Chapter 16 : General Methods To Investigate Microbial Symbioses

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This chapter presents methods that highlight two general areas of symbiosis research: (i) the detection and characterization of environmental symbioses and (ii) elucidation of the molecular and cellular mechanisms of symbiosis in model systems. The methods are described to demonstrate that an organism is symbiotic and examples of techniques used to elucidate the molecular, physiological, cellular, and evolutionary basis of symbiosis. Many symbioses, however, present additional challenges to fulfilling Koch’s postulates. The study of bacteria-phage symbioses is important for our understanding of bacterial evolution and emergence of disease as well as providing powerful experimental models for the study of symbiosis in a test tube. The study of presently uncultured symbioses, for which either cultivation of the symbiont or live maintenance of the host is intractable or has not been attempted, can be accomplished using many recent advances in molecular biology and biochemistry, as well as more traditional methodologies. Molecular characterization of the phallodrine oligochaete symbiosis, using 16S rRNA (PCR) characterization and fluorescence in situ hybridization (FISH) microscopy, determined that the primary symbiont was a unique member of the and clustered with other known chemoautotrophic symbionts. Two established models for symbioses are the legume-rhizobia and light organ-, which share all of the above attributes. The chapter describes a few of the many potential monoxenic symbiotic models that contribute greatly to our understanding of the conserved and diverse mechanisms in which symbioses are initiated, maintained, and evolved.

Citation: Ciche T, Goffredi S. 2007. General Methods To Investigate Microbial Symbioses, p 394-420. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch16

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Denaturing Gradient Gel Electrophoresis
Confocal Laser Scanning Microscopy
Type III Secretion System
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Image of FIGURE 1

Labeling bacteria with mini-TnKSGFP. (A) pURR25 (D. Lies and D. Newman, Caltech) containing the mini-TnKSGFP where GFP expression is driven from the constitutive Plac promoter (PA1/04/03) on a mobilizable ( ) suicide plasmid ( ). (B) pUX-BF13 containing the genes () encoding the Tntransposase on a mobilizable suicide plasmid similar to pURR25. (C) Triparental mating introduces pURR25 (mini-TnKSGFP) and pUX-BF13 (Tntransposase) into the recipient cells, which allows the mini-TnKSGFP to transpose, usually in an site, downstream of , of the recipient's genome. (D) Because pURR25 and pUX-BF13 cannot replicate in recipient cells and BW29427 donor cells require DAP for growth, plating on kanamycin- and streptomycin-containing media selects for recipient cells containing the mini-TnKSGFP.

Citation: Ciche T, Goffredi S. 2007. General Methods To Investigate Microbial Symbioses, p 394-420. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch16
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Image of FIGURE 2

Transposon mutagenesis of symbiotic bacteria. (A) pURE10 is a mobilizable suicide plasmid as described in Fig. 1 and contains a hyperactive transposon (HiGm) similar to pSC189 ( ) with replacing of pSC189. (B) BW29427 containing pURE10 is mated with symbiont recipient cells. Transconjugants containing the HiGm are selected for on media containing Gm and lacking DAP.

Citation: Ciche T, Goffredi S. 2007. General Methods To Investigate Microbial Symbioses, p 394-420. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch16
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Image of FIGURE 3

Creating unmarked deletions using SOE PCR and allelic exchange. (A) Two fragments (∼0.75 to 1.5 kb) are amplified by PCR using primers that are complementary (Arev/Bfor) and that have EagI sequences attached (Afor/Brev). The PCR fragments are purified by agarose gel electrophoresis (AGE). (B) Each fragment is then combined and fused by SOEing ( ). (C) The fused PCR products are then digested with EagI and then cloned into the NotI site of the suicide plasmid pWM91 creating pWM91Δ, which is transformed into BW29427 and mated with symbiotic bacteria. Transconjugants containing pWM91Δinserted into are selected based on their resistance to ampicillin, creating a merodiploid strain. Strains that have excised pWM91 are selected for by plating on media containing 5% sucrose. A portion of the sucrose-resistant strains have only the deleted . These are screened by PCR. The presence of the wt can be determined by using one primer for the deleted sequence and another flanking it. Afor and Bfor can also be used to verify the presence of the deletion.

Citation: Ciche T, Goffredi S. 2007. General Methods To Investigate Microbial Symbioses, p 394-420. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch16
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Examples of microbial symbioses

Citation: Ciche T, Goffredi S. 2007. General Methods To Investigate Microbial Symbioses, p 394-420. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch16

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