Type IV secretion system effector sabotages multiple defense systems in a competing bacterium.

Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system (T4SS) from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.

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.

Soil bacterium manipulates antifungal weapons by sensing intracellular type IVA secretion system effectors of a competitor.

Soil beneficial bacteria can effectively inhibit bacterial pathogens by assembling contact-dependent killing weapons, such as the type IVA secretion system (T4ASS). It's not clear whether these antibacterial weapons are involved in biotrophic microbial interactions in soil. Here we showed that an antifungal antibiotic 2,4-DAPG production of the soil bacterium, Pseudomonas protegens can be triggered by another soil bacterium, Lysobacter enzymogenes, via T4ASS by co-culturing on agar plates to mimic cell-to-cell contact. We demonstrated that the induced 2,4-DAPG production of P. protegens is achieved by intracellular detection of the T4ASS effector protein Le1519 translocated from L. enzymogenes. We defined Le1519 as LtaE (Lysobacter T4E triggering antifungal effects), which specifically stimulates the expression of 2,4-DAPG biosynthesis genes in P. protegens, thereby protecting soybean seedlings from infection by the fungus Rhizoctonia solani. We further found that LtaE directly bound to PhlF, a pathway-specific transcriptional repressor of the 2,4-DAPG biosynthesis, then activated the 2,4-DAPG production. Our results highlight a novel pattern of microbial interspecies and interkingdom interactions, providing a unique case for expanding the diversity of soil microbial interactions.

Assembly of an active microbial consortium by engineering compatible combinations containing foreign and native biocontrol bacteria of kiwifruit.

Assembling functional bacterial biocontrol consortia is expected to expand the scope and efficiency of biocontrol agents. Generally, bacterial interspecies interactions lead to incompatibility events, as bacteria can produce antibacterial compounds and/or assemble contact-dependent killing (CDK) devices. Here, we aimed to assemble a bacterial consortium comprising Lysobacter enzymogenes OH11 and Bacillus safensis ZK-1 for the synergistic control of bacterial and fungal diseases of kiwifruit. ZK-1, a native kiwifruit biocontrol bacterium, is effective against Pseudomonas syringae pv. actinidiae (Psa) that causes bacterial kiwifruit canker, but has weak antifungal activity. OH11 is a foreign kiwifruit biocontrol agent with strong antifungal activity. While OH11 was unable to produce anti-Gram-negative metabolites, this strain could utilize type IV secretion system as an antibacterial CDK weapon. We first observed that OH11 could inhibit growth of ZK-1 by generating diffusible anti-Gram-positive antibiotic WAP-8294A2, whereas ZK-1 failed to generate diffusible antibacterial compound to inhibit growth of OH11. To disrupt this interspecies incompatibility, we generated a transgenic OH11-derived strain, OH11W, by deleting the WAP-8294A2 biosynthetic gene and found that OH11W did not kill ZK-1. We further observed that when OH11W and ZK-1 were co-inoculated on agar plates, no CDK effect was observed between them, whereas co-culture of OH11W or ZK-1 with Psa on agar plates resulted in Psa killing, suggesting L. enzymogenes and B. safensis assemble antibacterial CDK weapons against bacterial pathogens, and these CDK weapons did not affect the compatibility between OH11W and ZK-1. Based on these findings, we assembled an OH11W/ZK-1 dependent consortium that was shown to be functional in controlling bacterial canker and several representative fungal diseases of kiwifruit.

The transcriptional regulators of virulence for Pseudomonas aeruginosa: Therapeutic opportunity and preventive potential of its clinical infections.

In Pseudomonas aeruginosa (P. aeruginosa), transcription factors (TFs) are important mediators in the genetic regulation of adaptability and pathogenicity to respond to multiple environmental stresses and host defences. The P. aeruginosa genome harbours 371 putative TFs; of these, about 70 have been shown to regulate virulence-associated phenotypes by binding to the promoters of their target genes. Over the past three decades, several techniques have been applied to identify TF binding sites on the P. aeruginosa genome, and an atlas of TF binding patterns has been mapped. The virulence-associated regulons of TFs show complex crosstalk in P. aeruginosa's regulatory network. In this review, we summarise the recent literature on TF regulatory networks involved in the quorum-sensing system, biofilm formation, pyocyanin synthesis, motility, the type III secretion system, the type VI secretion system, and oxidative stress responses. We discuss future perspectives that could provide insights and targets for preventing clinical infections caused by P. aeruginosa based on the global regulatory network of transcriptional regulators.

