OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9

Abstract

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem−1 and weaker phem−2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the −35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem−1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5′-untranslated region (5′-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.

Urease Expression in Pathogenic Yersinia enterocolitica Strains of Bio-Serotypes 2/O:9 and 1B/O:8 Is Differentially Regulated by the OmpR Regulator

“…The in vitro interaction between phosphorylated OmpR (OmpR-P) and the promoters of selected genes was examined using EMSAs, as described previously (Jaworska et al, 2018), with some modifications. The primers listed in Supplementary Table S2 were used in PCRs with Y. enterocolitica Ye9N genomic DNA to amplify DNA fragments comprising the regulatory regions of the operons ureABC (−305 to + 7 relative to the A of the ATG start codon of ureA), ureEF (−352 to + 33 relative to the A of the ATG start codon of ureE), ureGD (−272 to + 33 relative to the A of the ATG start codon of ureG), and gene ureR-like (−310 to + 52 relative to the A of the ATG start codon of ureR-like).…”

Mechanisms Of Functioning And Control Of Heme Uptake In Gram-Negative Pathogenic Bacteria

“…Po przedostaniu się do cytoplazmy, hem może być degradowany w wyniku aktywności białka HemS, bądź przechowywany w komórce patogenu. [14]. Wszystkie cząsteczki transporterów hemu bakterii Gram-ujemnych tworzą w błonie zewnętrznej strukturę β-baryłki zbudowanej z 22 łańcuchów aminokwasowych przyjmujących formę antyrównoległych β-kartek.…”

“…U Y. enterocolitica miejsce wiązania dla tego represora pokrywa się z sekwencją -10 promotora operonu hemPRSTUV-1, jak również hemPRST-2. Wykazano, iż Fur kontroluje w sposób negatywny aktywność obydwu operonów w warunkach nadmiaru żelaza w środowisku [14]. Dodatkowy, wewnętrzny promotor przed genami hemSTUV zidentyfikowano u Y. enterocolitica, Y. pestis oraz Y. pseudotuberculosis, ale transkrypcja tego operonu pozostaje niezależna od Fur [13,28,29].…”

“…Analizy ekspresji promotorów dwóch operonów hemPRSTUV-1 i hemPRST-2 kodujących składowe systemu transportu hemu u Y. enterocolitica wykazały, iż OmpR, obok Fur, hamuje transkrypcję obu operonów poprzez bezpośrednie oddziaływanie z DNA w obrębie motywu -35 sekwencji promotorowej. Ponadto, wpływ OmpR na aktywność promotora hemPRSTUV-1 obserwowano zarówno w warunkach niedoboru, jak i nadmiaru żelaza [14].…”

“…Taki typ termosensora zidentyfikowano także w genie chuA receptora hemu E. coli UPEC oraz udowodniono termoregulację syntezy receptora ChuA, co sugeruje iż ten mechanizm potranskrypcyjnej regulacji może występować także w innych gatunkach bakterii [61]. Chociaż w 5'UTR mRNA hemR Y. enterocolitica zidentyfikowano ciąg FourU to poziom HemR w komórce tego patogenu nie podlega termoregulacji [14].…”