Dataset Information

IscR regulation of capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

ABSTRACT: IscR, an Fe-S cluster-containing transcriptional factor, regulates genes involved in various cellular processes. In response to environmental stimuli such as oxidative stress and iron levels, IscR switches between its holo and apo forms to regulate various targets. IscR binding sequences are classified into two types: the type 1 IscR box that is specific for holo-IscR binding, and the type 2 IscR box that binds holo- and apo-IscR. Studying Klebsiella pneumoniae CG43S3, we have previously shown that iron availability regulates capsular polysaccharide (CPS) biosynthesis and iron-acquisition systems. The present study investigated whether IscR is involved in this regulation. Compared with that in CG43S3, the amount of CPS was decreased in AP001 (?iscR) or AP002 (iscR3CA), a CG43S3-derived strain expressing mutated IscR mimicked apo-IscR, suggesting that only holo-IscR activates CPS biosynthesis. Furthermore, a promoter-reporter assay verified that the transcription of cps genes was reduced in AP001 and AP002. Purified IscR::His6, but not IscR3CA::His6, was also found to bind the predicted type 1 IscR box specifically in the cps promoter. Furthermore, reduced siderophore production was observed in AP004 (?fur-?iscR) but not in AP005 (?fur-iscR3CA), implying that apo-IscR activates iron acquisition. Compared with those in AP004, mRNA levels of three putative iron acquisition systems (fhu, iuc, and sit) were increased in AP005, and both purified IscR::His6 and IscR3CA::His6 bound the predicted type 2 IscR box in the fhuA, iucA, and sitA promoters, whereas IscR3CA::His6 displayed a lower affinity. Finally, we analyzed the effect of external iron levels on iscR expression. The transcription of iscR was increased under iron-depleted conditions as well as in AP001 and AP002, suggesting an auto-repression exerted by apo-IscR. Our results show that in K. pneumoniae, IscR plays a dual role in the regulation of CPS biosynthesis and iron-acquisition systems in response to environmental iron availability.

SUBMITTER: Wu CC

PROVIDER: S-EPMC4169559 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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IscR regulation of capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

Wu Chien-Chen CC Wang Chien-Kuo CK Chen Yu-Ching YC Lin Tien-Huang TH Jinn Tzyy-Rong TR Lin Ching-Ting CT

PloS one 20140919 9

IscR, an Fe-S cluster-containing transcriptional factor, regulates genes involved in various cellular processes. In response to environmental stimuli such as oxidative stress and iron levels, IscR switches between its holo and apo forms to regulate various targets. IscR binding sequences are classified into two types: the type 1 IscR box that is specific for holo-IscR binding, and the type 2 IscR box that binds holo- and apo-IscR. Studying Klebsiella pneumoniae CG43S3, we have previously shown t ...[more]

PMID: 25237815

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Project description:BackgroundThe capsular polysaccharide (CPS) and iron acquisition systems are important determinants of Klebsiella pneumoniae infections, and we have previously reported that the ferric uptake repressor (Fur) can play dual role in iron acquisition and CPS biosynthesis. In many bacteria, Fur negatively controls the transcription of the small non-coding RNA RyhB to modulate cellular functions and virulence. However, in K. pneumoniae, the role played by RyhB in the Fur regulon has not been characterised. This study investigated Fur regulation of ryhB transcription and the functional role of RyhB in K. pneumoniae.ResultsDeletion of fur from K. pneumoniae increased the transcription of ryhB; the electric mobility shift assay and the Fur-titration assay revealed that Fur could bind to the promoter region of ryhB, suggesting that Fur directly represses ryhB transcription. Additionally, in a ?fur strain with elevated CPS production, deletion of ryhB obviously reduced CPS production. The following promoter-reporter assay and quantitative real-time PCR of cps genes verified that RyhB activated orf1 and orf16 transcription to elevate CPS production. However, deletion of ryhB did not affect the mRNA levels of rcsA, rmpA, or rmpA2. These results imply that Fur represses the transcription of ryhB to mediate the biosynthesis of CPS, which is independent of RcsA, RmpA, and RmpA2. In addition, the ?fur strain's high level of serum resistance was attenuated by the deletion of ryhB, indicating that RyhB plays a positive role in protecting the bacterium from serum killing. Finally, deletion of ryhB in ?fur reduced the expression of several genes corresponding to 3 iron acquisition systems in K. pneumoniae, and resulted in reduced siderophore production.ConclusionsThe regulation and functional role of RyhB in K. pneumoniae is characterized in this study. RyhB participates in Fur regulon to modulate the bacterial CPS biosynthesis and iron acquisition systems in K. pneumoniae.

Project description:K. pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. Although elevated blood glucose levels cause various immune problems, its effects on K. pneumoniae virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for K. pneumoniae virulence, in response to exogenous glucose. We found that K. pneumoniae produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of cyaA or crp, which respectively encode adenylate cyclase and cAMP receptor protein in K. pneumoniae, markedly increased CPS production, while deletion of cpdA, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that K. pneumoniae CPS biosynthesis is controlled by the cAMP-dependent carbon catabolite repression (CCR). To investigate the underlying mechanism, quantitative real-time PCR and promoter-reporter assays were used to verify that the transcription of CPS biosynthesis genes, which are organized into 3 transcription units (orf1-2, orf3-15, and orf16-17), were activated by the deletion of crp. Sequence analysis revealed putative CRP binding sites located on P(orf3-15) and P(orf16-17), suggesting direct CRP-cAMP regulation on the promoters. These results were then confirmed by electrophoretic mobility shift assay. In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA). The deletion of rcsA in mutation of crp partially reduced CPS biosynthesis and the transcription of orf1-2 but not of orf3-15 or orf16-17. These results suggest that RcsA participates in the CRP-cAMP regulation of orf1-2 transcription and influences CPS biosynthesis. Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing. We here provide evidence that K. pneumoniae increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR.

Dataset Information

IscR regulation of capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

Publications

IscR regulation of capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

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