Quorum quenching by a type IVA secretion system effector.

Proteobacteria primarily utilize acyl-homoserine lactones (AHLs) as quorum-sensing signals for intra-/interspecies communication to control pathogen infections. Enzymatic degradation of AHL represents the major quorum-quenching mechanism that has been developed as a promising approach to prevent bacterial infections. Here we identified a novel quorum-quenching mechanism revealed by an effector of the type IVA secretion system (T4ASS) in bacterial interspecies competition. We found that the soil antifungal bacterium Lysobacter enzymogenes OH11 (OH11) could use T4ASS to deliver the effector protein Le1288 into the cytoplasm of another soil microbiome bacterium Pseudomonas fluorescens 2P24 (2P24). Le1288 did not degrade AHL, whereas its delivery to strain 2P24 significantly impaired AHL production through binding to the AHL synthase PcoI. Therefore, we defined Le1288 as LqqE1 (Lysobacter quorum-quenching effector 1). Formation of the LqqE1-PcoI complex enabled LqqE1 to block the ability of PcoI to recognize/bind S-adenosy-L-methionine, a substrate required for AHL synthesis. This LqqE1-triggered interspecies quorum-quenching in bacteria seemed to be of key ecological significance, as it conferred strain OH11 a better competitive advantage in killing strain 2P24 via cell-to-cell contact. This novel quorum-quenching also appeared to be adopted by other T4ASS-production bacteria. Our findings suggest a novel quorum-quenching that occurred naturally in bacterial interspecies interactions within the soil microbiome by effector translocation. Finally, we presented two case studies showing the application potential of LqqE1 to block AHL signaling in the human pathogen Pseudomonas aeruginosa and the plant pathogen Ralstonia solanacearum.

A Monoclonal Antibody-Based Immunochromatographic Test Strip and Its Application in the Rapid Detection of Cucumber Green Mottle Mosaic Virus.

Two specific monoclonal antibodies (mAbs) were screened, and an immunochromatographic strip (ICS) test for rapid and specific detection of cucumber green mottle mosaic virus (CGMMV) was developed. The coat protein of CGMMV was heterologously expressed as an immunogen, and specific capture mAb 2C9 and the detection mAb 4D4 were screened by an uncompetitive immunoassay. The test and control lines on the nitrocellulose membrane were coated with the purified 2C9 and a goat anti-mouse IgG, respectively, and a nanogold probe combined with 4D4 was applied to the conjugate pad. Using these mAbs, a rapid and sensitive ICS was developed. Within the sandwich mode of 2C9-CGMMV-4D4, the test line showed a corresponding positive relationship with CGMMV in infected samples. The ICS test had a detection limit of 1:5000 (w/v) for CGMMV in samples and was specific for CGMMV, with no observed cross-reaction with TMV or CMV.

Lysobacter enzymogenes prevents Phytophthora infection by inhibiting pathogen growth and eliciting plant immune responses.

The Phytophthora pathogen causes enormous damage to important agricultural plants. This group of filamentous pathogens is phylogenetically distant from fungi, making them difficult to control by most chemical fungicides. Lysobacter enzymogenes OH11 (OH11) is a biocontrol bacterium that secretes HSAF (Heat-Stable Antifungal Factor) as a broad-spectrum antifungal weapon. Here, we showed that OH11 could also control a variety of plant Phytophthora diseases caused by three major oomycetes (P. sojae, P. capsici and P. infestans). We provided abundant evidence to prove that OH11 protected host plants from Phytophthora pathogen infection by inhibiting mycelial growth, digesting cysts, suppressing cyst germination, and eliciting plant immune responses. Interestingly, the former two processes required the presence of HSAF, while the latter two did not. This suggested that L. enzymogenes could prevent Phytophthora infection via multiple previously unknown mechanisms. Therefore, this study showed that L. enzymogenes could serve as a promising alternative resource for promoting plant resistance to multiple Phytophthora pathogens.

