Klebsiella pneumoniae is the second-most common opportunistic pathogen in clinical practice and has developed resistance to potent antibacterial drugs such as carbapenems. Therefore, developing safe and effective strategies for the prevention and treatment of K. pneumoniae infections remains a critical challenge. In this study, a strain named Tie-10 isolated from Antarctic samples demonstrated potent antibacterial activity against K. pneumoniae, which was subsequently identified as Bacillus nakamurai. The fermentation medium and culture conditions were systematically optimized through single-factor experiments, orthogonal array testing, and response surface methodology. The optimal medium composition was determined to be beef extract, peptone, and KNO₃. The culture conditions included a time of 24 h, temperature of 37 °C, pH of 7.0, and bottling volume of 80 mL. Antagonistic experiments demonstrated that the crude extract of B. nakamurai Tie-10 exhibited significant inhibitory activity against K. pneumoniae. The alkaline protease (AKP) assay demonstrated that the crude extract effectively disrupted the cellular integrity of K. pneumoniae, a finding further corroborated by scanning electron microscopy (SEM) analysis. Furthermore, the crude extract significantly inhibited extracellular protease secretion in K. pneumoniae, downregulated the expression of virulence-associated genes, and effectively disrupted biofilm formation. The study presented innovative strategies for the management and containment of K. pneumoniae infections.

Butanol dehydrogenase (BDH) plays a significant role in the biosynthesis of butanol in bacteria by catalyzing butanal conversion to butanol at the expense of the NAD(P)H cofactor. BDH is an attractive enzyme for industrial application in butanol production; however, its molecular function remains largely uncharacterized. In this study, we found that Fusobacterium nucleatum YqdH (FnYqdH) converts aldehyde into alcohol by utilizing NAD(P)H, with broad substrate specificity toward aldehydes but not alcohols. An in vitro metal ion substitution experiment showed that FnYqdH has higher enzyme activity in the presence of Co²⁺. Crystal structures of FnYqdH, in its apo and complexed forms (with NAD and Co²⁺), were determined at 1.98 and 2.72 Å resolution, respectively. The crystal structure of apo- and cofactor-binding states of FnYqdH showed an open conformation between the nucleotide binding and catalytic domain. Key residues involved in the catalytic and cofactor-binding sites of FnYqdH were identified by mutagenesis and microscale thermophoresis assays. The structural conformation and preferred optimal metal ion of FnYqdH differed from that of TmBDH (homolog protein of FnYqdH). Overall, we proposed an alternative model for putative proton relay in FnYqdH, thereby providing better insight into the molecular function of BDH.