Characterization of Edwardsiella tarda rpoN: roles in σ70 family regulation, growth, stress adaption and virulence toward fish

Abstract

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN (+), the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria-Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ(70) class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Comparative analysis of sigma factors RpoS, FliA, and RpoN in Edwardsiella tarda

“…Moreover, the motility of ∆fliA was not enhanced after prolonged growth for 48 h (data not shown). This was also observed for ∆rpoN in our previous research (Wang et al 2012). In contrast, the rpoS mutant showed normal motility, which was highly similar to the wild-type strain (Fig.…”

“…Therefore, FliA is a potential virulence factor due to its essential roles in flagellar filament structure and biofilm formation. RpoN was originally found in some enteric bacteria, such as Escherichia, but is D r a f t 4 RpoN plays an important role in the control of virulence genes and is essential for resistance to H 2 O 2 , starvation, high osmotic pressure, and acid (Wang et al 2012). In this study, we constructed rpoS and fliA deletion mutants and complemented strains.…”

Zebrafish as a model for zoonotic aquatic pathogens

“…However, a strain with a deletion of both catalase genes showed a 100-fold increase in LD 50 (Xiao et al, 2012). Similarly, Wang et al (2012) showed that deletion of the rpoN gene, which encodes an alternative sigma factor implicated in multiple species as a transcriptional regulator of virulence genes, had an 8-fold increase in LD 50 compared to infection with the wild type strain (Wang et al, 2012b). The Δ rpoN strain also had reduced ability to survive peroxide, acid, osmotic, and starvation stresses and produced less biofilm and chondroitinase, all of which could be implicated in virulence (Wang et al, 2012b).…”

σN‐dependent control of acid resistance and the locus of enterocyte effacement in enterohemorrhagic Escherichia coli is activated by acetyl phosphate in a manner requiring flagellar regulator FlhDC and the σS antagonist FliZ

“…; Wang et al. ). For most pathogens, the mechanism underlying σ N ‐dependent regulation of pathogenesis remains unknown; two exceptions to this include Borrelia burgdorferi , and to a lesser extent, enterohemorrhagic Escherichia coli (EHEC).…”

“…Alternative sigma factor N (r N ) when bound to RNA polymerase directs the transcription of genes for carbon and nitrogen metabolism, stress fitness, and regulation (Reitzer and Schneider 2001). In an increasing number of bacterial pathogens, r N also regulates genes for virulence and transmission, and is required for complete in vivo disease progression (Okada et al 2008;Barchiesi et al 2009;Albert-Weissenberger et al 2010;Damron et al 2012;Iyer and Hancock 2012;Mills et al 2012;Sheng et al 2012;Wang et al 2012). For most pathogens, the mechanism underlying r N -dependent regulation of pathogenesis remains unknown; two exceptions to this include Borrelia burgdorferi, and to a lesser extent, enterohemorrhagic Escherichia coli (EHEC).…”