Twitching Motility Assays of Lysobacter enzymogenes OH11 Under a Light Microscope.

Bacterial twitching motility is a peculiar way of adherence and surface translocation on moist solid or semisolid surfaces. Although the twitching motility has been detected in various flagellated bacteria, such as Pseudomonas aeruginosa, it has been rarely detected in flagella-less bacteria like Lysobacter enzymogenes, a natural predator of filamentous fungi. Here, by using a strain OH11 of L. enzymogenes as a model system, we describe a convenient method for observing the twitching motility, with fewer steps and better repetition than conventional methods. This new method provides important technical support for the motile study of Lysobacter.

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.

A Novel and Efficient Platform for Discovering Noncanonical Quorum-Quenching Proteins.

Quorum sensing (QS) is a well-known chemical signaling system responsible for intercellular communication that is widespread in bacteria. Acyl-homoserine lactone (AHL) is the most-studied QS signal. Previously, bacterially encoded AHL-degrading enzymes were considered to be canonical quorum-quenching proteins that have been widely used to control pathogenic infections. Here, we report a novel platform that enabled the efficient discovery of noncanonical AHL quorum-quenching proteins. This platform initially asked bacteriologists to carry out comparative genomic analyses between phylogenetically related AHL-producing and non-AHL-producing members to identify genes that are conservatively shared by non-AHL-producing members but absent in AHL-producing species. These candidate genes were then introduced into recombinant AHL-producing E. coli to screen for target proteins with the ability to block AHL production. Via this platform, we found that non-AHL-producing Lysobacter containing numerous environmentally ubiquitous members encoded a conserved glycosyltransferase-like protein Le4759, which was experimentally shown to be a noncanonical AHL-quenching protein. Le4759 could not directly degrade exogenous AHL but rather recognized and altered the activities of multiple AHL synthases through protein-protein interactions. This versatile capability enabled Le4759 to block specific AHL synthase such as CarI from Pectobacterium carotovorum to reduce its protein abundance to suppress AHL synthesis, thereby impairing bacterial infection. Thus, this study provided bacteriologists with a unique platform to discover noncanonical quorum-quenching proteins that could be developed as promising next-generation drug candidates to overcome emerging bacterial antibiotic resistance. IMPORTANCE Targeting and blocking bacterial quorum sensing (QS), the process known as quorum quenching (QQ) is an effective mean to control bacterial infection and overcome the emerging antibiotic resistance. Previously, diverse QS signal-degradation enzymes are identified as canonical QQ proteins. Here, we provided a novel and universal platform that enabled to discover previously unidentified noncanonical QQ proteins that were unable to degrade acyl-homoserine lactone (AHL) but could block AHL generation by recognizing multiple AHL synthases via direct protein-protein interactions. Our findings are believed to trigger broad interest for bacteriologists to identify potentially widely distributed noncanonical QQ proteins that have great potential for developing next-generation anti-infectious drugs.

Identification of atypical T4SS effector proteins mediating bacterial defense.

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in Escherichia coli to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri. We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L. enzymogenes and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter.

The ubiquitous Wsp (wrinkly spreader phenotype) chemosensory system and DSF (diffusible signal factor) quorum sensing are two important chemically associated signaling systems that mediate bacterial communications between the host and environment. Although these two systems individually control biofilm formation in pathogenic bacteria via the ubiquitous second messenger c-di-GMP, their crosstalk mechanisms remain elusive. Here we present a scenario from the plant-beneficial and antifungal bacterium Lysobacter enzymogenes OH11, where biofilm formation favors the colonization of this bacterium in fungal hyphae. We found that the Wsp system regulated biofilm formation via WspR-mediated c-di-GMP signaling, whereas DSF system did not depend on the enzymatic activity of RpfG to regulate biofilm formation. We further found that WspR, a diguanylate cyclase (DGC) responsible for c-di-GMP synthesis, could directly bind to one of the DSF signaling components, RpfG, an active phosphodiesterase (PDE) responsible for c-di-GMP degradation. Thus, the WspR-RpfG complex represents a previously undiscovered molecular linker connecting the Wsp and DSF systems. Mechanistically, RpfG could function as an adaptor protein to bind and inhibit the DGC activity of unphosphorylated WspR independent of its PDE activity. Phosphorylation of WspR impaired its binding affinity to RpfG and also blocked the ability of RpfG to act as an adaptor protein, which enabled the Wsp system to regulate biofilm formation in a c-di-GMP-dependent manner by dynamically integrating the DSF system. Our findings demonstrated a previously uncharacterized mechanism of crosstalk between Wsp and DSF systems in plant-beneficial and antifungal bacteria.

