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 (ΔiscR) or AP002 (iscR 3CA), 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 (Δfur-ΔiscR) but not in AP005 (Δfur-iscR 3CA), 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.
Highlights
Klebsiella pneumoniae is a rod-shaped, gram-negative bacterium that causes community-acquired diseases including pneumonia, bacteremia, septicemia, and urinary and respiratory tract infections that occurr in immune-compromised patients [1]
Cluster-dependent manner To study whether IscR regulates K. pneumoniae capsular polysaccharide (CPS) biosynthesis, we determined the amounts of K2 CPS in CG43S3 and AP001 (DiscR) strains
Tightly controlling CPS biosynthesis is critical for successful infection by K. pneumoniae [10,11,25]
Summary
Klebsiella pneumoniae is a rod-shaped, gram-negative bacterium that causes community-acquired diseases including pneumonia, bacteremia, septicemia, and urinary and respiratory tract infections that occurr in immune-compromised patients [1]. Pyogenic liver abscess isolates often carry heavy CPS loads that protect bacteria from phagocytosis and from being killed by serum factors [5,6]. Our previous studies have demonstrated that CPS biosynthesis in K. pneumoniae is repressed in iron-replete conditions, and this regulation is controlled by an iron uptake regulator (Fur) [10]. Under iron-replete conditions, dimeric Fur in complex with Fe(II) indirectly activates K. pneumoniae CPS biosynthesis through transcriptional factors RmpA and RcsA and a small non-coding RNA, RyhB [10,11]. The transcription of cps genes is directly regulated by RmpA and RcsA but appears to be indirectly regulated by RyhB. These findings indicate that environmental iron availability influences K. pneumoniae CPS biosynthesis through multiple regulators
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