Recombinant expression and enzymatic analysis of essential enzymes Slt and MltA from Francisella tularensis

Francisella tularensis is a Gram-negative bacterium which is highly infectious and poses a significant risk to public health as a Tier 1 Select Agent. Despite their essential roles in cell growth and division, the peptidoglycan cell wall and its associated remodeling enzymes remain poorly understood in F. tularensis. Lytic transglycosylases (LTs) are a class of enzymes critical for cell division and peptidoglycan recycling that represent attractive targets for novel antibiotics and inhibitors. Notably, the F. tularensis genome encodes only two LTs, MltA and Slt, both of which are essential for growth and contribute to virulence yet have not been biochemically characterized. In this study, we report the first expression, purification, and enzymatic characterization of recombinant His-tagged MltA (rMltA) and Slt (rSlt). rMltA was readily expressed in Escherichia coli and purified using affinity chromatography. However, rSlt proved refractory to conventional expression, and purification methods required extensive optimization to obtain soluble protein, including expression in SHuffle T7 E. coli and modified purification conditions. Both rMltA and rSlt were soluble in citric acid buffer at pH 5 and exhibited enzymatic activity in a fluorescent glycanase assay. rMltA displayed robust activity and quantitative kinetic analysis yielded a K_m of 98.67 μM, whereas rSlt exhibited weaker but detectable activity. These results establish the first biochemical framework for studying the LT enzymes of F. tularensis and lay the foundation for future mechanistic studies and inhibitor screening to develop countermeasures for treatment of tularemia.