Transmission Electron Micrographs of Negatively Stained Salmonella typhimurium Flagella and Fimbriae and Campylobacter jejuni Flagella
Salmonella typhimurium and Campylobacter jejuni
are gram-negative pathogens responsible for many food-poisoning
outbreaks across the United States. Gram-negative bacteria have various
filamentous protein structures that extend from their outer membrane.
The most commonly encountered structures are flagella that provide
motility and chemotaxic response. Figure 1 is a transmission electron
micrograph of a single negatively stained S. typhimurium organism
with two flagella (white arrows) exiting from opposite poles of the
bacterium. The light-colored circles in the background of the
preparation are protein aggregates from the culture media. While S. typhimurium
organisms are typically peritrichous bacteria (i.e., bacteria with the
surface covered with flagella), a cell with limited flagella expression
was chosen to more clearly demonstrate the presence of other structures
radiating from the outer membrane of the cell (see Fig. 3 or enlarge
Fig. 1).
The flagellum filament itself is made up of the protein flagellin which can have different protein structural orientations depending on the bacteria. This is illustrated in Fig. 2, a panel of transmission electron micrographs depicting negatively stained flagella from S. typhimurium (Fig. 2A) and C. jejuni (Fig. 2B). In the case of S. typhimurium, flagellin is present in a helical arrangement (Fig. 2A, white arrow), while in C. jejuni flagellin appears to have an elongated, more filamentous appearance (Fig. 2B, white arrow).
Fimbriae are also frequently observed radiating from the outer membrane of gram-negative bacteria. These structures allow for bacterial attachment to other cells and surfaces. Figure 3 is an enlargement of Fig. 1 showing numerous fimbriae (white arrows) radiating from the outer membrane of a S. typhimurium bacterium. The inset depicts the area on the bacterium from which the enlargement is made. Of note, the presence of fimbriae on Campylobacter has not been demonstrated (5).
Methods
Bacterial cultures were field isolates obtained from the intestine of a chicken (S. typhimurium) and a dairy cow (C. jejuni). Both isolates were prepared from overnight cultures with S. typhimurium grown in trypic soy broth while C. jejuni was grown in Bolton’s broth without antibiotics under microaerophilic conditions (1). Aliquots of each species were negatively stained on formvar coated copper grids (Ted Pella Inc., Redding, CA) using either freshly prepared 1% uranyl acetate or 1% uranyl formate. Grids were floated on the culture media for up to 1 minute; then, negative stain was applied for several seconds and subsequently decanted. Stained grids were allowed to air dry. Negative stained grids were examined using a Hitachi H7000 transmission electron microscope and images were recorded using conventional electron imaging film.
Discussion
Flagella, composed of arrays of the protein flagellin, are substantially longer and larger in diameter than fimbriae, measuring approximately 10 µm in length and 20 nm in width. Flagella rotate, creating thrust that allows for bacterial motility and, as the case for both species presented in this report, have been identified as a virulence factor (2, 4). Due to sequence similarity between flagellar coiled-coil domains, S. typhimurium was thought to be a model for all bacterial flagellar filaments. However, it has been determined that C. jejuni and several related species have a different flagellar structure due in part to a difference in the number of protofilaments within each flagellum (3). Transmission electron micrographs show that while S. typhimurium flagella appear to have a helical protein arrangement, Campylobacter flagella can exhibit a parallel protein structure (6). Fimbriae are present as short protein tubes that extend from the bacterial surface of many species. These extensions allow for bacterial attachment to surfaces and cells. While fimbriae are coded by many genes, they appear to have the same general structure when viewed by transmission electron microscopy, varying primarily in length and diameter. These differences may be of key importance in developing efficient medications against these prevalent enteric pathogens.
References
1. Dhillon, A. S., H. L. Shivaprasad, D. Schaberg, F. Wier, S. Weber, and D. Bandli. 2006. Campylobacter jejuni infection in broiler chickens. Avian Dis. 50:55–58.
2. Dreyfus, G., A. W. Williams, I. Kawagishi, and R. M. Macnab. 1993. Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens. J. Bacteriol. 175:3131–3138.
3. Galkin, V. E., X. Yu, J. Bielnicki, J. Heuser, C. P. Ewing, P. Guerry, and E. H. Egelman. 2008. Divergence of quaternary structures among bacterial flagellar filaments. Science 320:382–385.
4. Guerry, P. 2007. Campylobacter flagella: not just for motility. Trends Microbiol. 15:456–461. http://www.sciencedirect.com/science/article/pii/S0966842X07001746.
5. Konkel, M. E., M. R. Monteville, V. Rivera-Amill, and L. A. Jones. 2001. The pathogenesis of Campylobacter jejuni-mediated enteritis. Curr. Issues Intest. Microbiol. 2:55–71.
6. McCoy, E. C., D. Doyle, H. Wiltberger, K. Burda, and A. J. Winter. 1975. Flagellar ultrastructure and flagella-associated antigens of Campylobacter fetus. J. Bacteriol. 122:307–315.
