Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca²⁺- and Mg²⁺-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.

Fusarium pseudograminearum (Fpg) is a significant pathogen responsible for fusarium crown rot (FCR) in wheat (Triticum aestivum L.), a disease with devastating impacts on crop yield. The utilization of biocontrol bacteria to combat fungal diseases in plants is a cost-effective, eco-friendly, and sustainable strategy. In this trial, an endophytic bacterial species, designated as SW, was isolated from the roots of wheat. The strain exhibited potent antagonistic effects against Fpg and reduced the FCR disease severity index by 76.07 ± 0.33% in a greenhouse pot trial. Here, 10⁶ colony-forming units (CFUs)/mL of the SW strain was determined to be the minimum dose required to exhibit the antagonism against Fpg. The strain was identified as Bacillus atrophaeus using genome sequencing and comparison with type strains in the NCBI database. Whole-genome sequencing analysis revealed that SW harbors genes for siderophores, antifungal metabolites, and antibiotics, which are key contributors to its antagonistic activity. Additionally, the strain’s ability to utilize various carbon and nitrogen sources, successfully colonize wheat root tissues as an endophyte, and form biofilms are critical attributes for promoting plant growth. In summary, these findings demonstrate the ability of Bacillus atrophaeus to control FCR disease in wheat in a sustainable agricultural setting.

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa , respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis . Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis . We further identified YPK_0952 as a DNase dependent on Mg ²⁺ , Ni ²⁺ , Mn ²⁺ , and Co ²⁺ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis . IMPORTANCE: Bacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis . YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli ) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.

Fusarium pseudograminearum , a soil-borne fungus, is the cause of the devastating wheat disease known as wheat crown rot (WCR). The persistence of this pathogen in the soil and crop residues contributes to the increased occurrence and severity of WCR. Therefore, developing effective strategies to prevent and manage WCR is of great importance. In this study, we isolated a bacterial strain, designated as SR9, from the stem of wheat, that exhibited potent antagonistic effects against F. pseudograminearum , as well as the biocontrol efficacy of SR9 on WCR was quantified at 83.99% ± 0.11%. We identified SR9 as Pseudomonas khavaziana and demonstrated its potential as a plant probiotic. SR9 displayed broad-spectrum antagonism against other fungal pathogens, including Neurospora dictyophora , Botrytis californica , and Botryosphaeria dothidea . Whole-genome sequencing analysis revealed that SR9 harbored genes encoding various cell wall-degrading enzymes, cellulases, and lipases, along with antifungal metabolites, which are responsible for its antagonistic activity. Gene knockout and quantitative PCR analyses reveal that phenazine is the essential factor for antagonism. SR9 possessed genes related to auxin synthesis, flagellar biosynthesis, biofilm adhesion, and the chemotaxis system, which play pivotal roles in plant colonization and growth promotion; we also evaluated the effects of SR9 on plant growth in wheat and Arabidopsis . Our findings strongly suggest that SR9 holds great promise as a biocontrol agent for WCR in sustainable agriculture. IMPORTANCE: The escalating prevalence of wheat crown rot, primarily attributed to Fusarium pseudograminearum , necessitates the development of cost-effective and eco-friendly biocontrol strategies. While plant endophytes are recognized for their biocontrol potential, reports on effective strains targeting wheat crown rot are sparse. This study introduces the Pseudomonas khavaziana SR9 strain as an efficacious antagonist to the wheat crown rot pathogen Fusarium pseudograminearum . Demonstrating a significant reduction in wheat crown rot incidence and notable plant growth promotion, SR9 emerges as a key contributor to plant health and agricultural sustainability. Our study outlines a biological approach to tackle wheat crown rot, establishing a groundwork for innovative sustainable agricultural practices.

Synthetic biology seeks to build predictable, programmable biological systems. We developed a blue-light-inducible T7RNAP system with dual-input regulation to enable precise spatiotemporal gene control, which is vital for biomanufacturing, therapy, and microbial engineering. We optimized it by replacing RBS sequences, testing tandem T7 promoters, and evaluating split-T7RNAP variants. Expression and bactericidal efficacy were assessed via fluorescent output and real-time growth curves under blue light. RBS variants caused up to 50-fold differences in expression. Three tandem T7 promoters provided the best balance between yield and fidelity. Integration of a benzoate-responsive module enabled 4.5-fold repression at 3 mM benzoate, demonstrating effective chemical off-switching without compromising light induction. This system combines blue light precision with environmental responsiveness, offering non-invasive, on-demand activation for antimicrobial therapy or spatial bioproduction. The benzoate-triggered off-switch is especially valuable for ecological applications such as biocontainment or bioremediation, where gene expression must shut down upon detection of pollutants, for example, aromatic hydrocarbons. Its orthogonal, modular design supports context-dependent control, making it ideal for environmental biosensors, programmable probiotics, and smart antimicrobial delivery in complex ecosystems.