Aug 2026· Journal of Bacteriology· 0 citations· 30 references
Medicine
TL;DR
Salmonella can assemble functional flagella, even when the acetylglucosaminidase activity of FlgJ is genetically inactivated, showing 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.
Abstract
ABSTRACT 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. IMPORTANCE Bacterial 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. Bacterial 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.
Analysis of oligomeric organization and structural plasticity of the periplasmic domain of polar-flagellum FliL from Vibrio alginolyticus and compared with detergent-solubilized full-length pofFliL suggests that such plasticity enables FliL to act as an adaptable scaffold for stator engagement and mechanosensitive remodeling of the motor.
Tatsuro Nishikino, N. Takekawa, Raymond N. Burton-Smith et al.· Molecular Microbiology· 0 citations
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations
Many bacteria assemble multiple flagella, although building flagella imposes a substantial biosynthetic and energetic cost. We used the peritrichously flagellated model organism Salmonella enterica to quantify how flagellar abundance affects bacterial growth, proteome allocation, and motility. For this, we generated genetically modified strains with inducible or constitutive expression of the flagellar master regulator flhDC, resulting in a panel of strains ranging from nearly non-flagellated to hyperflagellated cells. We found that higher flagellar investment reduced growth rate and redirected proteome allocation, with an expansion of the flagellar sector occurring largely at the expense of the ribosomal sector. Growth analyses of flagellar assembly mutants, combined with cost modeling, suggested that flagellin biosynthesis dominated the energetic burden, whereas motor rotation contributed a smaller additional cost. Despite the associated cost, increased flagellation improved soft-agar spreading, single-cell swimming speed, effective diffusivity, and competitive fitness in spatially structured environments. A coarse-grained proteome-allocation model parametrized from these data reproduced the observed growth penalties, while simulations of navigation in dynamic chemical gradients predicted that motility benefits saturate near a flagellar investment of 3% of proteome mass. Beyond this point, rising biosynthetic costs outweigh diminishing motility gains. In summary, these results support a quantitative cost-benefit model in which heterogeneous, spatially structured environments favor an intermediate number of flagella by balancing motility benefits against the biosynthetic costs of building and operating multiple flagella.
María Giralt-Zúñiga, Michael Jahn, Joshua L. Franklin et al.· bioRxiv· 0 citations
Key mechanistic steps coordinating ATP-driven MreB polymerization and turnover are established and provide a basis for a complete MreB assembly–disassembly cycle and for further elucidating how MreB dynamics contribute to cell-wall organization.
Alba de San Eustaquio-Campillo, C. Cornilleau, Sana Afensiss et al.· bioRxiv· 0 citations
Contact with solid surfaces activates signaling pathways that promote biofilm formation in many bacteria. The alphaproteobacterium Caulobacter crescentus uses its flagellum to sense surfaces and responds by synthesizing an adhesive called the holdfast. The C. crescentus surface sensing pathway can be activated by mutating genes required for the assembly of the flagellum or genes required for chemotaxis. However, flagellar assembly and chemotaxis mutations activate distinct surface sensing pathways that differ in the activation of the diguanylate cyclase PleD. Here, we used a genome-wide screen to identify cmrA (CCNA_02061) as a crucial determinant of hyperadhesion in the chemotaxis mutant ΔcheYII. Genetic analysis showed that cmrA is important for activation of PleD in a context-specific manner. It is dispensable in wild-type and late-stage flagellar (ΔflgH) mutant backgrounds but promotes adhesion in early-stage flagellar assembly (ΔfliF), chemotaxis (ΔcheYII) and stator (ΔmotB) mutant backgrounds. Fluorescently tagged CmrA displays a mostly cytoplasmic localization in genetic backgrounds where cmrA is dispensable for adhesion but localizes to the cell pole in backgrounds where it regulates adhesion. Structural modeling indicates that CmrA is a degenerate, catalytically inactive GGDEF/EAL domain containing protein, but cmrA alleles with mutated conserved c-di-GMP coordinating residues are unable to support hyperadhesion. Our results indicate that altering the directional switching of MotAB stators recruits CmrA to the cell pole where it activates PleD to drive surface adaptation. Ultimately, this work underscores the complexity of flagellar surface sensing by highlighting how the many rotational states of the motor stimulate distinct but overlapping c-di-GMP signaling pathways Importance Bacteria often transition from a free-swimming state to form surface-attached communities called biofilms. The flagellum allows bacteria to sense surface contact and activate biofilm formation, yet how distinct structural states of this complex machine trigger surface sensing remains poorly understood. In this study, we identify CmrA as a key signaling link that senses flagellar motor disruption and activates second-messenger signaling to promote cell adhesion in Caulobacter crescentus. Our findings demonstrate that bacterial surface sensing is not a simple binary switch. Instead, distinct mechanical perturbations to the flagellum engage specialized, overlapping signaling pathways to fine-tune surface adaptation. Understanding these nuanced pathways will inform strategies to manipulate biofilm formation for human benefit.
Rachel I. Salemi, David M. Hershey· bioRxiv· 0 citations
ABSTRACT Capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Yet, the mechanism linking CPS to peptidoglycan (PG) remains unclear. Here, we identified a strong negative genetic interaction between the genes encoding the putative capsule ligase CpsA and the WalK histidine kinase, a component of the WalRK two-component system regulating cell wall homeostasis. In the absence of cpsA, capsule polymers compete with wall teichoic acids for ligase activity to PG. This induces cell wall stress and is sensed by the WalRK system. Overexpression of the PG hydrolase pcsB or disruption of the PG-modifying enzymes pgdA and oatA(adr) restored growth of strains lacking cpsA and walK. Furthermore, CpsA overproduction compensates for the loss of other LytR-Cps2A-Psr (LCP) ligases, suggesting it can support capsule and wall teichoic acid syntheses. These findings support the model that LCP ligases are semi-redundant, although they may install secondary polymers on a different residue of PG. This work also suggests that WalRK signaling compensates for reduced capsule and WTA attachment by positively regulating PG hydrolases. IMPORTANCE Streptococcus pneumoniae causes more than half a million deaths annually. A powerful public health tool for controlling pneumococcal infections is vaccination against the protective capsule. Yet, the mechanisms by which the capsule layer attaches to the underlying cell wall remain poorly defined. This study shows that the conserved capsule gene CpsA is not strictly required for capsule attachment but instead works together with other LytR‑CpsA‑Psr (LCP) ligases. However, it requires the essential WalRK signaling system to maintain cell envelope integrity. Defects in LCP activity are alleviated by WalRK‑driven upregulation of peptidoglycan hydrolases, overexpression of PcsB, or inactivating peptidoglycan modifications that limit hydrolysis. These findings reveal coordination among flux to capsule synthesis, secondary wall polymer attachment, and cell wall remodeling. Streptococcus pneumoniae causes more than half a million deaths annually. A powerful public health tool for controlling pneumococcal infections is vaccination against the protective capsule. Yet, the mechanisms by which the capsule layer attaches to the underlying cell wall remain poorly defined. This study shows that the conserved capsule gene CpsA is not strictly required for capsule attachment but instead works together with other LytR‑CpsA‑Psr (LCP) ligases. However, it requires the essential WalRK signaling system to maintain cell envelope integrity. Defects in LCP activity are alleviated by WalRK‑driven upregulation of peptidoglycan hydrolases, overexpression of PcsB, or inactivating peptidoglycan modifications that limit hydrolysis. These findings reveal coordination among flux to capsule synthesis, secondary wall polymer attachment, and cell wall remodeling.
J. Zik, Zeyu Fu, M. Price et al.· mBio· 0 citations