Dissecting the molecular dance: c-di-GMP, cAMP-CRP, and VfmH collaboration in pectate lyase regulation for Dickeya dadantii-unveiling the soft rot pathogen's strategy.

IMPORTANCE: Bacteria respond to environmental changes and adapt to host systems. The response regulator VfmH of the Vfm quorum sensing system regulates a crucial virulence factor, pectate lyase (Pel), in Dickeya dadantii. At high c-di-GMP concentrations, VfmH binds c-di-GMP, resulting in the loss of its activation property in the Pel and virulence regulation in D. dadantii. VfmH binds to c-di-GMP via three conserved arginine residues, and mutations of these residues eliminate the c-di-GMP-related phenotypes of VfmH in Pel synthesis. Our data also show that VfmH interacts with CRP to regulate pelD transcription, thus integrating cyclic AMP and c-di-GMP signaling pathways to control virulence in D. dadantii. We propose that VfmH is an important intermediate factor incorporating quorum sensing and nucleotide signaling pathways for the collective regulation of D. dadantii pathogenesis.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

Aureobasidium pullulans from the Fire Blight Biocontrol Product, Blossom Protect, Induces Host Resistance in Apple Flowers.

Fire blight, caused by Erwinia amylovora, is a devastating disease of apple. Blossom Protect, a product that contains Aureobasidium pullulans as the active ingredient, is one of the most effective biological controls of fire blight. It has been postulated that the mode of action of A. pullulans is to compete against and antagonize epiphytic growth of E. amylovora on flowers, but recent studies have found that flowers treated with Blossom Protect harbored similar to or only slightly reduced E. amylovora populations compared with nontreated flowers. In this study, we tested the hypothesis that A. pullulans-mediated biocontrol of fire blight is the result of induced host resistance. We found that PR genes in the systemic acquired resistance pathway, but not genes in the induced systemic resistance pathway, were induced in hypanthial tissue of apple flowers after the Blossom Protect treatment. Additionally, the induction of PR gene expression was coupled with an increase of plant-derived salicylic acid in this tissue. After inoculation with E. amylovora, PR gene expression was suppressed in nontreated flowers, but in flowers pretreated with Blossom Protect, the heightened PR expression offset the immune repression caused by E. amylovora, and prevented infection. Temporal and spatial analysis of PR gene induction showed that induction of PR genes occurred 2 days after the Blossom Protect treatment, and required direct flower-yeast contact. Finally, we observed deterioration of the epidermal layer of the hypanthium in some of the Blossom Protect-treated flowers, suggesting that PR gene induction in flowers may be a result of pathogenesis by A. pullulans.

Potential reservoirs of antimicrobial resistance in livestock waste and treated wastewater that can be disseminated to agricultural land.

Livestock manure, dairy lagoon effluent, and treated wastewater are known reservoirs of antibiotic resistance genes (ARGs), antibiotic-resistant bacteria (ARB), and virulence factor genes (VFGs), and their application to agricultural farmland could be a serious public health threat. However, their dissemination to agricultural lands and impact on important geochemical pathways such as the nitrogen (N) cycle have not been jointly explored. In this study, shotgun metagenomic sequencing and analyses were performed to examine the diversity and composition of microbial communities, ARGs, VFGs, and N cycling genes in different livestock manure/lagoon and treated wastewater collected from concentrated animal feeding operations (CAFOs) and a municipal wastewater treatment plant along the west coast of the United States. Multivariate analysis showed that diversity indices of bacterial taxa from the different microbiomes were not significantly different based on InvSimpson (P = 0.05), but differences in ARG mechanisms were observed between swine manure and other microbiome sources. Comparative resistome profiling showed that ARGs in microbiome samples belonged to four core resistance classes: aminoglycosides (40-55 %), tetracyclines (30-45 %), beta-lactam-resistance (20-35 %), macrolides (18-30 %), and >50 % of the VFGs that the 24 microbiomes harbored were phyletically affiliated with two bacteria, Bacteroidetes fragilis and Enterobacter aerogenes. Network analysis based on Spearman correlation showed co-occurrence patterns between several genes such as transporter-gene and regulator, efflux pump and involved-in-polymyxin- resistance, aminoglycoside, beta-lactam, and macrolide with VFGs and bacterial taxa such as Firmicutes, Candidatus Themoplasmatota, Actinobacteria, and Bacteroidetes. Metabolic reconstruction of metagenome-assembled genome (MAGs) analysis showed that the most prevalent drug resistance mechanisms were associated with carbapenem resistance, multidrug resistance (MDR), and efflux pump. Bacteroidales was the main taxa involved in dissimilatory nitrate reduction (DNRA) in dairy lagoon effluent. This study demonstrates that the dissemination of waste from these sources can increase the spread of ARGs, ARB, and VFGs into agricultural lands, negatively impacting both soil and human health.

