Salmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conserved Salmonella enterica serovar Typhimurium (S Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support the Escherichia coli model, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. The S Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. The S Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example, tolQ, tolR, tolA, and cpoB mutants were significantly more attenuated than ybgC, tolB, and pal mutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary for S Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotes S Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

Edwardsiella ictaluri is a Gram-negative intracellular facultative pathogen causing enteric septicemia of channel catfish (ESC). The Tol system, consisting of four envelope proteins TolQ, TolR, TolA, and TolB, are required for colicin import and contributes to bacterial virulence in several pathogenic bacteria. However, the Tol system and its importance in E. ictaluri virulence have not been investigated. Here we present construction and evaluation of the E. ictaluri TolQ, TolR and TolQR mutants (EiΔtolQ, EiΔtolR, and EiΔtolQR). The Tol mutants were developed using in-frame gene deletion and their attenuation and vaccine efficacy were determined in catfish fingerlings. The EiΔtolQ, EiΔtolR, and EiΔtolQR mutants showed reduced virulence in catfish (28.93%, 19.70%, and 39.82% mortality, respectively) compared to wild type (46.91% mortality). Further, vaccination with these mutants protected catfish against subsequent wild-type infection. This study suggests that the Tol system contributes to E. ictaluri virulence in catfish.

Protein expression and fatty acid profiles of biofilm cells of chlorhexidine-tolerant Delftia acidovorans (MIC = 15 µg/ml) and its chlorhexidine-susceptible mutant (MIC = 1 µg/ml) were investigated. The chlorhexidine-susceptible mutant (MT51) was derived from the parental strain (WT15) using Tn5 transposon mutagenesis. The disrupted gene was identified as tolQ, a component of the tolQRAB gene cluster known to be involved in outer membrane stability. Proteomic responses of biofilm cells were compared by differential in-gel electrophoresis following exposure to chlorhexidine at sub-MIC (10 µg/ml) and above-MIC (30 µg/ml) concentrations. Numerous changes in protein abundance were observed in biofilm cells following chlorhexidine exposure, suggesting that molecular changes occurred during adaptation to chlorhexidine. Forty proteins showing significant differences (≥1.5-fold; P < 0.05) were identified by mass spectrometry and were associated with various functions, including amino acid and lipid biosynthesis, protein translation, energy metabolism, and stress-related functions (e.g., GroEL, aspartyl/glutamyl-tRNA amidotransferase, elongation factor Tu, Clp protease, and hydroxymyristoyl-ACP dehydratase). Several proteins involved in fatty acid synthesis were affected by chlorhexidine, in agreement with fatty acid analysis, wherein chlorhexidine-induced shifts in the fatty acid profile were observed in the chlorhexidine-tolerant cells, primarily the cyclic fatty acids. Transmission electron microscopy revealed more prominent changes in the cell envelope of chlorhexidine-susceptible MT51 cells. This study suggests that multiple mechanisms involving both the cell envelope (and likely TolQ) and panmetabolic regulation play roles in chlorhexidine tolerance in D. acidovorans. IMPORTANCE Delftia acidovorans has been associated with a number of serious infections, including bacteremia, empyema, bacterial endocarditis, and ocular and urinary tract infections. It has also been linked with a variety of surface-associated nosocomial infections. Biofilm-forming antimicrobial-resistant D. acidovorans strains have also been isolated, including ones displaying resistance to the common broad-spectrum agent chlorhexidine. The mechanisms of chlorhexidine resistance in D. acidovorans are not known; hence, a chlorhexidine-susceptible mutant of the tolerant wild-type strain was obtained using transposon mutagenesis, and the proteome and ultrastructural changes of both strains were compared under chlorhexidine challenge.

Mutations involving the Tol-Pal complex of Escherichia coli result in a subtle phenotype in which cells chain when grown under low-salt conditions. Here, the nonpolar deletion of individual genes encoding the cytoplasmic membrane-associated components of the complex (TolQ, TolR, TolA) produced a similar phenotype. Surprisingly, the overexpression of one of these proteins, TolQ, resulted in a much more overt phenotype in which cells occurred as elongated rods coupled in long chains when grown under normal salt conditions. Neither TolR nor TolA overexpression produced a phenotype, nor was the presence of either protein required for the TolQ-dependent phenotype. Consistent with their native membrane topology, the amino-terminal domain of TolQ specifically associated in vivo with the periplasmic domain of FtsN in a cytoplasm-based two-hybrid analysis. Further, the concomitant overexpression of FtsN rescued the TolQ-dependent phenotype, suggesting a model wherein the overexpression of TolQ sequesters FtsN, depleting this essential protein from the divisome during Gram-negative cell division. The role of the Tol-Pal system in division is discussed.