Deciphering FUR-regulated gene networks in Klebsiella pneumoniae using FUR knockout mutant

Klebsiella pneumoniae is a nosocomial pathogen causing life-threatening infections worldwide. It possesses many virulence properties and the ferric uptake regulator (FUR) is the transcription factor that regulates the expression of various genes in K. pneumoniae . FUR binds to a conserved 19 bp sequence, called the FUR box, for either positive or negative regulation of the gene. FUR alters gene expression according to iron availability and also participates in virulence, colonization & toxin secretion in K. pneumoniae . FUR deletion mutant is an interesting model to understand the virulence factors. In the present study, we identified the genes containing putative FUR boxes (>50% sequence homology) from the genome of K. pneumoniae M33. FUR deletion mutant for K. pneumoniae M33 was created using lambda red recombinase system using the plasmids- pACBSR-hyg, pMDIAI, and pFLP-hyg. The mutant (M33∆FUR) was characterized using various assays like CAS assay, string test, crystal violet assay to access siderophore production, capsule synthesis and biofilm formation respectively. FUR box was present upstream of 18 genes with distinct functions. In the FUR knockout mutant, M33∆FUR an increase in expression of iron transport and siderophore related genes feoC, fhuA, fepB and fes was observed whereas genes feoA, cirA, fecA, fepA and entC were down-regulated. Genes related to biofilm ( fimA and mrkD ) were downregulated whereas genes related to capsular polysaccharide ( rcsA and rcsB ) were upregulated upon FUR deletion. Among other genes murG and sucA were downregulated and priB and srlB were upregulated upon FUR deletion. This study shows that FUR regulates many genes involved in virulence, either positively or negatively. • FUR deletion mutant for K. pneumoniae M33 was created using lambda red recombinase system • FUR deletion mutant (M33∆FUR) was characterized using various assays to access its siderophore production (CAS assay), capsule synthesis (string test) and biofilm formation (crystal violet assay) respectively. • Location of FUR box in genome of K. pneumoniae M33 was observed, and it was found to be present upstream of 18 genes. • FUR regulates the gene involved in virulence, either positively or negatively.