Search

Every microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant-pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome.

Bacterial pathogens in theRalstonia solanacearumspecies complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments like biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem.IMPORTANCEPlant pathogenicRalstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels.Ralstoniacells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. Virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant pathogenicRalstoniacan use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenicRalstoniaspecies. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct micro-niches in the same habitat.

Adhesins (adhesive proteins) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogenRalstonia solanacearum(Rs) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putativeRsadhesin genes were upregulated in a ΔphcAquorum sensing mutant that cannot respond to high cell densities:radA(Ralstoniaadhesin),rcpA(Ralstoniacollagen-likeprotein), andrcpB. Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages whenRsexperiences a low cell density. During root colonizationRsupregulatedrcpAandrcpB, but notradA, relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with ΔrcpA/Band ΔradArevealed that all three adhesins helpRsattach to tomato seedling roots. Biofilm assays on abiotic surfaces found thatRsdoes not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However,Rsdid not require the adhesins for later disease stagesin planta, including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitnessin planta. Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease.ImportanceEvery microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant pathogenic bacteriumRalstonia solanacearumcan stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of several afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three individual plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to bacterial success. Combined with tissue-specific regulatory data, this work indicates thatR. solanacearumdeploys distinct adhesins that help it succeed at different stages of plant pathogenesis.Research AreasPlant Microbiology, Host-Microbial Interactions, Microbial Pathogenesis

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development., Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by certain bacteria and fungi that establish symbiotic relationships with plants. Here, the authors show that LCOs are produced also by many other, non-symbiotic fungi, and regulate fungal growth and development.

There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an anti-activator that tightly controls that activity of the nitrogen fixation specific transcriptional activator, NifA, in response to redox, nitrogen, and carbon status. While several studies reported mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonia, knowledge about the specific determinants for this ammonia-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonia accumulated in liquid culture (~ 12 mM). The ammonia excretion phenotype is solely associated with deletions of NifL domains combined with the insertion of a promoter sequence in the opposite orientation to nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonia excreted by affecting rnf1 gene expression as an additional requirement for ammonia excretion. These ammonia-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines the discreet determinants that bring about A. vinelandii ammonia excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

The pectinolytic genus Dickeya (formerly Erwinia chrysanthemi) comprises numerous pathogenic species which cause diseases in various crops and ornamental plants across the globe. Their pathogenicity is governed by complex multi-factorial processes of adaptive virulence gene regulation. Extracellular polysaccharides and lipopolysaccharides present on bacterial envelope surface play a significant role in the virulence of phytopathogenic bacteria. However, very little is known about the genomic location, diversity, and organization of the polysaccharide and lipopolysaccharide biosynthetic gene clusters in Dickeya. In the present study, we report the diversity and structural organization of the group 4 capsule (G4C)/O-antigen capsule, putative O-antigen lipopolysaccharide, enterobacterial common antigen, and core lipopolysaccharide biosynthesis clusters from 54 Dickeya strains. The presence of these clusters suggests that Dickeya has both capsule and lipopolysaccharide carrying O-antigen to their external surface. These gene clusters are key regulatory components in the composition and structure of the outer surface of Dickeya. The O-antigen capsule/group 4 capsule (G4C) coding region shows a variation in gene content and organization. Based on nucleotide sequence homology in these Dickeya strains, two distinct groups, G4C group I and G4C group II, exist. However, comparatively less variation is observed in the putative O-antigen lipopolysaccharide cluster in Dickeya spp. except for in Dickeya zeae. Also, enterobacterial common antigen and core lipopolysaccharide biosynthesis clusters are present mostly as conserved genomic regions. The variation in the O-antigen capsule and putative O-antigen lipopolysaccharide coding region in relation to their phylogeny suggests a role of multiple horizontal gene transfer (HGT) events. These multiple HGT processes might have been manifested into the current heterogeneity of O-antigen capsules and O-antigen lipopolysaccharides in Dickeya strains during its evolution.

Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

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.

Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence.

The plant wilt pathogen Ralstonia solanacearum thrives in the water-transporting xylem vessels of its host plants. Xylem is a relatively nutrient-poor, high-flow environment but R. solanacearum succeeds there by tuning its own metabolism and altering xylem sap biochemistry. Flow influences many traits that the bacterium requires for pathogenesis. Most notably, a quorum sensing system mediates the pathogen's major transition from a rapidly dividing early phase that voraciously consumes diverse food sources and avidly adheres to plant surfaces to a slower-growing late phase that can use fewer nutrients but produces virulence factors and disperses effectively. This review discusses recent findings about R. solanacearum pathogenesis in the context of its flowing in planta niche, with emphasis on R. solanacearum metabolism in plants.

Virulence assays are powerful tools to study microbial pathogenesis in vivo. Good assays track disease development and, coupled with targeted mutagenesis, can identify pathogen virulence factors. Disease development in plants is extremely sensitive to environmental factors such as temperature, atmospheric humidity, and soil water level, so it can be challenging to standardize conditions to achieve consistent results. Here, we present optimized and validated experimental conditions and analysis methods for nine assays that measure specific aspects of virulence in the phytopathogenic bacterium Ralstonia solanacearum, using tomato as the model host plant.

Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression.

Summary The type III secretion system (T3SS) is a major virulence factor in many Gram-negative bacterial pathogens and represents a particularly appealing target for antimicrobial agents. Previous studies have shown that the plant phenolic compound p-coumaric acid (PCA) plays a role in the inhibition of T3SS expression of the phytopathogen Dickeya dadantii 3937. This study screened a series of derivatives of plant phenolic compounds and identified that trans-4-hydroxycinnamohydroxamic acid (TS103) has an eight-fold higher inhibitory potency than PCA on the T3SS of D. dadantii. The effect of TS103 on regulatory components of the T3SS was further elucidated. Our results suggest that TS103 inhibits HrpY phosphorylation and leads to reduced levels of hrpS and hrpL transcripts. In addition, through a reduction in the RNA levels of the regulatory small RNA RsmB, TS103 also inhibits hrpL at the post-transcriptional level via the rsmB-RsmA regulatory pathway. Finally, TS103 inhibits hrpL transcription and mRNA stability, which leads to reduced expression of HrpL regulon genes, such as hrpA and hrpN. To our knowledge, this is the first inhibitor to affect the T3SS through both the transcriptional and post-transcriptional pathways in the soft-rot phytopathogen D. dadantii 3937.

Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence.

Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans -2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmB Ea -RsmA Ea system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora . In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.

Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa , an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS , which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.