subtilis from the native spoVAEa locus and super resolution rescan confocal microscopy (RCM) was used to analyze SpoVAEa-SGFP2 location in spores.Īs mentioned above, GRs are present in foci in the dormant spore IM, and our previous work found that B. In the current work, we created a SpoVAEa-SGFP2 reporter protein. In contrast, by averaging hundreds of consecutive images, previous work indicated that SpoVAEa seems uniformly distributed throughout the IM 17. ![]() Notably, all GRs in spores are in a complex in the IM that is generally present in only one or two immobile spots/spore termed germinosomes 14, 15, 16, 17. subtilis spores have an estimated level of ~ 7000 molecules of SpoVA proteins, which is 7–10 times higher than the level of GRs 13. Somehow there must be signal transduction from activated GRs to the SpoVA channel, but while there is some evidence for physical interaction between GRs and some SpoVA proteins 6, 12, how signal transduction takes place is not clear. SpoVAD and -Ea are hydrophilic proteins, located on the outer surface of the IM. SpoVAA, -B, -C, -Eb and -F are transmembrane IM proteins, one of which, SpoVAC, is a pressure-sensitive membrane channel protein 6, 7, 10, 11. The SpoVA channel has seven subunits, A, B, C, D, Eb, Ea and F 6, 9. Multiple signals can trigger the opening of the SpoVA channel, including activated germinant receptors (GRs), hydrolysis of the cortex PG and high hydrostatic pressure 2. When the latter is complete the core water content has returned to the levels observed in vegetative cells 2, 8. Upon release of the Ca 2+-DPA, water is taken up and hydrolysis of the PG cortex begins. ![]() This channel functions to release the large pool of the 1:1 complex of Ca 2+ and dipicolinic acid (Ca 2+-DPA) from the spore core during germination 5, 8. The SpoVA channel is located in the IM, and has a crucial role in spore germination 3, 4, 5, 6, 7. Despite their potential long period of dormancy, these spores can be revived or germinated by many environmental cues, primarily small molecules termed germinants that signal the presence of a growth friendly environment, and they then resume cell growth 2. From the outside in, these spore layers are the coat, outer membrane (OM), peptidoglycan (PG) cortex, germ cell wall, inner membrane (IM) and core 2. Spores in particular are capable of maintaining metabolic dormancy for very long times by protecting their chromosomal DNA through its location in the low water environment of the spore core and its surrounding by multiple protective macromolecular layers 2. Notably, it can generate different subsets of cells, such as persisters, spores, and biofilms to promote survival in harsh environmental conditions 1. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.īacillus subtilis, the model Gram-positive bacterium, has multiple complex responses to environmental stress and nutrient depletion. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 ☌ treatments induce the appearance of phase-grey-like spores. We also tested the response of SpoVAEa and the IM to thermal treatments at 40–80 ☌. These observed events were highly related to spores’ rapid phase darkening, which is considered as due to rapid Ca 2+DPA release. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. ![]() The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. ![]() It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. However, while the GRs’ spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca 2+-DPA from the spore core during germination. The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores.
0 Comments
Leave a Reply. |