ABSTRACTBolA has been characterized as an important transcriptional regulator, which is induced in the stationary phase of growth and is often associated with bacterial virulence. This study was initiated to elucidate the role of the BolA in the virulence of K. pneumoniae. Using a mouse infection model, we revealed bolA mutant strain yielded significantly decreased bacterial loads in the liver, spleen, lung, and kidney, and failed to form liver abscesses. Gene deletion demonstrated that the bolA was required for siderophore production, biofilm formation, and adhesion to human colon cancer epithelial cells HCT116. Quantitative reverse transcriptase PCR (RT-qPCR) indicated that BolA could impact the expression of pulK, pulF, pulE, clpV, vgrG, entE, relA, and spoT genes on a genome-wide scale, which are related to type II secretion system (T2SS), type VI secretion system (T6SS), guanosine tetraphosphate (ppGpp), and siderophore synthesis and contribute to fitness in the host. Furthermore, the metabolome analysis showed that the deletion of the bolA gene led to decreased pools of five metabolites: biotin, spermine, cadaverine, guanosine, and flavin adenine dinucleotide, all of which are involved in pathways related to virulence and stress resistance. Taken together, we provided evidence that BolA was a significant virulence factor in the ability of K. pneumoniae to survive, and this was an important step in progress to an understanding of the pathways underlying bacterial virulence.IMPORTANCE BolA has been characterized as an important transcriptional regulator, which is induced in the stationary phase of growth and affects different pathways directly associated with bacterial virulence. Here, we unraveled the role of BolA in several phenotypes associated with the process of cell morphology, siderophore production, biofilm formation, cell adhesion, tissue colonization, and liver abscess. We also uncovered the importance of BolA for the success of K. pneumoniae infection and provided new clues to the pathogenesis strategies of this organism. This work constitutes a relevant step toward an understanding of the role of BolA protein as a master regulator and virulence factor. Therefore, this study is of great importance for understanding the pathways underlying K. pneumoniae virulence and may contribute to public health care applications.