Identification of non-canonical antagonistic bacteria via interspecies contact-dependent killing.

BACKGROUND: Canonical biocontrol bacteria were considered to inhibit pathogenic bacteria mainly by secreting antibiotic metabolites or enzymes. Recent studies revealed that some biocontrol bacteria can inhibit pathogenic bacteria through contact-dependent killing (CDK) mediated by contact-dependent secretion systems. The CDK was independent of antibiotic metabolites and often ignored in normal biocontrol activity assay. RESULTS: In this study, we aimed to use a pathogen enrichment strategy to isolate non-canonical bacteria with CDK ability. Rhizosphere soil samples from Chinese cabbage showing soft rot symptom were collected and Pectobacterium carotovorum subsp. carotovorum (Pcc), the pathogen of cabbage soft rot, were added into these samples to enrich bacteria which attached on Pcc cells. By co-culture with Pcc, four bacteria strains (named as PcE1, PcE8, PcE12 and PcE13) showing antibacterial activity were isolated from Chinese cabbage rhizosphere. These four bacteria strains showed CDK abilities to different pathogenic bacteria of horticultural plants. Among them, PcE1 was identified as Chryseobacterium cucumeris. Genome sequencing showed that PcE1 genome encoded a type VI secretion system (T6SS) gene cluster. By heterologous expression, four predicted T6SS effectors of PcE1 showed antibacterial activity to Escherichia coli. CONCLUSION: Overall, this study isolated four bacteria strains with CDK activity to various horticultural plant pathogens, and revealed possible involvement of T6SS of Chryseobacterium cucumeris in antibacterial activity. These results provide valuable insight for potential application of CDK activity in biocontrol bacteria. © 2024 Society of Chemical Industry.

Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome.

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae, from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity in vitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.

Loss of Flagella-Related Genes Enables a Nonflagellated, Fungal-Predating Bacterium To Strengthen the Synthesis of an Antifungal Weapon.

Loss of flagellar genes causes a nonmotile phenotype. The genus Lysobacter consists of numerous environmentally ubiquitous, nonflagellated bacteria, including Lysobacter enzymogenes, an antifungal bacterium that is beneficial to plants. L. enzymogenes still has many flagellar genes on its genome, although this bacterium does not engage in flagella-driven motility. Here, we report that loss of certain flagellar genes allows L. enzymogenes to strengthen its evolutionarily gained capacity in fungal killing. To clarify why this bacterium loses flagellar genes during the evolutionary process, we cloned several representative flagellar genes from Xanthomonas oryzae, a flagellated, phylogenetically related species of Lysobacter, and introduced them individually into L. enzymogenes to mimic genomic reacquisition of lost flagellar genes. Heterogeneous expression of the three X. oryzae flagellar structural genes (Xo-motA, Xo-motB, Xo-fliE) and one flagellar regulatory gene (Xo-fleQ) remarkably weakened the bacterial capacity to kill fungal pathogens by impairing the synthesis of an antifungal weapon, known as the heat-stable antifungal factor (HSAF). We further investigated the underlying mechanism by selecting Xo-FleQ as the representative because it is a master transcription factor responsible for flagellar gene expression. Xo-FleQ inhibited the transcription of operon genes responsible for HSAF synthesis via direct binding of Xo-FleQ to the promoter region, thereby decreasing HSAF biosynthesis by L. enzymogenes. These observations suggest a possible genome and function coevolution event, in which an antifungal bacterium deletes certain flagellar genes in order to enhance its ability to kill fungi. IMPORTANCE It is generally recognized that flagellar genes are commonly responsible for the flagella-driven bacterial motility. Thus, finding nonflagellated bacteria partially or fully lost flagellar genes is not a surprise. However, the present study provides new insights into this common idea. We found that loss of either certain flagellar structural or regulatory genes (such as motA, motB, fliE, and fleQ) allows a nonflagellated, antifungal bacterium (L. enzymogenes) to stimulate its fungal-killing capacity, outlining a genome-function coevolution event, where an antifungal bacterium "smartly" designed its genome to "delete" crucial flagellar genes to coordinate flagellar loss and fungal predation. This unusual finding might trigger bacteriologists to reconsider previously ignored functions of the lost flagellar genes in any nonflagellated, pathogenic, or beneficial bacteria.