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Chapter 27 : Selection of Signature-Tagged Mutants in

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Abstract:

is a facultative intracellular bacillus that causes nosocomial and community-acquired pneumonia and, rarely, extrapulmonary infections in humans. Signature-tagged mutagenesis employs uniquely tagged transposons that are used to randomly mutagenize a bacterial chromosome and create a library. A library of 700 mutant clones created by signature-tagged mutagenesis was screened using this negative-selection strategy in , a free-living amoeba that may serve as an environmental reservoir of legionellae. The efficiency of invasion was studied by incubating strains grown to postexponential phase with , using gentamicin to kill extracellular organisms and then determining remaining intracellular CFU. The behavior of the mutants was also examined in human macrophages derived from peripheral blood mononuclear cells (PBMCs) and in phorbol myristate acetate (PMA)-differentiated U-937 cells. In contrast, all of the mutants replicated within PMA-differentiated U-937 cells, indicating that U-937 cells are deficient in killing compared with freshly isolated macrophages. The homologous gene in serovar Typhimurium is linked to virulence since mutants are attenuated in their ability to kill mice. Two mutants, STM-1 and STM-4, appeared to be deficient in alone, and two other mutants, STM-2 and STM-5, led to intracellular replication defects that were observed in both and .

Citation: Tompkins L, Ross J, Falkow S, Polesky A. 2002. Selection of Signature-Tagged Mutants in , p 152-160. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch27

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Infection and Immunity
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Aromatic Amino Acid Biosynthesis
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Type II Secretion System
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Type IV Secretion Systems
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Figures

Image of FIGURE 1
FIGURE 1

Invasion and intracellular growth of strains in and at 37°C. After growth to postexponential phase in BΥΕ-MOPS, wild-type , Salt, STM-1, -2 and -3 (A) and STM-4, -5 and -6 (B) were incubated for 2 h with . After growth in BYE-ACES, wild-type , Salt, STM-2 and -3 (C), and STM-5 and -6 (D) were incubated with for 2 h. The initial time point ( = 0 h) is the log CFU ml of input bacteria. The arrow at = 4 h, after 2 h gentamicin killing of extracellular bacteria, represents invasion. Subsequent time points represent intracellular replication. Reprinted from ( ) with permission of the publisher.

Citation: Tompkins L, Ross J, Falkow S, Polesky A. 2002. Selection of Signature-Tagged Mutants in , p 152-160. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch27
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Image of FIGURE 3
FIGURE 3

Comparison of invasion and attachment frequencies. Comparison of wild-type invasion of after growth in BΥΕ-ACES or ?ΥΕ-MOPS broths (A). Ratio of mutant to wild-type invasion frequencies in , after growth in BΥΕ-ACES broth (circle) and in BYE-MOPS broth (square) (B). Comparison of wild-type and invasion of after growth in ?ΥΕ-ACES or BΥΕ-MOPS (C). Ratio of to wild-type attachment and invasion frequencies with or without centrifugation of bacteria onto (D). Parts of this figure reprinted from ( ) with permission of the publisher.

Citation: Tompkins L, Ross J, Falkow S, Polesky A. 2002. Selection of Signature-Tagged Mutants in , p 152-160. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch27
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Image of FIGURE 2
FIGURE 2

Invasion and intracellular growth of strains in PBMC-derived macrophages and PMA-differentiated U-937 cells at 37°C. After growth to postexponential phase in BΥΕ-ACES, wild-type , Salt, and STM-2 (A) and STM-3, -5, and -6 (B) were incubated with PBMC-derived macrophages for 2 h. After growth to postexponential phase in BΥΕ-MOPS, wild-type , the salt resistant mutant, STM-1, -2, and -3 (C) and STM-4, -5, and -6 (D) were incubated with PMA-differentiated U-937 cells for 1 h. The initial time point ( = 0 h) is the log CFU ml of input bacteria. The arrow represents invasion. Subsequent time points represent intracellular replication. Reprinted from ( ) with permission of the publisher.

Citation: Tompkins L, Ross J, Falkow S, Polesky A. 2002. Selection of Signature-Tagged Mutants in , p 152-160. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch27
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References

/content/book/10.1128/9781555817985.chap27
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Tables

Generic image for table
TABLE 1

Summary of the STM phenotypes

Citation: Tompkins L, Ross J, Falkow S, Polesky A. 2002. Selection of Signature-Tagged Mutants in , p 152-160. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch27
Generic image for table
TABLE 2

Sequence similarities of genes identified by signature-tagged mutagenesis

Citation: Tompkins L, Ross J, Falkow S, Polesky A. 2002. Selection of Signature-Tagged Mutants in , p 152-160. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch27

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