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Chapter 45 : Identification of Translocated Substrates of the Dot/Icm System without the use of Eukaryotic Host Cells

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Identification of Translocated Substrates of the Dot/Icm System without the use of Eukaryotic Host Cells, Page 1 of 2

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Abstract:

This chapter describes alternate approaches that take advantage of special properties of strains that allowed the formulation of simple screens using toothpicks and petri dishes containing bacteriological medium. Developing readouts in the microorganism that allow screens on solid medium has allowed a number of translocated substrates to be identified. Interestingly, it was found that mutants producing low levels of DotA were able to grow on agar plates. This important finding allowed considerable manipulation of phenotypes, because the behavior of mutants that would otherwise be inviable could now be evaluated during intracellular growth. The great majority of mutations, in fact, appeared to directly affect the assembly of the Dot/Icm system or disrupt function of DotL, and many of the impaired proteins are involved in either membrane biogenesis or membrane protein folding. In several T4SSs,Mob protein can be transferred into recipient cells in the absence of DNA mobilization, indicating that protein translocation into bacteria can occur in the same fashion that is observed when the pathogen contacts a mammalian cell. Furthermore, elimination of multiple paralogs of a single gene family does not solve the redundancy issue, suggesting that a similar amino acid sequence does not necessarily mean the proteins have similar functions. Development of functional and biochemical assays for these proteins will be necessary to sort out their roles in intracellular growth.

Citation: R. Isberg R, Machner M. 2006. Identification of Translocated Substrates of the Dot/Icm System without the use of Eukaryotic Host Cells, p 169-176. 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.ch45

Key Concept Ranking

Type III Secretion System
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Type IV Secretion Systems
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Figures

Image of FIGURE 1
FIGURE 1

Isolation of miniTn, a mutation that eliminates a translocated substrate of Dot/Icm (from [ ]). Insertion mutations were generated in an strain that has a defective Dot/Icm transporter due to a mutation in the gene. These strains were then patched onto bacteriological plates and allowed to grow. Strains of high viability were then patched onto a plate seeded with with a mobilizable plasmid that encodes an intact gene to allow mating and movement of the gene into the mutagenized strains. Such strains will now have an intact Dot/Icm translo-cator, and should grow like wild-type strains unless the insertion mutation has caused a growth defect when there is a Dot/Icm system present. These mutants with lowered plating efficiency in the presence of an intact Dot/Icm will make a smaller patch on selective media than do strains that can tolerate an intact translocator. Charcoal-yeast extract plates showing examples of mutants that fail to tolerate the presence of the Dot/Icm system.

Citation: R. Isberg R, Machner M. 2006. Identification of Translocated Substrates of the Dot/Icm System without the use of Eukaryotic Host Cells, p 169-176. 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.ch45
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Image of FIGURE 2
FIGURE 2

Identification of translocated substrates of Dot/Icm by transfer between bacterial strains (from [ ]). Fusions of DNA to the gene encoding Cre were constructed and were used to test the ability of the resulting hybrid protein to translocate into a tester strain. Translocation of a hybrid protein from the donor strain can be detected by the generation of kanamycin-resistant (Kan) exconjugants. These strains are recombinants that result from removal of a floxed transcriptional terminator located between a promoter and the kan gene found on a construction in the recipient bacterial strain. In contrast, in the absence of Cre, bacteria harboring the intact reporter are unable to grow on media containing kanamycin and sucrose. The translocation of Cre hybrid protein into the recipient strain leads to the excision, via recombination at the sites, of the DNA fragment that confers sucrose sensitivity and reconstitution of a functional translational fusion. S. D., Shine-Dalgarno sequence; neomycin phosphotransferase; ⋄, transcriptional terminator. Arrows indicate the promoter and sites.

Citation: R. Isberg R, Machner M. 2006. Identification of Translocated Substrates of the Dot/Icm System without the use of Eukaryotic Host Cells, p 169-176. 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.ch45
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References

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Tables

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TABLE 1

Translocated substrates identified by the Cre-Lox interbacterial screen

Citation: R. Isberg R, Machner M. 2006. Identification of Translocated Substrates of the Dot/Icm System without the use of Eukaryotic Host Cells, p 169-176. 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.ch45

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