Genetic and Environmental Investigation of a Novel Phenylamino Acetamide Inhibitor of the Pseudomonas aeruginosa Type III Secretion System.

Traditional antibiotics target essential cellular components or metabolic pathways conserved in both pathogenic and nonpathogenic bacteria. Unfortunately, long-term antibiotic use often leads to antibiotic resistance and disruption of the overall microbiota. In this work, we identified a phenylamino acetamide compound, named 187R, that strongly inhibited the expression of the type III secretion system (T3SS) encoding genes and the secretion of the T3SS effector proteins in Pseudomonas aeruginosa. T3SS is an important virulence factor, as T3SS-deficient strains of P. aeruginosa are greatly attenuated in virulence. We further showed that 187R had no effect on bacterial growth, implying a reduced selective pressure for the development of resistance. 187R-mediated repression of T3SS was dependent on ExsA, the master regulator of T3SS in P. aeruginosa. The impact of 187R on the host-associated microbial community was also tested using the Arabidopsis thaliana phyllosphere as a model. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the application of 187R had little impact on the composition and function of microbial community compared to the antibiotic streptomycin. Together, these results suggested that compounds that target virulence factors could serve as an alternative strategy for disease management caused by bacterial pathogens. IMPORTANCE New antimicrobial therapies are urgently needed, since antibiotic resistance in human pathogens has become one of the world's most urgent public health problems. Antivirulence therapy has been considered a promising alternative for the management of infectious diseases, as antivirulence compounds target only the virulence factors instead of the growth of bacteria, and they are therefore unlikely to affect commensal microorganisms. However, the impacts of antivirulence compounds on the host microbiota are not well understood. We report a potent synthetic inhibitor of the P. aeruginosa T3SS, 187R, and its effect on the host microbiota of Arabidopsis. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the impacts of the antivirulence compound on the composition and function of host microbiota were limited. These results suggest that antivirulence compounds can be a potential alternative method to antibiotics.

The phytopathogen Dickeya dadantii 3937 cpxR locus gene participates in the regulation of virulence and the global c-di-GMP network.

Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress and responds by up-regulating envelope protein factors and down-regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS-inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c-di-GMP regulators were also up-regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c-di-GMP phenotypes in biofilm formation and swimming. Increased production of cellular c-di-GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c-di-GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.

Quorum-Sensing Master Regulator VfmE Is a c-di-GMP Effector That Controls Pectate Lyase Production in the Phytopathogen Dickeya dadantii.

Dickeya dadantii is a phytopathogenic bacterium that causes diseases on a wide range of host plants. The pathogen secretes pectate lyases (Pel) through the type II secretion system (T2SS) that degrades the cell wall in host plants. The virulence of D. dadantii is controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP), and the homeostasis of c-di-GMP is maintained by a number of diguanylate cyclases and phosphodiesterases. Deletion of a phosphodiesterase ecpC repressed pelD transcription, and such repression can be suppressed by an additional deletion in vfmE. VfmE is an AraC type of transcriptional regulator in the Vfm quorum-sensing system. Our results suggest that VfmE is a c-di-GMP effector that functions as an activator of pel at low c-di-GMP concentrations and a repressor of pel at high c-di-GMP concentrations through regulation of the transcriptional activator SlyA. Multiple sequence alignment with known c-di-GMP effectors identified an RWIWR motif in VfmE that we demonstrate is required for the c-di-GMP binding. Mutation of R93D in the RxxxR motif eliminates the c-di-GMP-related phenotypes in Pel activity. Our results show that VfmE is not only a quorum-sensing regulator but also a c-di-GMP effector, suggesting that D. dadantii integrates the c-di-GMP signaling network with the Vfm quorum-sensing pathway during environmental adaptation. IMPORTANCE How bacteria integrate environmental cues from multiple sources to appropriately regulate adaptive phenotypes is a central question in microbiology. In Dickeya dadantii, the quorum-sensing regulator VfmE controls the key virulence factor pectate lyase (Pel). Here, we demonstrate that VfmE also binds to c-di-GMP, resulting in VfmE functioning as an activator of pel at low c-di-GMP concentrations and repressor of pel at high c-di-GMP concentrations. The RWIWR motif in VfmE is required for c-di-GMP binding, and mutation of the motif in the mutant R93D eliminates the c-di-GMP-related phenotypes in Pel activity. We propose that VfmE is an important mediator to integrate quorum-sensing signals with c-di-GMP to collectively regulate D. dadantii pathogenesis.

