Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination

Abstract

The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB -in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme's hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD 600 fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore's ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination : the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

Subcellular Localization of a Germiantion-specific Cortex-lytic Enzyme, SleB, of Bacilli during Sporulation

“…The fluorescence of SleB 1-108 -GFP was localized around the spore's periphery, as about 75% of the dormant spores were surrounded by SleB 1-108 -GFP. Previous works have shown that SleB is translocated by a secretion signal peptide from the forespore compartment and located in the spore cortex of the dormant spore (Moriyama et al, 1999, Chirakkal et al, 2002. Thus, the peripheral location of SleB 1-108 -GFP suggests the deposition of the fusion protein in the spore cortex synthesized between the inner and the outer forespore membranes, which is consistent with that of wildtype SleB.…”

“…From the results that SleB could not be detected immunochemically in the spore ingredients of ypeB mutant, Chirakkal et al concluded that YpeB might be required for the localization and/or stabilization of SleB (Chirakkal et al, 2002). However, our results suggested that SleB after translocation across the inner forespore membrane could be localized in the spore cortex mediated by the interaction between the peptidoglycan binding motif of SleB and the muramic δ-lactam in the spore cortex.…”

In Vitro Studies of Peptidoglycan Binding and Hydrolysis by the Bacillus anthracis Germination-Specific Lytic Enzyme SleB

“…The only muropeptides detected in supernatants untreated by muramidase were the anhydromuropeptides aG7a and aG7b ( Fig. 3E and F and Table 3), which were the only two products previously found to depend on in vivo SleB action (6,8,16,17). Exposure to SleB 125-253 led to an almost imperceptible decrease of insoluble cortex ( Fig.…”

Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores

“…Following the activation of these receptors, spores release dipicolinic acid, calcium and amino acids (Moir, 2006;Setlow, 2003). Subsequently, the core of the spores becomes hydrated, and the spore cortex and sporespecific proteins are hydrolysed (Chirakkal et al, 2002;Sanchez-Salas et al, 1992;Setlow & Waites, 1976). Approximately 30 min after addition of germinants, the newly formed vegetative cells start to divide (Levinson & Hyatt, 1966;Setlow, 2003).…”