Chapter 15 : Microbial Channels: Forbidden Fruit from Missense Rather than Nonsense

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Microbial Channels: Forbidden Fruit from Missense Rather than Nonsense, Page 1 of 2

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The majority of transport processes undertaken by bacterial cells are unregulated-that is, apart from regulation of their transcription, there is no active regulation of the solute translocation event. Systems that play major roles in homeostasis must, by their specific roles, have their activity curtailed such that they are “fired up” when the cellular homeostasis is perturbed. The current understanding of microbial channels derives extensively from the study of missense mutations selected by specific screens rather than from the explosion of genomic information. Electrophiles are reactive molecules that are attacked by lone pairs of electrons on organic molecules. Suppressor analysis of the missense mutations in the Ktn domain generated the first insights into the relationship between KefF and KefC. Ktn domains regulate a wide variety of types of transporters and channels. The first insights into these proteins arose from the study of missense mutations of KefC, making this strategy truly a pivotal point for the analysis of regulation of ion flux. Analysis of the mutant locus revealed two missense mutations, L565Q and G922S, of which the latter is responsible for the phenotype. The discovery of both glutathione-gated potassium efflux systems that regulate cytoplasmic pH during electrophile challenge and mecha-nosensitive channels was essentially predicated on the isolation of missense mutations that modified their activity.

Citation: Booth I. 2011. Microbial Channels: Forbidden Fruit from Missense Rather than Nonsense, p 143-152. In Maloy S, Hughes K, Casadesús J (ed), The Lure of Bacterial Genetics. ASM Press, Washington, DC. doi: 10.1128/9781555816810.ch15
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Image of FIGURE 1

Response of homeostatic systems to perturbation. (a) Initially the system (see below for definition) is active at a very low level, which may be very close to zero (e.g., KefC in the absence of an electrophile or a mechanosensitive channel at low bilayer tension). On imposition of a perturbation (*) the system rises to a new level of activity (b) and retains that activity as long as the perturbing signal is present (c) but declines if the immediate activation is sufficient to restore normal conditions in the cytoplasm (d). Intermediate time courses and kinetics of decay from the activated state (e) can also be encountered. For example, KefC activity would follow (c) if cells were exposed to excess NEM (>100 µM), but activity would decay if a low concentration of NEM (e.g., 10 µM) was added to cells and would follow (e) at intermediate concentrations ( ). Although not immediately obvious, the graph also represents the pattern of gene expression seen for a system with feedback. If feedback is fully implemented, curve (d) applies, but if the feedback loop is broken, gene expression may follow curve (c). In some homeostatic systems, such as the ProP and ProU betaine transporters, the activity/time profile is a combination of both activation and gene expression with the final shape of the curve determined by the availability of betaine or proline to be transported (relief of stress) and the osmotic shift ( ).

Citation: Booth I. 2011. Microbial Channels: Forbidden Fruit from Missense Rather than Nonsense, p 143-152. In Maloy S, Hughes K, Casadesús J (ed), The Lure of Bacterial Genetics. ASM Press, Washington, DC. doi: 10.1128/9781555816810.ch15
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Image of FIGURE 2

Suppressor effects on KefC activity. Suppressors were isolated from two independent missense mutations, V427A in the Rossman fold and R416S in the GSH-binding pocket ( ) of KefC. Their influence on KefC activity was determined by creating the mutations in the cloned gene that had the wild-type sequence (black bars), an R416S mutation (open bars), or D264A (gray bars). The D264A mutation lies in the loop between transmembrane helices and gives rise to high rates of spontaneous activity in the absence of electrophiles but retains control by GSH and activation by electrophiles. R416S defines part of the GSH-binding site and is not inhibited by glutathione but is activated by electrophiles. Three suppressors (G526V, A522V, and E520G) reside in the hinge region of the Ktn domain, whereas M558R lies immediately adjacent to the GSH-binding pocket. The data represent the first order rate constant for potassium efflux from cells elicited by exposure to NEM (unpublished data).

Citation: Booth I. 2011. Microbial Channels: Forbidden Fruit from Missense Rather than Nonsense, p 143-152. In Maloy S, Hughes K, Casadesús J (ed), The Lure of Bacterial Genetics. ASM Press, Washington, DC. doi: 10.1128/9781555816810.ch15
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