Expression of the Type III Secretion System Genes in Epiphytic Erwinia amylovora Cells on Apple Stigmas Benefits Endophytic Infection at the Hypanthium.

Erwinia amylovora causes fire blight on rosaceous plants. One of the major entry points of E. amylovora into hosts is flowers, where E. amylovora proliferates epiphytically on stigmatic and hypanthium surfaces and, subsequently, causes endophytic infection at the hypanthium. The type III secretion system (T3SS) is an important virulence factor in E. amylovora. Although the role of T3SS during endophytic infection is well characterized, its expression during epiphytic colonization and role in the subsequent infection is less understood. Here, we investigated T3SS gene expression in epiphytic E. amylovora on stigma and hypanthium of apple flowers under different relative humidities (RH). On stigma surfaces, T3SS was expressed in a high percentage of E. amylovora cells, and its expression promoted epiphytic growth. On hypanthium surfaces, however, T3SS was expressed in fewer E. amylovora cells than on the stigma, and displayed no correlation with epiphytic growth, even though T3SS expression is essential for infection. E. amylovora cells grown on stigmatic surfaces and then flushed down to the hypanthium displayed a higher level of T3SS expression than cells grown on the hypanthium surface alone. Furthermore, E. amylovora cells precultured on stigma had a higher potential to infect flowers than E. amylovora cells precultured in a T3SS-repressive medium. This suggests that T3SS induction during the stigmatic epiphytic colonization may be beneficial for subsequent infection. Finally, epiphytic expression of T3SS was influenced by RH. Higher percentage of stigmatic E. amylovora cells expressed T3SS under high RH than under low RH.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An improved, versatile and efficient modular plasmid assembly system for expression analyses of genes in Xanthomonas oryzae.

Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) infect rice, causing bacterial blight and bacterial leaf streak, respectively, which are two economically important bacterial diseases in paddy fields. The interactions of Xoo and Xoc with rice can be used as models for studying fundamental aspects of bacterial pathogenesis and host tissue specificity. However, an improved vector system for gene expression analysis is desired for Xoo and Xoc because some broad host range vectors that can replicate stably in X. oryzae pathovars are low-copy number plasmids. To overcome this limitation, we developed a modular plasmid assembly system to transfer the functional DNA modules from the entry vectors into the pHM1-derived backbone vectors on a high-copy number basis. We demonstrated the feasibility of our vector system for protein detection, and quantification of virulence gene expression under laboratory conditions and in association with host rice and nonhost tobacco cells. This system also allows execution of a mutant complementation equivalent to the single-copy chromosomal integration system and tracing of pathogens in rice leaf. Based on this assembly system, we constructed a series of protein expression and promoter-probe vectors suitable for classical double restriction enzyme cloning. These vector systems enable cloning of all genes or promoters of interest from Xoo and Xoc strains. Our modular assembly system represents a versatile and highly efficient toolkit for gene expression analysis that will accelerate studies on interactions of X. oryzae with rice.

Soil salinity, pH, and indigenous bacterial community interactively influence the survival of E. coli O157:H7 revealed by multivariate statistics.

