In a recent study published in Cell, researchers elucidated mechanisms by which a bacterial organism and plant pathogen, Pseudomonas syringae, kills amoeba predators. The process involves a chemical sensing (radar) system, wherein the bacterium releases a compound (syringafactin) that the predator modifies.
The modified compound then binds to a chemical radar regulator (CraR), causing the secretion of an amoebicidal agent (pyrofactin). The system enhances the ability of the plant pathogen to infect plants. The findings highlight the evolutionary pressure exerted by predation on bacteria.
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Introduction
Predation is a significant evolutionary force in ecosystems that affects the macroscopic and microbial worlds. In plants, predation by amoebas enhances nutrient recycling and controls plant pathogens.
P. syringae are devastating, Gram-negative pathogens that target economically relevant plants and are arduous to control. Such pathogenic bacteria constantly encounter predators.
Consequentially, many bacteria possess powerful biosynthetic machinery that enables the production of compounds used as chemical armaments against predators.
These compounds are crucial for their survival in competitive environments. Analyzing bacterial evasion strategies can improve the understanding of microbial community interactions and inform agricultural strategy-makers.
About the Study
In the present study, researchers explore interactions between the plant pathogenic bacterium Pseudomonas syringae and the amoeba Polysphondylium pallidum, focusing on mechanisms used by the bacterium to kill its predator.
The team collected soil samples from the Thuringian Forest, focusing on areas rich in organic matter. They isolated amoebae from the samples to obtain corresponding cell cultures. High-performance liquid chromatography (HPLC) enabled comparison of the profiles of the culture extracts.
The researchers also isolated bacteria to obtain genomic deoxyribonucleic acid DNA (gDNA) for whole-genome sequencing. Genetic analyses identified genes related to pyrofactin production in the P. syringae SZ47 strain. Reverse-transcription-polymerase chain reactions (RT-PCR) amplified specific genes involved in the chemical system of predator killing.
The team conducted resazurin assays to assess the viability of amoebae. In addition, they calculated half-maximal inhibitory concentration (IC50) values based on fluorescence intensity to ensure reliability.
The researchers generated gene deletion mutants in the two phylogenetically identical strains of Pseudomonas syringae, SZ47 and SZ57. Plaque assays revealed the edibility of these strains, assessing the survival of bacterial strains after predation by amoeba.
The researchers expressed Cra proteins in Escherichia coli to investigate the role of Cra in predator killing. They conducted feeding experiments in vitro and in vivo to assess the interactions between bacterial strains and amoebae.
Further analyses included nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), pathogenicity analyses, and molecular docking. NMR revealed structural differences between various compounds.
In molecular docking experiments, the team docked the structures into the CraR protein to study the interactions with ligands. MALDI-MSI imaging revealed the molecular exchange between predator and prey. Pathogenicity assays evaluated the infectivity of P. syringae strains on Arabidopsis thaliana.
Results
The study reveals that P. syringae utilizes a chemical radar system to detect and kill its amoebal predators. This system includes P. syringae generating a compound named syringafactin, which serves as a signal to sense the presence of predators.
On contact with P. syringae, the amoeba modifies the syringafactin by deacylating it. This modification is crucial since the deacylated form of syringafactin can bind to the chemical radar regulator.
Amoebal predators convert the diffusible syringafactins into lipophilic octapeptides and heptapeptides by diacylation. The binding of deacylated syringafactin to the chemical radar regulatory activates the transcription of genes responsible for synthesizing pyrofactins, compounds that can kill the amoebae.
The findings highlight a defense mechanism that allows the bacteria to respond effectively to predation.
The chemical regulator enables Pseudomonas syringae to infect the Arabidopsis thaliana plant even in amoebal presence. Therefore, the chemical radar not only aids in predator detection but also plays a role in the bacterium's ability to thrive in plant environments.
The two closely related strains of P. syringae studied possess different anti-predator traits. SZ47 secretes natural products that kill P. pallidum, unlike SZ57. Thus, SZ57 is edible to P. pallidum, while SZ47 is not, regardless of culture conditions.
However, both strains can infect and proliferate in Arabidopsis thaliana leaves. Both strains produce syringafactins, but SZ47 produces pyrofactins when co-cultured. Pyrofactins A and C from SZ47 exhibit potent amoebicidal activity, while syringafactins do not. Mutant strains lacking syringafactins are edible to predators and lose their ability to swarm.
The study shows that Pseudomonas syringae uses a chemical sensing system to destroy amoebal predators. This system involves syringafactin production by the bacterium, syringafactin diacylation by the amoeba, and amoeba killing upon pyrofactin release by the bacterium after deacylated syringafactin binding with CraR.
This mechanism is reminiscent of a radar system that emits electromagnetic radiation (syringafactin) and detects the reflected waves (deacyl syringafactin) by a foreign object (predatory amoeba).
Future studies could explore the chemical-sensing mechanism across different predators and investigate alternate sensing mechanisms that could reveal biological molecules with promising potential to expand the toolkit for managing pests and diseases.
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