Abstract
The flagellum is required for bacterial swimming and swarming motility. In the biphasic Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellar filament is build up by two distinct monomeric subunits, flagellin FliC and FljB. S. Typhimurium has the ability to switch between two flagellins, FliC and FljB, in a phase-variable manner. The switch to FliC is called phase H1 and considered important for bacterial growth and survival in the spleen in a murine infection model of typhoid fever. Flagellin is secreted as monomeric subunits, but the majority of flagellin is polymerized upon secretion as the flagellar filament. Salmonella flagellin has traditionally been isolated through a process involving multiple steps of centrifugation and acid treatment. Here, we delineate a simplified protocol for preparing Salmonella´s flagellin for analytical purpose to determine the amount of flagellin without the aid of antibodies. The growth conditions used were stationary phase, logarithmic phase and a low oxygen and high salt condition mimicking the gastrointestinal tract. Flagellin expression of other source organisms, such as other serovars of Salmonella enterica and Escherichia coli, including flagellar phase- or genetic variants can be analysed. Flagellin expression analysis complements flagella-associated phenotype analysis such as swimming and swarming behaviour.
Keywords: Bacteria, Salmonella, Flagellin, Flagellar phase variation
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Coomassie brilliant blue stained 12% SDS PAGE of flagellar preparations from Salmonella enterica serovar Typhimurium UMR1 wild type and isogenic flagellin mutants. M. PageRuler prestained protein ladder 1. Wild type: ATCC14028-1s Nalr; 2. UMR1 fliC5050::MudJ fljB5001::MudCmr; 3. UMR1 fljB5001::MudCmr; 4. UMR1 fliC5050::MudJ. Molecular weight of flagellin: FliC: 52 kDa, FljB: 53 kDa
Notes
Recipes
Acknowledgments
The development of this protocol was supported by research grants from Karolinska Institutet Doctoral funding (project no. C112130052) and the Swedish Research Council Natural Sciences and Engineering (project no. 621-2010-5755). The authors have no conflict of interest to declare. The protocol described above is adapted from one reported previously (Le Guyon et al., 2014; Ahmad et al., 2013). Kelly T. Hughes kindly provided fliC5050::MudJ and fljB5001::MudCmr mutant alleles in S. Typhimurium LT2.
References
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