FlgJ cell-wall hydrolyzing activity enhances, but is not required, for flagellum assembly in Salmonella enterica

Penetration of the peptidoglycan (PG) layer by the nascent flagellar rod is a critical step in basal body assembly and has long been attributed to the acetylglucosaminidase activity of the flagellar rod cap protein FlgJ. Previous work in Salmonella enterica suggested that occasional preexisting openings in the PG layer allow some flagella to assemble in the absence of the FlgJ enzymatic activity. More recently, studies in Bacillus subtilis demonstrated that membrane mobility of nascent flagellar structures enables rod penetration without dedicated PG hydrolysis. Here, we revisited the requirement for FlgJ acetylglucosaminidase activity in S. enterica by testing whether inhibition of class 3 flagellar gene expression by the anti-σ28 factor FlgM contributes to the flagellation defect of FlgJ catalytic mutants. Consistent with previous studies, loss of FlgJ acetylglucosaminidase activity did not abolish flagellar assembly but instead reduced its efficiency, resulting in a heterogeneous population in which many cells assembled functional basal bodies and flagella. Deletion of flgM significantly increased both the proportion of flagellated cells and the number of flagellar filaments per cell, indicating that reduced class three gene expression contributes substantially to the observed defect. These findings support a model in which FlgJ enzymatic activity enhances the efficiency of local PG remodeling but is not essential for rod penetration or basal body assembly. Our results demonstrate that acetylglucosaminidase-independent flagellar rod growth occurs in S. enterica, although less efficiently than in organisms that lack FlgJ-like PG hydrolases, highlighting the evolutionary diversity of mechanisms that accommodate flagellar assembly through the bacterial cell wall.IMPORTANCEBacterial flagella allow pathogens, such as Salmonella, to navigate complex environments and invade host cells. During flagellar assembly, the basal body must traverse the peptidoglycan layer, a step-long thought to require the dedicated cell wall-degrading activity of FlgJ. Here, we show that Salmonella can assemble functional flagella, even when the acetylglucosaminidase activity of FlgJ is genetically inactivated. Although these mutants produce fewer flagella per cell, the assembly pathway remains active, and enhanced σ²⁸-dependent gene expression partially restores flagellation. These findings reveal that early steps in flagellar assembly are more diverse than previously thought and that differences in cell wall structure between gram-positive and -negative bacteria likely determine whether a cell wall hydrolyzing activity is required for flagellum assembly.