Identification and characterization of a new iron‐dependent sRNA in Klebsiella oxytoca (LB204)

Abstract

Klebsiella sp. are responsible for many serious hospital‐acquired infections such as pneumonia, sepsis, urinary tract infections, and hemorrhagic colitis. Because iron is an essential nutrient required by virtually all organisms, the ability to acquire iron correlates with virulence and pathogenesis of bacterial infections. The discovery of the regulatory small RNA (sRNA) ryhB in Escherichia coli helped to identify similar iron‐regulated sRNA targets in other bacteria. We have identified a homologous gene, krrF, in Klebsiella oxytoca via in silico methods. We first confirmed the expression of this gene by applying non‐quantitative RT‐PCR. After proving krrF transcription, we demonstrated that its expression is dependent on extracellular iron levels by performing qRT‐PCR on RNA isolated from wild type K. oxytoca cultures grown in media with varying concentrations of iron. Results indicated that krrF transcription increased in cultures with lower concentrations of iron and in cultures to which an iron chelator was added, while cultures with the highest iron concentrations showed decreased krrF transcription. Moreover, we performed binding assays using purified K. oxytoca Ferric Uptake Regulator (Fur), which showed affinity of the Fur protein for the krrF gene promoter. In conclusion, we present krrF from K. oxytoca, a new sRNA likely regulated by the Fur protein in response to iron levels in the medium.