Complexities of biotic-abiotic interactions in soils result in the lack of integrated understanding of environmental variables that restrict the survival of shiga toxin-producing E. coli O157:H7. Herein, we reanalyzed previously published data and highlighted the influence of soil abiotic factors on E. coli O157:H7 survivability and elucidated how these factors took effect indirectly through affecting indigenous bacterial community. Interaction network analysis indicated salinity and pH decreased the relative abundances of some bacterial taxa (e.g., Acidobacteria_Gp4, Acidobacteria_Gp6, and Deltaproteobacteria) which were positively correlated with the survival of E. coli O157:H7 in soils, and vice versa (e.g., Gammaproteobacteria and Flavobacteria) (P < 0.05). An array of multivariate statistical approaches including partial Mantel test, variation partition analysis (VPA), and structural equation model (SEM) further confirmed that biotic and abiotic factors interactively shaped the survival profile of E. coli O157:H7. This study revealed that some bacterial taxa were correlated with survival of E. coli O157:H7 directly, and salinity and pH could affect E. coli O157:H7 survival through changing these bacterial taxa. These findings suggest that salinity in soil might benefit the control of fecal pathogenic E. coli invasion, while soil acidification caused by anthropogenic influences could potentially increase the persistence of E. coli O157:H7 in agro-ecosystem.

Innovation and Application of the Type III Secretion System Inhibitors in Plant Pathogenic Bacteria.

Many Gram-negative pathogenic bacteria rely on a functional type III secretion system (T3SS), which injects multiple effector proteins into eukaryotic host cells, for their pathogenicity. Genetic studies conducted in different host-microbe pathosystems often revealed a sophisticated regulatory mechanism of their T3SSs, suggesting that the expression of T3SS is tightly controlled and constantly monitored by bacteria in response to the ever-changing host environment. Therefore, it is critical to understand the regulation of T3SS in pathogenic bacteria for successful disease management. This review focuses on a model plant pathogen, Dickeyadadantii, and summarizes the current knowledge of its T3SS regulation. We highlight the roles of several T3SS regulators that were recently discovered, including the transcriptional regulators: FlhDC, RpoS, and SlyA; the post-transcriptional regulators: PNPase, Hfq with its dependent sRNA ArcZ, and the RsmA/B system; and the bacterial second messenger cyclic-di-GMP (c-di-GMP). Homologs of these regulatory components have also been characterized in almost all major bacterial plant pathogens like Erwiniaamylovora, Pseudomonassyringae, Pectobacterium spp., Xanthomonas spp., and Ralstonia spp. The second half of this review shifts focus to an in-depth discussion of the innovation and development of T3SS inhibitors, small molecules that inhibit T3SSs, in the field of plant pathology. This includes T3SS inhibitors that are derived from plant phenolic compounds, plant coumarins, and salicylidene acylhydrazides. We also discuss their modes of action in bacteria and application for controlling plant diseases.

Functional relationships between aboveground and belowground spinach (Spinacia oleracea L., cv. Racoon) microbiomes impacted by salinity and drought.

Salinity is a major problem facing agriculture in arid and semiarid regions of the world. This problem may vary among seasons affecting both above- and belowground plant microbiomes. However, very few studies have been conducted to examine the influence of salinity and drought on microbiomes and on their functional relationships. The objective for the study was to examine the effects of salinity and drought on above- and belowground spinach microbiomes and evaluate seasonal changes in their bacterial community composition and diversity. Furthermore, potential consequences for community functioning were assessed based on 16S V4 rRNA gene profiles by indirectly inferring the abundance of functional genes based on results obtained with Piphillin. The experiment was repeated three times from early fall to late spring in sand tanks planted with spinach (Spinacia oleracea L., cv. Racoon) grown with saline water of different concentrations and provided at different amounts. Proteobacteria, Cyanobacteria, and Bacteroidetes accounted for 77.1% of taxa detected in the rhizosphere; Proteobacteria, Bacteroidetes, and Actinobacteria accounted for 55.1% of taxa detected in soil, while Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria accounted for 55.35% of taxa detected in the phyllosphere. Salinity significantly affected root microbiome beta-diversity according to weighted abundances (p = 0.032) but had no significant effect on the relative abundances of microbial taxa (p = 0.568). Pathways and functional genes analysis of soil, rhizosphere, and phyllosphere showed that the most abundant functional genes were mapped to membrane transport, DNA repair and recombination, signal transduction, purine metabolism, translation-related protein processing, oxidative phosphorylation, bacterial motility protein secretion, and membrane receptor proteins. Monoterpenoid biosynthesis was the most significantly enriched pathway in rhizosphere samples when compared to the soil samples. Overall, the predictive abundances indicate that, functionally, the rhizosphere bacteria had the highest gene abundances and that salinity and drought affected the above- and belowground microbiomes differently.

