RESUMEN
Indole is well known as an interspecies signalling molecule to modulate bacterial physiology; however, it is not clear how the indole signal is perceived and responded to by plant growth promoting rhizobacteria (PGPR) in the rhizosphere. Here, we demonstrated that indole enhanced the antibiotic tolerance of Pseudomonas fluorescens 2P24, a PGPR well known for its biocontrol capacity. Proteomic analysis revealed that indole influenced the expression of multiple genes including the emhABC operon encoding a major multidrug efflux pump. The expression of emhABC was regulated by a TetR-family transcription factor EmhR, which was demonstrated to be an indole-responsive regulator. Molecular dynamics simulation showed that indole allosterically affected the distance between the two DNA-recognizing helices within the EmhR dimer, leading to diminished EmhR-DNA interaction. It was further revealed the EmhR ortholog in Pseudomonas syringae was also responsible for indole-induced antibiotic tolerance, suggesting this EmhR-dependent, indole-induced antibiotic tolerance is likely to be conserved among Pseudomonas species. Taken together, our results elucidated the molecular mechanism of indole-induced antibiotic tolerance in Pseudomonas species and had important implications on how rhizobacteria sense and respond to indole in the rhizosphere.
Asunto(s)
Pseudomonas fluorescens , Antibacterianos/farmacología , Indoles , Proteómica , Pseudomonas , Pseudomonas fluorescens/genéticaRESUMEN
Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the -35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation.IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Floroglucinol/análogos & derivados , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Vías Biosintéticas , Hidrolasas/genética , Hidrolasas/metabolismo , Floroglucinol/metabolismo , Pseudomonas fluorescens/enzimología , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Clostridium sordellii , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridium sordellii/química , Clostridium sordellii/metabolismo , Exotoxinas/metabolismo , Metaloproteasas/metabolismoRESUMEN
Clostridium novyi alpha-toxin (Tcnα) is a potent exotoxin that induces severe symptoms including gas gangrene, myositis, necrotic hepatitis, and sepsis. Tcnα binds to sulfated glycosaminoglycans (sGAG) for cell-surface attachment and utilizes low-density lipoprotein receptor (LDLR) for rapid entry. However, it was also shown that Tcnα may use alternative entry receptors other than LDLR. Here, we define that LRP1 and Megalin can also facilitate the cellular entry of Tcnα by employing reconstitutive LDLR family proteins. LDLR, LRP1, and Megalin recognize Tcnα via their ligand-binding domains (also known as LDL receptor type A repeats). Notably, LDLR and LRP1 have contrasting expression levels in many different cells, thus the dominant entry receptor for Tcnα could be cell-type dependent. These findings together increase our knowledge of the Tcnα actions and further help to understand the pathogenesis of C. novyi infection-associated diseases.
Asunto(s)
Toxinas Bacterianas , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Toxinas Bacterianas/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
Multidrug resistance (MDR) efflux pumps are involved in bacterial intrinsic resistance to multiple antimicrobials. Expression of MDR efflux pumps can be either constitutive or transiently induced by various environmental signals, which are typically perceived by bacterial two-component systems (TCSs) and relayed to the bacterial nucleoid, where gene expression is modulated for niche adaptation. Here, we demonstrate that RstA/RstB, a TCS previously shown to control acid-induced and biofilm-related genes in Escherichia coli, confers resistance to multiple antibiotics in Pseudomonas fluorescens by directly regulating the MDR efflux pumps EmhABC and MexCD-OprJ. Moreover, we show that phosphorylation of the conserved Asp52 residue in RstA greatly enhances RstA-DNA interaction, and regulation of the multidrug resistance by RstA/RstB is dependent on the phosphorylation of the RstA Asp52 residue by RstB. Proteome analysis reveals RstA/RstB also positively regulates the efflux pump MexEF-OprN and enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. Our results suggest that, by coupling the expression of multiple efflux pumps and anaerobic nitrate respiration, RstA/RstB could play a role in defense against nitrosative stress caused by anaerobic nitrate respiration. IMPORTANCE Microenvironmental hypoxia typically increases bacterial multidrug resistance by elevating expression of multidrug efflux pumps, but the precise mechanism is currently not well understood. Here, we showed that the two-component system RstA/RstB not only positively regulated expression of several efflux pumps involved in multidrug resistance, but also promoted expression of enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. These results suggested that, by upregulating expression of efflux pumps and pyoverdine biosynthesis-related enzymes, RstA/RstB could play a role in promoting bacterial tolerance to hypoxia by providing protection against nitrosative stress.