Factors Influencing Germination of Bacillus subtilis Spores via Activation of Nutrient Receptors by High Pressure

Abstract

Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl 2 .

Cooperativity Between Different Nutrient Receptors in Germination of Spores of Bacillus subtilis and Reduction of This Cooperativity by Alterations in the GerB Receptor

“…The fall in OD 600 in the absence of germinants was Յ7%/h. This latter fall in OD 600 was not due to spore germination, as determined by either flow cytometry (3,28) or phase-contrast microscopy, but was likely the result of spore aggregation and adsorbtion to test tube walls. The degree of spore germination was also assessed in many experiments by examination of spores by phasecontrast microscopy.…”

“…Spore preparations were free (Ն98%) of growing or sporulating cells, cell debris, or germinated spores as seen by phase-contrast microscopy and in some cases by flow cytometry after staining with Syto 16 (Molecular Probes, Eugene, OR) (3,28). Spore germination.…”

“…The degree of spore germination was also assessed in many experiments by examination of spores by phasecontrast microscopy. In some cases, the percentage of spore germination at various times was assessed by flow cytometry after staining of spores with Syto l6 (3,28). This assay was particularly useful for assessing very low rates of spore germination (3).…”

“…In some cases, the percentage of spore germination at various times was assessed by flow cytometry after staining of spores with Syto l6 (3,28). This assay was particularly useful for assessing very low rates of spore germination (3). In other experiments, the OD 270 of the supernatant fluid from germinating spore cultures was measured to assess the percentage of the spore's large depot of DPA that had been released, as described previously (4).…”

“…Rates of spore germination were routinely calculated from measurements of the fall in the OD 600 of cultures as described previously (4), taking a 60% fall in the OD 600 of as 100% germination. When germination rates were very slow, data from flow cytometry were used to calculate maximum rates of germination as described previously (3). All rates of germination presented are the averages of at least duplicate experiments, each of which gave a rate that was ՅϮ9% of the average value.…”