Tricarboxylic Acid (TCA) Cycle Enzymes and Intermediates Modulate Intracellular Cyclic di-GMP Levels and the Production of Plant Cell Wall-Degrading Enzymes in Soft Rot Pathogen Dickeya dadantii.

Dickeya dadantii is a plant-pathogenic bacterium that causes soft-rot in a wide range of plants. Although we have previously demonstrated that cyclic bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial secondary messenger, plays a central role in virulence regulation in D. dadantii, the upstream signals that modulate c-di-GMP remain enigmatic. Using a genome-wide transposon mutagenesis approach of a Δhfq mutant strain that has high c-di-GMP and reduced motility, we uncovered transposon mutants that recovered the c-di-GMP-mediated repression on swimming motility. A number of these mutants harbored transposon insertions in genes encoding tricarboxylic acid (TCA) cycle enzymes. Two of these TCA transposon mutants were studied further by generating chromosomal deletions of the fumA gene (encoding fumarase) and the sdhCDAB operon (encoding succinate dehydrogenase). Disruption of the TCA cycle in these deletion mutants resulted in reduced intracellular c-di-GMP and enhanced production of pectate lyases (Pels), a major plant cell wall-degrading enzyme (PCWDE) known to be transcriptionally repressed by c-di-GMP. Consistent with this result, addition of TCA cycle intermediates such as citrate also resulted in increased c-di-GMP levels and decreased production of Pels. Additionally, we found that a diguanylate cyclase GcpA was solely responsible for the observed citrate-mediated modulation of c-di-GMP. Finally, we demonstrated that addition of citrate induced not only an overproduction of GcpA protein but also a concomitant repression of the c-di-GMP-degrading phosphodiesterase EGcpB which, together, resulted in an increase in the intracellular concentration of c-di-GMP. In summary, our report demonstrates that bacterial respiration and respiration metabolites serve as signals for the regulation of c-di-GMP signaling.

Cell-length heterogeneity: a population-level solution to growth/virulence trade-offs in the plant pathogen Dickeya dadantii.

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10µm), the rest elongated into filamentous cells (>10µm). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed upregulated metabolic genes and increased growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential production of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.

Identification of c-di-GMP Signaling Components in Xanthomonas oryzae and Their Orthologs in Xanthomonads Involved in Regulation of Bacterial Virulence Expression.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. Xoo produces a range of virulence-related factors to facilitate its pathogenesis in rice, however, the regulatory mechanisms of Xoo virulence expression have been not fully elucidated. Recent studies have revealed that virulence factor production is regulated via cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway that is well-conserved in Xoo and other Xanthomonas species. A set of GGDEF, EAL, HD-GYP, and PilZ domain proteins with diverse signal sensory domains for c-di-GMP synthesis, hydrolysis, and binding is encoded in the Xoo genome. Bioinformatic, genetic, and biochemical analysis has identified an array of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), as well as degenerate GGDEF/EAL, PilZ domain proteins along with a transcription regulator. These signaling components have been characterized to regulate various bacterial cellular processes, such as virulence, exopolysaccharide (EPS) production, biofilm formation, motility, and adaptation at the transcriptional, post-translational, and protein-protein interaction levels. This review summarized the recent progress in understanding the importance and complexity of c-di-GMP signaling in regulating bacterial virulence expression, highlighting the identified key signal elements and orthologs found in Xanthomonads, discussing the diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs, and effectors, and further exploration of the new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in regulation of bacterial pathogenesis.

Transcriptional responses of Xanthomonas oryzae pv. oryzae to type III secretion system inhibitor ortho-coumaric acid.

