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 (ΔiscR) 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 (Δfur-ΔiscR) but not in AP005 (Δfur-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.

Figure 1. Holo-IscR positively regulates the biosynthesis of CPS.

(A) CPS levels of the WT, AP001, and AP002 strains grown in LB broth. (B) CPS levels in WT carrying pACYC184 and AP001 carrying pACYC184, pIscR, or pIscR_3CA were determined in LB. Bacterial glucuronic acid content was determined after 16 h of growth. (C) β-Galactosidase activities of K. pneumoniae AP006 and isogenic strains (AP007 and AP008) carrying the reporter plasmid pOrf12 (P_orf1-2::lacZ), pOrf315 (P_orf3-15::lacZ), or pOrf1617 (P_orf16-17::lacZ) were determined using log-phase cultures grown in LB medium. Error bars indicate standard deviations. *P<0.01 compared with the indicated groups.

Figure 2. IscR binds directly to P_galF.

(A) Diagrammatic representation of the galF loci. The primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the DNA amplified length. DNA probes are listed on the left. The box in grey indicates the predicted type 1 IscR box. The dashed box indicates the DNA sequence alignment among the predicted type 1 IscR box, the IscR binding motif, and the putative IscR binding sequence in P_galF, and the numbers denote the positions relative to the translational start site. Deletion of the predicted IscR box on PgalF-1* is indicated by a caret. (B) EMSA of IscR recombinant proteins and various DNA fragments of the upstream regions of galF. Different concentrations of purified IscR::His₆ or IscR_3CA::His₆ were incubated with 5 ng of DNA fragments, as indicated in the margin. After incubation at room temperature for 30 min, the mixtures were resolved on a 5% non-denaturing polyacrylamide gel. The gel was stained with SYBR Green I dye and photographed.

Figure 3. Deletion effect of iscR on K. pneumoniae susceptibility to normal human serum.

The susceptibility to normal human serum of each bacterial mutant (A) and the complement strains (B) indicated in the margin was determined. Bacterial serum resistance was determined using log-phase cultures grown in LB medium. *P<0.01 compared with the indicated groups.

Figure 4. Deletion of iscR decreases K. pneumoniae Δfur siderophore production assessed using CAS assay.

All assayed bacterial mutants (A) and the complement strains (B) are indicated. The halos around the colonies correspond to the iron-chelating activity of siderophores in bacteria were measured after 24 h of incubation at 37°C. The assay was independently repeated at least five times, and the differences among strains are consistent.

Figure 5. IscR_3CA::His₆ binds directly to P_fhuA, P_iucA, and P_sitA.

(A) DNA sequence alignment between the E. coli type 2 IscR box and the putative IscR binding sequence in the upstream regions of fhuA, iucA, and sitA. Positions identical to the consensus sequences are bolded. Diagrammatic representation of the fhuA (B), iucA (C), and sitA (D) loci. The large arrows represent the open reading frames. The primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the DNA amplified length. The predicted IscR boxes is deleted and indicated by a caret. The grey boxes indicate the predicted type 2 IscR box. Different concentrations of purified IscR_3CA::His₆ were incubated with 5 ng of various DNA fragments of the upstream regions of indicated genes. Following incubation at room temperature for 30 min, the mixtures were analysed on a 5% non-denaturing polyacrylamide gel. The gel was stained with SYBR Green I dye and photographed.

Figure 6. Regulation of K. pneumoniae iscR expression.

(A) β-Galactosidase activity of K. pneumoniae AP006 (ΔlacZ) carrying the reporter plasmid piscRZ15 (P_iscR::lacZ) were determined using log-phase cultures grown in the indicated concentrations of Dip. (B) The β-galactosidase activity of piscRZ15 was determined in the AP006 and isogenic strains, AP009 (ΔlacZ-Δfur), AP010 (ΔlacZ-Δfur-ΔryhB), AP007 (ΔlacZ-ΔiscR), and AP008 (ΔlacZ-iscR _3CA), using log-phase cultures grown in LB medium. (C) The β-galactosidase activity of piscRZ15 was determined in the AP006 and isogenic strains, AP007, AP008, and AP011 (ΔlacZ-Δfur-ΔiscR), using log-phase cultures grown in the indicated media. Error bars indicate standard deviations. *P<0.01 compared with the indicated group.

Figure 7. A proposed model for IscR regulation on the CPS biosynthesis and iron-acquisition genes in K. pneumoniae.

(A) Under iron-replete conditions, and when the cellular supply of Fe-S clusters is sufficient, the holo-IscR exerts an auto-repression as well as activates the K2 cps genes and the iron-acquisition genes; however, Fur in complex with Fe(II) exerts a stronger repression on these genes (red lines). (B) Under iron-deplete conditions, the apo-IscR accumulates and preferably activates the transcription of the iron-acquisition genes but not the cps genes. Upon the de-repression of Fur, both the transcription of the iron-acquisition and cps genes are increased.