The antifungal activity and mechanism of silver nanoparticles against four pathogens causing kiwifruit post-harvest rot.

Post-harvest rot causes enormous economic loss to the global kiwifruit industry. Currently, there are no effective fungicides to combat the disease. It is unclear whether silver nanoparticles (AgNPs) are effective in controlling post-harvest rot and, if so, what the underlying antifungal mechanism is. Our results indicated that 75 ppm AgNPs effectively inhibited the mycelial growth and spore germination of four kiwifruit rot pathogens: Alternaria alternata, Pestalotiopsis microspora, Diaporthe actinidiae, and Botryosphaeria dothidea. Additionally, AgNPs increased the permeability of mycelium's cell membrane, indicating the leakage of intracellular substance. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that AgNPs induced pathogen hypha shrinkage and distortion, as well as vacuolation in hypha cells, implying that AgNPs caused cellular and organelle structural degradation. The transcriptome sequencing of mycelium treated with AgNPs (24 h / 48 h) was performed on the Illumina Hiseq 4000 sequencing (RNA-Seq) platform. For the time points of 24 h and 48 h, AgNPs treatment resulted in 1,178 and 1,461 differentially expressed genes (DEGs) of A. alternata, 517 and 91 DEGs of P. microspora, 1,287 and 65 DEGs of D. actinidiae, 239 and 55 DEGs of B. dothidea, respectively. The DEGs were found to be involved in "catalytic activity," "small molecule binding," "metal ion binding," "transporter activity," "cellular component organization," "protein metabolic process," "carbohydrate metabolic process," and "establishment of localization." Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also revealed that "carbohydrate metabolism," "amino acid metabolism," "energy metabolism," and "xenobiotics biodegradation and metabolism" of "metabolism processes" were the most highly enriched pathways for these DEGs in four pathogens, with "cellular processes" being particularly enriched for B. dothidea. Furthermore, quantitative polymerase chain reactions (qPCRs) were used to validate the RNA-seq results. It was also confirmed that AgNPs could significantly reduce the symptoms of kiwifruit rot without leaving any Ag+ residue on the peel and flesh of kiwifruit. Our findings contributed to a better understanding of the antifungal effect and molecular mechanisms of AgNPs against pathogens causing kiwifruit post-harvest rot, as well as a new perspective on the application of this novel antifungal alternative to fruit disease control.

Molecular basis of the key regulator WRINKLED1 in plant oil biosynthesis.

Vegetable oils are not only major components of human diet but also vital for industrial applications. WRINKLED1 (WRI1) is a pivotal transcription factor governing plant oil biosynthesis, but the underlying DNA-binding mechanism remains incompletely understood. Here, we resolved the structure of Arabidopsis WRI1 (AtWRI1) with its cognate double-stranded DNA (dsDNA), revealing two antiparallel ß sheets in the tandem AP2 domains that intercalate into the adjacent major grooves of dsDNA to determine the sequence recognition specificity. We showed that AtWRI1 represented a previously unidentified structural fold and DNA-binding mode. Mutations of the key residues interacting with DNA element affected its binding affinity and oil biosynthesis when these variants were transiently expressed in tobacco leaves. Seed oil content was enhanced in stable transgenic wri1-1 expressing an AtWRI1 variant (W74R). Together, our findings offer a structural basis explaining WRI1 recognition and binding of DNA and suggest an alternative strategy to increase oil yield in crops through WRI1 bioengineering.