BACKGROUND: We previously identified a plant-derived phenolic compound ortho-coumaric acid (OCA) as an inhibitor of type III secretion system (T3SS) of Xanthomonas oryzae pv. oryzae (Xoo), the pathogen causing bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. However, the molecular mechanisms by which OCA suppresses T3SS and the transcriptional responses to the OCA treatments in Xoo remains unclear. RESULTS: The present study conducted the RNA-seq-based transcriptomic analysis to reveal changes in gene expression in Xoo in response to 30 min, 1 h, 3 h, and 6 h of OCA treatment. Results showed that OCA significantly inhibited the expression of T3SS genes after 30 min, and the inhibition also existed after 1 h, 3 h, and 6 h. After treatment for 30 min, membrane proteins in the functional category of cellular process was the predominant group affected, indicating that Xoo was in the early stress stage. Over time, more differentially-expressed genes (DEGs) gathered in the functional category of biological process. Analysis of common DEGs at all four of time points revealed the core elements of Xoo during the response to OCA treatment. Notable, a multidrug transporter cluster that consisted of a MarR-family protein (PXO_RS13760), a multidrug RND transporter (PXO_RS13755), a multidrug transporter (PXO_RS13750), and an MFS transporter (PXO_RS13745) were significantly up-regulated at all four of the time points. Although these three transporter genes were not upregulated by OCA in the PXO_RS13760 deletion mutant, the deficiency of PXO_RS13760 in Xoo did not affect T3SS transcript, and OCA still had the ability to inhibit the expression of T3SS in the mutant, suggesting that the MarR-family protein was involved in bacterial responses to OCA, but not direct OCA inhibition of T3SS in Xoo. CONCLUSIONS: We analyzed the transcriptome of Xoo during OCA treatment at both early and late stages, which revealed the landscape of Xoo responses to OCA at the whole-genome transcription level. A multidrug transporter cluster was identified to be involved in the response process, but had no direct relation to T3SS in Xoo.

A feed-forward signalling circuit controls bacterial virulence through linking cyclic di-GMP and two mechanistically distinct sRNAs, ArcZ and RsmB.

Dickeya dadantii is a plant pathogen that causes soft rot disease on vegetable and potato crops. To successfully cause infection, this pathogen needs to coordinately modulate the expression of genes encoding several virulence determinants, including plant cell wall degrading enzymes (PCWDEs), type III secretion system (T3SS) and flagellar motility. Here, we uncover a novel feed-forward signalling circuit for controlling virulence. Global RNA chaperone Hfq interacts with an Hfq-dependent sRNA ArcZ and represses the translation of pecT, encoding a LysR-type transcriptional regulator. We demonstrate that the ability of ArcZ to be processed to a 50 nt 3'- end fragment is essential for its regulation of pecT. PecT down-regulates PCWDE and the T3SS by repressing the expression of a global post-transcriptional regulator- (RsmA-) associated sRNA encoding gene rsmB. In addition, we show that the protein levels of two cyclic di-GMP (c-di-GMP) diguanylate cyclases (DGCs), GcpA and GcpL, are repressed by Hfq. Further studies show that both DGCs are essential for the Hfq-mediated post-transcriptional regulation on RsmB. Overall, our report provides new insights into the interplays between ubiquitous signalling transduction systems that were most studied independently and sheds light on multitiered regulatory mechanisms for a precise disease regulation in bacteria.

The phosphotransferase system gene ptsH plays an important role in MnSOD production, biofilm formation, swarming motility, and root colonization in Bacillus cereus 905.

The rhizosphere bacterium Bacillus cereus 905 is capable of promoting plant growth through effective colonization on plant roots. The sodA2-encoding manganese-containing superoxide dismutase (MnSOD2) is important for survival of B. cereus 905 in the wheat rhizosphere. However, the genes involved in regulating sodA2 expression and the mechanisms of rhizosphere colonization of B. cereus 905 are not well elucidated. In this study, we found that the deletion of the ptsH gene, which encodes the histidine-phosphorylatable protein (HPr), a component of the phosphotransferase system (PTS), causes a decrease of about 60% in the MnSOD2 expression. Evidences indicate that the ptsH dramatically influences resistance to oxidative stress, glucose uptake, as well as biofilm formation and swarming motility of B. cereus 905. Root colonization assay demonstrated that ΔptsH is defective in colonizing wheat roots, while complementation of the sodA2 gene could partially restore the ability in utilization of arabinose, a non-PTS sugar, and root colonization caused by the loss of the ptsH gene. In toto, based on the current findings, we propose that PtsH contributes to root colonization of B. cereus 905 through multiple indistinct mechanisms, involving PTS and uptake of PTS-sugars, up-regulation of MnSOD2 production, and promotion of biofilm formation and swarming motility.

