Phenotypic variation in the plant pathogenic bacterium Acidovorax citrulli.

Ram Kumar Shrestha,Einat Zelinger,Johannes Sikorski,Daria Makarovsky,Tally Rosenberg,June Kopelowitz,Saul Burdman,Noam Eckshtain-Levi,Dawn Arnold

doi:10.1371/journal.pone.0073189

Abstract

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.

Highlights

Bacteria have developed mechanisms that allow them to adapt and maintain cell viability in rapidly changing environments
The percentage of appearance of phenotypic variant (PV)-type colonies relative to the total number of colonies varied for the same treatment among multiple experiments, ranging from non-detected to 1.5%
Despite accumulated knowledge about phenotypic variation in environmental and animal pathogenic bacteria, still there is a major gap in our understanding whether this phenomenon has a major impact on bacterial fitness [10]

Summary

Introduction

Bacteria have developed mechanisms that allow them to adapt and maintain cell viability in rapidly changing environments These mechanisms, that are associated with genetic or epigenetic alterations and phenotypic variation, include so-called adaptive mutations and phase variation [1,2]. Phase variation has been defined as a random event that occurs at high frequency (much higher than spontaneous mutations), involves changes in the DNA and leads to a phenotypically heterogeneous population [9,10]. It has been studied extensively in animal pathogenic bacteria in relation to extracellular polysaccharide (EPS) and lipopolysaccharide (LPS) composition, surface proteins, toxins, flagella, colony morphology and more [11]

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