogenic potential. This study addresses a critical gap by developing a novel conditional plasmid system that enables gene deletion in genetically recalcitrant strains of F. nucleatum. We successfully deleted genes in the clinical strain CTI-2, the FNA strain 21_1A, and F. periodonticum for the first time. Our findings, particularly the varying behavior of the radD gene production in coaggregation across strains, underscore the complexity of F. nucleatum and the need for broader genetic studies. This work advances our understanding of F. nucleatum virulence at the strain level and provides a valuable tool for future bacterial genetics research.

microbiology↗

bioRxiv2024

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 knockin of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possible 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 nucleatums 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, while 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 knockin 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.

microbiology↗

bioRxiv2023

Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum

Gene inactivation via creating in-frame deletion mutations in Fusobacterium nucleatum is time-consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPRi system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX, and radD, pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing coaggregation. The system was then extended to probe essential genes bamA and ftsZ, vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ-targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains. IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPRi system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and sgRNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered resistant strains like CTI-2 and Fusobacterium periodontium. With CRISPRis potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.

microbiology↗