ABSTRACTHypermucoviscosity is a hallmark of hypervirulentKlebsiella pneumoniae(hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosityin vitroand virulencein vivoin a mouse pneumonia model. The ΔompRmutant lost its mucoidy but retained its capsule level and comparablermpADCexpression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in ΔompRmutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the ΔompRmutant, theatpoperon encoding F-type ATP synthase and thegcvTHPencoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of theatpoperon, not thegcvTHP, suggesting that OmpR regulates the expression of theatpoperon directly andgcvTHPindirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of ΔompRcells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity.IMPORTANCEHypermucoviscosity is a critical virulent factor forKlebsiella pneumoniaeinfections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity inK. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence ofK. pneumoniaein a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the ΔompRmutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the ΔompRmutant phenotype. Our findings provide new insights into the regulation ofK. pneumoniaehypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.