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Chapter 51 : Identification of a Cytotoxic Paralogue in a Multicopy Suppressor Screen using as a Selective Host

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Identification of a Cytotoxic Paralogue in a Multicopy Suppressor Screen using as a Selective Host, Page 1 of 2

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Abstract:

The IcmG and IcmS proteins are not constituents of the membrane-spanning part of the Icm/Dot T4SS; rather, these proteins bind to secreted effector proteins, thus possibly facilitating their translocation. Perhaps this specific function accounts for the only partial phenotype in the amoebae plate test (APT) of strains lacking IcmG or IcmS. We used and mutants to screen by the APT an genomic library for multicopy suppressors. Overexpression of but not its paralogue in an mutant strain was cytotoxic for . LcsC and its paralogue LpxB share 31% identity on an amino acid level and are 30% or 42% identical, respectively, to the LpxB orthologue from . A closer inspection revealed that this oxidoreductase is 39% identical to the GnnA protein of , which together with the GnnB protein catalyzes the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-GlcNAcN3. Finally, not only the glycosyl transferase gene but also the acyl trans-ferase genes and are present in at least two copies in the genome. The Icm/Dot T4SS is not only required for the upregulation of phagocytosis and intracellular replication of , but also for growth of in the presence of on agar plates. The APT established here may prove useful to discover other bacterial factors relevant for interactions with amoeba.

Citation: Albers U, Reus K, Hilbi H. 2006. Identification of a Cytotoxic Paralogue in a Multicopy Suppressor Screen using as a Selective Host, p 203-206. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch51

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Type IV Secretion Systems
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Figures

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FIGURE 1

Growth of on agar plates in the presence of amoebae: the APT. wild-type and mutant strains were spotted in serial dilutions onto CYE agar plates in presence or absence of 4 × 10 and incubated for 5 days at 30°C. The strains harbored either an empty vector (pMMB) or a complementing plasmid (). While wild-type and the complemented mutant strain grew robustly even at high dilutions, the and mutant strains showed a partial or complete growth defect in presence of amoebae. In the absence of amoebae, all bacterial strains grew equally well.

Citation: Albers U, Reus K, Hilbi H. 2006. Identification of a Cytotoxic Paralogue in a Multicopy Suppressor Screen using as a Selective Host, p 203-206. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch51
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Image of FIGURE 2
FIGURE 2

but not expressed in an mutant strain is cytotoxic for was infected at a multiplicity of infection of 50 with mutant strains (Δ) harboring a complementing plasmid (p), an empty plasmid (pBCR), or plasmids expressing (p) or (p) from the P promotor. Cyto-toxicity was quantified 2 days postinfection (30°C) by uptake of the fluorescent dye propidium iodide (PI). Bright field and fluorescence micrographs are shown.

Citation: Albers U, Reus K, Hilbi H. 2006. Identification of a Cytotoxic Paralogue in a Multicopy Suppressor Screen using as a Selective Host, p 203-206. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch51
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References

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