The flagellum filament itself is made up of the protein flagellin which can have different protein structural orientations depending on the bacteria. This is illustrated in Fig. 2, a panel of transmission electron micrographs depicting negatively stained flagella from S. typhimurium (Fig. 2A) and C. jejuni (Fig. 2B). In the case of S. typhimurium, flagellin is present in a helical arrangement (Fig. 2A, white arrow), while in C. jejuni flagellin appears to have an elongated, more filamentous appearance (Fig. 2B, white arrow).
Fimbriae are also frequently observed radiating from the outer membrane of gram-negative bacteria. These structures allow for bacterial attachment to other cells and surfaces. Figure 3 is an enlargement of Fig. 1 showing numerous fimbriae (white arrows) radiating from the outer membrane of a S. typhimurium bacterium. The inset depicts the area on the bacterium from which the enlargement is made. Of note, the presence of fimbriae on Campylobacter has not been demonstrated (5).
Methods
Bacterial cultures were field isolates obtained from the intestine of a chicken (S. typhimurium) and a dairy cow (C. jejuni). Both isolates were prepared from overnight cultures with S. typhimurium grown in trypic soy broth while C. jejuni was grown in Bolton’s broth without antibiotics under microaerophilic conditions (1). Aliquots of each species were negatively stained on formvar coated copper grids (Ted Pella Inc., Redding, CA) using either freshly prepared 1% uranyl acetate or 1% uranyl formate. Grids were floated on the culture media for up to 1 minute; then, negative stain was applied for several seconds and subsequently decanted. Stained grids were allowed to air dry. Negative stained grids were examined using a Hitachi H7000 transmission electron microscope and images were recorded using conventional electron imaging film.
Discussion
Flagella, composed of arrays of the protein flagellin, are substantially longer and larger in diameter than fimbriae, measuring approximately 10 µm in length and 20 nm in width. Flagella rotate, creating thrust that allows for bacterial motility and, as the case for both species presented in this report, have been identified as a virulence factor (2, 4). Due to sequence similarity between flagellar coiled-coil domains, S. typhimurium was thought to be a model for all bacterial flagellar filaments. However, it has been determined that C. jejuni and several related species have a different flagellar structure due in part to a difference in the number of protofilaments within each flagellum (3). Transmission electron micrographs show that while S. typhimurium flagella appear to have a helical protein arrangement, Campylobacter flagella can exhibit a parallel protein structure (6). Fimbriae are present as short protein tubes that extend from the bacterial surface of many species. These extensions allow for bacterial attachment to surfaces and cells. While fimbriae are coded by many genes, they appear to have the same general structure when viewed by transmission electron microscopy, varying primarily in length and diameter. These differences may be of key importance in developing efficient medications against these prevalent enteric pathogens.
References
1. Dhillon, A. S., H. L. Shivaprasad, D. Schaberg, F. Wier, S. Weber, and D. Bandli. 2006. Campylobacter jejuni infection in broiler chickens. Avian Dis. 50:55–58.
2. Dreyfus, G., A. W. Williams, I. Kawagishi, and R. M. Macnab. 1993. Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens. J. Bacteriol. 175:3131–3138.
3. Galkin, V. E., X. Yu, J. Bielnicki, J. Heuser, C. P. Ewing, P. Guerry, and E. H. Egelman. 2008. Divergence of quaternary structures among bacterial flagellar filaments. Science 320:382–385.
4. Guerry, P. 2007. Campylobacter flagella: not just for motility. Trends Microbiol. 15:456–461. http://www.sciencedirect.com/science/article/pii/S0966842X07001746.
5. Konkel, M. E., M. R. Monteville, V. Rivera-Amill, and L. A. Jones. 2001. The pathogenesis of Campylobacter jejuni-mediated enteritis. Curr. Issues Intest. Microbiol. 2:55–71.
6. McCoy, E. C., D. Doyle, H. Wiltberger, K. Burda, and A. J. Winter. 1975. Flagellar ultrastructure and flagella-associated antigens of Campylobacter fetus. J. Bacteriol. 122:307–315.
S. typhimurium negatively stained with 1% uranyl acetate.
This low magnification micrograph depicts a single bacterium possessing
multiple flagella; the arrow indicates one of these flagella. Protein aggregates from the media are visible as light-colored circles. Bar = 1 µm.
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(A) S. typhimurium flagellum, stained with 1% uranyl acetate, showing the helical nature of its flagella protein structure. (B) C. jejuni negatively stained with 1% uranyl formate, highlighting the parallel protein structure of its flagella. Bar = 50 nm.
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S. typhimurium enlarged from an area of Fig. 1 (indicated by
highlighted boxed area in the inset). Fimbriae, indicated by arrows,
are evident radiating from the outer membrane of the bacterium. Bar =
100 nm.
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