RyhB is involved in the Fur-regulated capsular polysaccharide biosynthesis and biofilm formation in Klebsiella pneumoniae CG43
Yi-Ming Hong, Chia Chi, Jing-Ciao Lin, Chien-Chen Wu, Ching-Ting Lin*
School of Chinese Medicine, China Medical University, Taichung, Taiwan. Republic of China
Backgrounds:
RyhB is a small non-coding RNA that controls gene expression at post-transcriptional level. In Escherichia coli, the ferric uptake repressor (Fur) represses the transcription of ryhB to mediate its downstream gene expression. However, biological role of RyhB in Klebsiella pneumoniae remains largely unknown. We have previously showed that Fur regulated the capsular polysaccharide (CPS) biosynthesis and iron acquisition in K. pneumoniae CG43. Besides, bioinformatic analysis revealed putative Fur and RcsAB binding sequences in the promoter region of ryhB. In this study, function and regulation of ryhB in K. pneumoniae CG43 was characterized.
Methods:
The effect of fur- or rcsB-deletion in K. pneumoniae CG43 on the promoter activity of ryhB was assessed by LacZ reporter assay. The direct binding of Fur or RcsB to PryhB
was detected by Fur titration assay or electrophoretic mobility shift assay. Deletion of ryhB in K. pneumoniae CG43 wild type, fur, and rcsB strains were respectively constructed, and then the mutants were subjected to various phenotypic analyses.
RyhB regulation on the expression of its target genes were analyzed by quantitative real-time PCR and two-dimensional SDS-PAGE.
Results:
Deletion of fur or rcsB in K. pneumoniae activated the promoter activity of ryhB.
Direct binding of Fur or RcsB to PryhB could also be observed. Bacterial iron acquisition, CPS biosynthesis, biofilm forming activity, and resistance to acid or oxidative stress were affected by the deletion of fur. However, the increased CPS biosynthesis and decreased biofilm forming activity in the fur strain were partially restored by the ryhB-deletion. Transcription of the biosynthesis and regulatory genes of CPS were analyzed infur and furryhB strains to clarify the regulatory role of ryhB on the CPS biosynthesis. Furthermore, comparative proteomic analysis between the fur and furryhB strains was performed to identify the RyhB-regulated genes in the Fur regulon.
Conclusion:
Both Fur and RcsB were showed to directly repress the transcription of ryhB. Besides iron acquisition and CPS biosynthesis, Fur also affected the bacterial biofilm forming activity and resistance to acid or oxidative stress in K. pneumoniae. RyhB was found to involve in the Fur-regulated CPS biosynthesis and biofilm formation.
