Monitoring the Kinetics of Uptake of a Nucleic Acid Dye during the Germination of Single Spores of Bacillus Species

Abstract

Dormant bacterial spores do not take up and bind nucleic acid dyes in the spore core but readily take up such dyes when they are fully germinated. We present a methodology that combines fluorescence microscopy, phase contrast microscopy, and laser tweezers Raman spectroscopy to monitor the kinetics of uptake of the nucleic acid dye SYTO 16 during germination of individual Bacillus cereus and Bacillus subtilis spores. The level of dye bound to nucleic acids of individual spores was measured by fluorescence emission, while changes in spore refractility and the level of the 1:1 chelate of dipicolinic acid and Ca(2+) (CaDPA) were monitored by phase contrast microscopy and Raman spectroscopy, respectively. The results obtained include (1) during nutrient germination, SYTO 16 began to enter the spore core and bind to nucleic acids just when spores had released all CaDPA and continued until hydrolysis of spores' peptidoglycan cortex was complete; (2) during germination with exogenous CaDPA, rapid SYTO 16 uptake began only 2-7 min after complete release of endogenous CaDPA for both B. cereus and B. subtilis spores; (3) the rate but not the timing of dye uptake and the maximum level of dye bound to nucleic acid were increased during nutrient germination of B. subtilis spores lacking ~75% of the DNA binding proteins that normally saturate dormant spore DNA; (4) SYTO 16-DNA binding was not observed during nutrient germination of B. subtilis spores lacking the protease that degrades spores' DNA binding proteins, even after cortex hydrolysis; (5) SYTO 16 uptake by germinating B. subtilis spores lacking the cortex-lytic enzyme (CLE) CwlJ was low, again even after cortex hydrolysis, although SYTO 16 uptake by germinating spores lacking the other redundant CLE SleB was even higher than in germinating wild-type spores; and (6) there was no SYTO 16 uptake by germinating spores that lacked both CwlJ and SleB, even after CaDPA release. These results suggest that during spore germination SYTO 16 uptake is minimal until CaDPA has been released and DNA binding proteins have been degraded and further that CLEs' degradation of the spore cortex plays a crucial role in uptake of this dye.

Effects of Cortex Peptidoglycan Structure and Cortex Hydrolysis on the Kinetics of Ca 2+ -Dipicolinic Acid Release during Bacillus subtilis Spore Germination

“…However, it was also observed with wild-type spores that a high EDTA concentration lowered the ⌬T release values of individual spores germinating with L-valine almost 3-fold (Table 4). This is consistent with a role for CaDPA released from an individual spore in triggering cortex hydrolysis by CwlJ and thus further stimulating the rate of CaDPA release from that spore, since DPA is not as effective in activating CwlJ as CaDPA (7,11,13,20). Effects of cortex structure or degradation on kinetic parameters of individual spores' germination with dodecylamine (Table 5) were generally similar to those with L-valine.…”

“…The effects of CwlJ and SleB on the ⌬T release values of individual B. subtilis spores germinating with nutrient germinants have been seen previously (7,13,20). However, it was also observed with wild-type spores that a high EDTA concentration lowered the ⌬T release values of individual spores germinating with L-valine almost 3-fold (Table 4).…”

“…Previous work has found that cwlJ and cwlJ sleB spores germinate more slowly than wild-type spores with nutrient germinants and in particular take significantly longer to release CaDPA, while sleB spores germinate essentially identically to wildtype spores (7,13,20). However, the germination of CLEdeficient spores with the nonnutrient germinant dodecylamine has not been studied.…”

“…The most dramatic initial event in spore germination triggered by both types of germinants is the release of the dormant spores' large CaDPA depot. This release generally begins after a lag period of many minutes, the precise timing of which is quite variable for individual spores in populations, although total CaDPA release generally takes only a few minutes once it has begun (4,6,7,13,25,29,32,33). CaDPA release is followed by degradation of the spore cortex, and with spores of Bacillus species this degradation is carried out by two cortex-lytic enzymes (CLEs), CwlJ and SleB, that are redundant in that either enzyme can catalyze cortex hydrolysis and thus completion of spore germination.…”

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

“…Dormant spores are known to appear phase bright, whereas fully germinated spores are associated with a phase dark state [42]. For germinated spores, the loss of refractivity is associated with full DPA release and protoplast hydration [43]. It could be seen that an increase in ethanol concentration is associated with an increase of the number of wild type and cotE gerE spores that appear phase grey (Fig.…”