Functional characterization of a putative DNA methyltransferase, EadM, in Xanthomonas axonopodis pv. glycines by proteomic and phenotypic analyses

Sang-Wook Han,Jungkwan Lee,Hye-Jee Park,Boknam Jung

doi:10.1038/s41598-019-38650-3

Sang-Wook Han, Jungkwan Lee + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-38650-3

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Journal: Scientific Reports	Publication Date: Feb 21, 2019
Citations: 18	License type: open-access

Affiliation: Chung-Ang University, Dong-A University

Abstract

Xanthomonas axonopodis pv. glycines (Xag) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag. An EadM-overexpressing strain, Xag(EadM), was less virulent than the wild-type carrying an empty vector, Xag(EV). Interestingly, the viable cell numbers of Xag(EadM) were much lower (10-fold) than those of Xag(EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag(EadM). Additionally, Xag(EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag(EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag(EadM). As in Xag, Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

Highlights

Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative bacterium causing bacterial pustule disease on soybean, which is one of the most serious diseases and that reduces the yield and quality of the crop[1]
We checked the expression of eadM gene in Xag(EV) and Xag(EadM) using quantitative PCR (Supplementary Fig. 2)
It is generally known that DNA methyltransferases influence the cell growth rate by affecting replication mechanisms in bacteria

Summary

Introduction

Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative bacterium causing bacterial pustule disease on soybean, which is one of the most serious diseases and that reduces the yield and quality of the crop[1]. Glycines (Xag) is a Gram-negative bacterium causing bacterial pustule disease on soybean, which is one of the most serious diseases and that reduces the yield and quality of the crop[1]. Xag mutants that cannot synthesize diffusible signal factors showed reduced virulence on soybean leaves[10]. The roles of DNA methyltransferases involved in virulence or other mechanisms have not been reported in Xag. DNA methyltransferase is an enzyme which transfers methyl groups from S-adenosyl-L-methionine to specific nucleotides. Bacterial DNA modified by methyltransferases are involved in virulence and diverse cellular mechanisms in animal-associated bacteria. In Streptococcus mutans which causes tooth decay, DNA methylation regulates the expression of mutacin production and virulence genes[17]. The functions of DNA methyltransferases are poorly understood in plant pathogenic bacteria

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