Diguanylate Cyclase and Phosphodiesterase Interact To Maintain the Specificity of Cyclic di-GMP Signaling in the Regulation of Antibiotic Synthesis in Lysobacter enzymogenes.

Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. A large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs), and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components employ various methods to avoid undesired cross talk to maintain signaling specificity. The synthesis of the antibiotic HSAF (heat-stable antifungal factor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE, LchP, and a c-di-GMP effector, Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, that participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP display local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. IMPORTANCE Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases [DGCs]), c-di-GMP-degrading enzymes (phosphodiesterases [PDEs]), and c-di-GMP effectors. These components deploy various methods to avoid undesired cross talk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes. We provide direct evidence to show that the DGC and PDE form a complex and also indirect evidence to argue that they may balance a local c-di-GMP pool to control antibiotic production. These results represent an important finding regarding the mechanism of a DGC and PDE pair to control the expression of specific c-di-GMP signaling pathways.

Bacterial quorum sensing quenching activity of Lysobacter leucyl aminopeptidase acts by interacting with autoinducer synthase.

Acyl-homoserine lactone (AHL) is the most studied autoinducer in gram-negative bacteria controlling infections of various pathogens. Quenching of AHL signaling by inhibiting AHL synthesis or AHL-receptor binding via small molecular chemicals or enzymatically degrading AHL is commonly used to block bacterial infections. Here, we describe a new quorum-quenching strategy that directly "acquires" bacterial genes/proteins through a defined platform. We artificially expressed a typical AHL synthase gene pcoI from the biocontrol Pseudomonas fluorescens 2P24 in the antifungal bacterium Lysobacter enzymogenes OH11 lacking AHL production. This step led to the discovery of multiple PcoI interacting protein candidates from L. enzymogenes. The individual expression of these candidate genes in 2P24 led to the identification of Le0959, which encodes leucyl aminopeptidase, an effective protein that inhibits AHL synthesis in 2P24. Therefore, we define Le0959 as LqqP (Lysobacterquorum-quenching protein). The expression of pcoI in E. coli could produce AHL, and the introduction of lqqP into E. coli expressing pcoI could prevent the production of AHL. LqqP directly binds to PcoI, and this protein-protein binding reduced the abundance of free PcoI (capable of AHL synthesis) in vivo, thereby blocking PcoI-dependent AHL production. Overall, this study highlights the discovery of LqqP in quenching AHL quorum sensing by binding to AHL synthase via developing a previously-uncharacterized screening technique for bacterial quorum quenching.

The PdeK-PdeR two-component system promotes unipolar localization of FimX and pilus extension in Xanthomonas oryzae pv. oryzicola.

Bacterial type IV pili (T4P) contribute to virulence and can be rapidly extended and retracted to mediate twitching motility. T4P biogenesis, which is normally limited to the cell poles, is regulated by extracellular stimuli and internal signals such as cyclic di-GMP (c-di-GMP). The c-di-GMP­binding protein FimX interacts with the T4P assembly complex and, when intracellular c-di-GMP concentrations are low, assumes a unipolar localization and promotes T4P biogenesis. Here, we demonstrated that FimX formed a complex with the two-component system consisting of the histidine kinase PdeK and its downstream response regulator PdeR. This complex promoted T4P assembly in the phytopathogen Xanthomonas oryzae pv. oryzicola and virulence in rice. PdeK and the c-di-GMP phosphodiesterase activity of PdeR were required for the unipolar localization of FimX, leading to T4P extension. High amounts of c-di-GMP reduced the affinity of FimX for PdeR in vitro, consistent with FimX promoting T4P extension only under conditions of low c-di-GMP. We propose that low intracellular amounts of c-di-GMP created by PdeR facilitate the recruitment of FimX to the leading pole of motile cells. Our findings indicate that the PdeK-PdeR two-component system connects environmental cues to second messenger turnover, resulting in a change in the intracellular concentration of c-di-GMP that promotes T4P biogenesis and virulence.

Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system.

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell-cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.