Xanthomonas oryzae pv. oryzae Response Regulator TriP Regulates Virulence and Exopolysaccharide Production Via Interacting With c-di-GMP Phosphodiesterase PdeR.

PdeR, a response regulator of the two-component system (TCS) with the cognate histidine kinase PdeK, has been shown to be an active phosphodiesterase (PDE) for intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) turnover and positively regulates the virulence of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice. To further reveal the key components and pathways involved in the PdeR-mediated c-di-GMP regulation of virulence, 16 PdeR-interacting proteins were identified, using the yeast two-hybrid (Y2H) assay. Among them, PXO_04421 (named as TriP, a putative transcriptional regulator interacting with PdeR) was verified via Y2H and glutathione-S-transferase pull-down assays, and its regulatory functions in bacterial virulence and exopolysaccharide (EPS) production were assessed by biochemical and genetic analysis. The REC domain of TriP specifically interacted with the EAL domain of PdeR. TriP promoted the PDE activity of PdeR to degrade c-di-GMP in the presence of PdeK. In-frame deletion in triP abolished the polar localization of PdeR in the cell. Notably, the ∆triP mutant showed significantly reduced virulence on susceptible rice leaves and impaired EPS production compared with wild type, whereas the double mutant ∆triP∆pdeR, like ∆pdeR, caused shorter lesion lengths and produced less EPS than ∆triP. In addition, cross-complementation showed in trans expression of pdeR in ∆triP restored its EPS production to near wild-type levels but not vice versa. Taken together, our results suggest that TriP is a novel regulator that is epistatic to PdeR in positively regulating virulence expression in X. oryzae pv. oryzae.

Phosphodiesterase EdpX1 Promotes Xanthomonas oryzae pv. oryzae Virulence, Exopolysaccharide Production, and Biofilm Formation.

In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, there are over 20 genes encoding GGDEF, EAL, and HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on the characterization of an EAL domain protein, EdpX1. Deletion of the edpX1 gene resulted in a 2-fold increase in the intracellular c-di-GMP levels, which were restored to the wild-type levels in the complemented ΔedpX1(pB-edpX1) strain, demonstrating that EdpX1 is an active phosphodiesterase (PDE) in X. oryzae pv. oryzae. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that the E153A mutation at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that the ΔedpX1 mutant was severely impaired in causing disease symptoms. In trans expression of wild-type edpX1, but not edpX1E153A, was able to complement the weakened virulence phenotype. These results indicated that an active EAL domain is required for EdpX1 to regulate the virulence of X. oryzae pv. oryzae. We then demonstrated that the ΔedpX1 mutant was defective in secreting exopolysaccharide (EPS) and forming biofilms. The expression of edpX1 in the ΔedpX1 mutant, but not edpX1E153A, restored the defective phenotypes to near-wild-type levels. In addition, we observed that EdpX1-green fluorescent protein (EdpX1-GFP) exhibited multiple subcellular localization foci, and this pattern was dependent on its transmembrane (TM) region, which did not seem to directly contribute to the regulatory function of EdpX1. Thus, we concluded that EdpX1 exhibits PDE activity to control c-di-GMP levels, and its EAL domain is necessary and sufficient for its regulation of virulence in X. oryzae pv. oryzae.IMPORTANCE Bacteria utilize c-di-GMP as a second messenger to regulate various biological functions. The synthesis and degradation of c-di-GMP are catalyzed by GGDEF domains and an EAL or HD-GYP domain, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome of X. oryzae pv. oryzae PXO99A, 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling in X. oryzae pv. oryzae, the enzymatic activities and regulatory functions of each GGDEF, EAL, and HD-GYP domain protein need to be elucidated. In this study, we showed that the EAL domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.