Rhizobacteria Impact Colonization of Listeria monocytogenes on Arabidopsis thaliana Roots

In spite of its relevance as a foodborne pathogen, we have limited knowledge about Listeria monocytogenes in the environment. L. monocytogenes outbreaks have been linked to fruits and vegetables; thus, a better understanding of the factors influencing its ability to colonize plants is important. We tested how environmental factors and other soil- and plant-associated bacteria influenced L. monocytogenes\u27 ability to colonize plant roots using Arabidopsis thaliana seedlings in a hydroponic growth system. We determined that the successful root colonization of L. monocytogenes 10403S was modestly but significantly enhanced by the bacterium being pregrown at higher temperatures, and this effect was independent of the biofilm and virulence regulator PrfA. We tested 14 rhizosphere-derived bacteria for their impact on L. monocytogenes 10403S, identifying one that enhanced and 10 that inhibited the association of 10403S with plant roots. We also characterized the outcomes of these interactions under both coinoculation and invasion conditions. We characterized the physical requirements of five of these rhizobacteria to impact the association of L. monocytogenes 10403S with roots, visualizing one of these interactions by microscopy. Furthermore, we determined that two rhizobacteria (one an inhibitor, the other an enhancer of 10403S root association) were able to similarly impact 10 different L. monocytogenes strains, indicating that the effects of these rhizobacteria on L. monocytogenes are not strain specific. Taken together, our results advance our understanding of the parameters that affect L. monocytogenes plant root colonization, knowledge that may enable us to deter its association with and, thus, downstream contamination of, food crops. IMPORTANCE Listeria monocytogenes is ubiquitous in the environment, being found in or on soil, water, plants, and wildlife. However, little is known about the requirements for L. monocytogenes\u27 existence in these settings. Recent L. monocytogenes outbreaks have been associated with contaminated produce; thus, we used a plant colonization model to investigate factors that alter L. monocytogenes\u27 ability to colonize plant roots. We show that L. monocytogenes colonization of roots was enhanced when grown at higher temperatures prior to inoculation but did not require a known regulator of virulence and biofilm formation. Additionally, we identified several rhizobacteria that altered the ability of 11 different strains of L. monocytogenes to colonize plant roots. Understanding the factors that impact L. monocytogenes physiology and growth will be crucial for finding mechanisms (whether chemical or microbial) that enable its removal from plant surfaces to reduce L. monocytogenes contamination of produce and eliminate foodborne illness

Natural variation in a short region of the Acidovorax citrulli type III‐secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.United States-Israel Binational Agriculture Research and Development (BARD) Fund S-5023-17

Phenotypic variation in the plant pathogenic bacterium Acidovorax citrulli.

Acidovorax citrulli causes bacterial fruit blotch (BFB) of cucurbits, a disease that threatens the cucurbit industry worldwide. Despite the economic importance of BFB, little is known about pathogenicity and fitness strategies of the bacterium. We have observed the phenomenon of phenotypic variation in A. citrulli. Here we report the characterization of phenotypic variants (PVs) of two strains, M6 and 7a1, isolated from melon and watermelon, respectively. Phenotypic variation was observed following growth in rich medium, as well as upon isolation of bacteria from inoculated plants or exposure to several stresses, including heat, salt and acidic conditions. When grown on nutrient agar, all PV colonies possessed a translucent appearance, in contrast to parental strain colonies that were opaque. After 72 h, PV colonies were bigger than parental colonies, and had a fuzzy appearance relative to parental strain colonies that are relatively smooth. A. citrulli colonies are generally surrounded by haloes detectable by the naked eye. These haloes are formed by type IV pilus (T4P)-mediated twitching motility that occurs at the edge of the colony. No twitching haloes could be detected around colonies of both M6 and 7a1 PVs, and microscopy observations confirmed that indeed the PVs did not perform twitching motility. In agreement with these results, transmission electron microscopy revealed that M6 and 7a1 PVs do not produce T4P under tested conditions. PVs also differed from their parental strain in swimming motility and biofilm formation, and interestingly, all assessed variants were less virulent than their corresponding parental strains in seed transmission assays. Slight alterations could be detected in some DNA fingerprinting profiles of 7a1 variants relative to the parental strain, while no differences at all could be seen among M6 variants and parental strain, suggesting that, at least in the latter, phenotypic variation is mediated by slight genetic and/or epigenetic alterations

PVs of <i>A. citrulli</i> lack the ability to produce type IV pili (T4P) in NA medium.

<p><b>A</b>. Binocular observation of 72-h colonies of parental strains M6, variants M6V1 and M6V2 and two T4P (<i>pilM</i>^-) mutants of strain M6, M6-2-23 and M6-2g-84. <b>B</b>. Transmission electron microscopy of parental strains M6 and 7a1, and PVs of both strains, following growth for 72 h on NA plates. Solid and dashed arrows indicate T4P and polar flagella, respectively. No T4P could be detected in PVs of all variants and no polar flagella could be detected in PVs of strain 7a1. Bars, 200 ηm.</p

Phenotypic variation in <i>Acidovorax citrulli</i>.

<p><b>A</b>. A phenotypic variant (PV) colony of strain M6 after 72 h of growth in NA. PV colonies of strain 7a1 have a similar appearance when observed at the naked eye. <b>B</b>. Binocular observation of 72-h colonies (grown on NA) of parental strains M6 and 7a1 and two variants of each strain, M6V1 and M6V2, and 7a1V1 and 7a1V2 (the same magnification was used for all colonies). <b>C</b>. Phenotypic variation during growth of an <i>A. citrulli</i> M6 colony.</p

Biofilm formation ability of strains M6 and 7a1 and their corresponding variants.

<p>Cultures were grown in XVM2 medium for 48 hours in polystyrene ELISA plates at 28 ^°C. Formed biofilms at the medium/air interface were stained with 0.01% crystal violet stain. Stained biofilms were resuspended with ethanol and quantitatively estimated by optical density measurements at 595 nm. Top, middle and bottom panels show the results of three independent experiments (first, second, third; with five replicates per strain in the first experiment, and ten replicates per strain in second and third experiments). The box-and-whisker plots indicate minimum, first quartile, median, third quartile, and maximum values. Different letters indicate statistically significant differences (p < 0.05; for details see Appendix S1) among strains in each experiment.</p

Estimation of colony forming units (CFU) per colonies of strains M6 and 7a1 and their corresponding variants, after 72 h of growth on NA.

<p>CFU/colony was determined following serial dilution plating of five colonies per strain. Top, middle, and bottom panels show the results of three independent experiments (first, second, third) for each strain and corresponding variants (5 replicates per strain per experiment). Each dot indicates the CFU of a single colony. The box-and-whisker plots indicate minimum, first quartile, median, third quartile, and maximum values. Different letters indicate statistically significant differences (p < 0.05; for details see Appendix S1) among strains in each experiment.</p

Phenotypic variants M6V1 and M6V2 possess increased swimming motility than the parental strain.

<p>Swimming assays were performed on soft NA plates (0.3% agar). Swimming areas were measured after 24 hours of incubation at 28°C. Each triangle represents a replicate from two independent experiments (first and second experiment replicates, represented by black and white triangles, respectively). The box-and-whisker plots indicate minimum, first quartile, median, third quartile, and maximum values, summarized from the two experiments. Different letters indicate statistically significant differences (p < 0.001; for details see Appendix S1) among strains.</p

Ice nucleation in a Gram-positive bacterium isolated from precipitation depends on a polyketide synthase and non-ribosomal peptide synthetase

International audienceEarth’s radiation budget and frequency and intensity of precipitation are influenced by aerosols with ice nucleation activity (INA), i.e., particles that catalyze the formation of ice. Some bacteria, fungi, and pollen are among the most efficient ice nucleators but the molecular basis of INA is poorly understood in most of them. Lysinibacillus parviboronicapiens ( Lp ) was previously identified as the first Gram-positive bacterium with INA. INA of Lp is associated with a secreted, nanometer-sized, non-proteinaceous macromolecule or particle. Here a combination of comparative genomics, transcriptomics, and a mutant screen showed that INA in Lp depends on a type I iterative polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS). Differential filtration in combination with gradient ultracentrifugation revealed that the product of the PKS-NRPS is associated with secreted particles of a density typical of extracellular vesicles and electron microscopy showed that these particles consist in “pearl chain”-like structures not resembling any other known bacterial structures. These findings expand our knowledge of biological INA, may be a model for INA in other organisms for which the molecular basis of INA is unknown, and present another step towards unraveling the role of microbes in atmospheric processes