Cortex Hydrolysis Research Articles

In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168.

Germination of endospores of Bacillus subtilis involves the activities of several germination-specific lytic enzymes, including glucosaminidase and lytic transglycosylase. Another non-hydrolytic activity, likely to be due to an epimerase, also occurs. The effect of pH on enzyme activities and the overall germination rate was measured. Optimal germination occurred between pH 7-9; however, optimum glucosaminidase and epimerase activities were noted at pH 5. Conversely, the lytic transglycosylase activity was greatest at pH 8. Treatment of spores (15 min) with heat (90 degrees C) or NaOH (0.25 M) led to impaired cortex hydrolysis/modification, but with <20% loss in viability. Analysis of muropeptides in the germination exudate revealed a reduction of >85% in glucosaminidase and epimerase products, when compared to untreated spores. Conversely, lytic transglycosylase activity was increased by alkali or heat treatment, which was possibly due to increased substrate availability. FB101 (sleB) spores, which lack lytic transglycosylase activity, showed 90-fold greater loss in viability than the wild-type after 1 h at 90 degrees C. Similarly, 97% of FB101 (sleB) spores were unable to form a colony on nutrient agar after 130 min exposure to 0.25 M NaOH at 4 degrees C, whereas the wild-type was unaffected. This demonstrates the crucial role of the lytic transglycosylase in cortex hydrolysis of damaged spores. The respective targets of heat and alkali in spores and their role during germination are discussed.

Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate.

Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca(2+)-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca(2+)-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca(2+)-DPA. Our findings thus define a mechanism for Ca(2+)-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.

A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterization and nucleotide sequence of the corresponding gene, sleL.

The exudate of germinated spores of B. cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330-5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of N-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL.

The role of peptidoglycan structure and structural dynamics during endospore dormancy and germination.

Dormant, bacterial endospores are the most resistant living structures known. The spore cell wall (cortex) maintains dormancy, core dehydration, and heat resistance. The cortex peptidoglycan has a unique, spore specific structure that enables it to fulfill its role. The cross-linking index of spore cortex peptidoglycan is very low, occurring at only 2.9% of the muramic acid residues compared to 33% in vegetative cells. The level of cross-linking of the cortex may be important in maintaining spore dormancy and heat resistance. Approximately 50% of the muramic acid residues in spore cortex are substituted with muramic delta-lactam. This modification is spore specific and is the major characteristic feature of the cortex. The muramic delta-lactam has no apparent role in establishing core dehydration, maintaining dormancy or heat resistance. However, the muramic delta-lactam residues are necessary for spore cortex hydrolysis during germination. They constitute part of the substrate recognition profile of the germination specific lytic enzymes (GSLEs) which are responsible for cortex hydrolysis. Germination results in loss of dormant spore properties and hydrolysis of the cortex is essential for later germination events and outgrowth. Application of muropeptide analysis to determine peptidoglycan structural dynamics during germination has revealed an unexpected degree of complexity in peptidoglycan hydrolysis. At least three hydrolytic activities, an N-acetyl glucosaminidase, a lytic transglycosylase and a possible amidase, are involved. A non-hydrolytic activity, likely to be an epimerase of muramic acid also occurs early during germination. The lytic transglycosylase generates anhydro-muropeptides which are released during germination and may be recycled during outgrowth to form part of the new vegetative cell wall.

Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores.

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic delta-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.

細菌胞子発芽の分子機構 コルテックス分解過程を中心にして

細菌胞子発芽の分子機構 コルテックス分解過程を中心にして

Autolysins during sporulation of Bacillus subtilis 168

Conditions for zymographic detection of a 41-kDa spore cortex hydrolysis-specific autolysin, A6, from Bacillus subtilis 168 were optimised. A6 was present during sporulation from stages II–IV and remained active in the dormant spore. Its expression was controlled by the mother cell-specific early-sporulation sigma factor σ E. The characteristic muramic acid δ-lactam of spore cortical peptidoglycan was not necessary for cortex hydrolysis by A6, but it may be important in the inability of the major vegetative autolysin LytC to digest wild-type cortex. Two other minor autolysins were also observed during sporulation. The possible physiological significance of these observations is discussed.

The use of inhibitors to identify early events during Bacillus megaterium KM spore germination.

The germination response of spores of Bacillus megaterium KM, as measured by loss of A600, is more than 95% inhibited by 1 mM-HgCl2. Two Hg2+-sensitive sites (referred to as 'sites I and II') have been identified during germination. Site I represents a pre-commitment event and can be protected from HgCl2 by 50 mM-D-alanine, whereas site II represents a post-commitment event and is not D-alanine-protectable. At 1 mM-HgCl2, 25% of the spore population becomes committed to germinate, but an A600 loss of less than 5% occurs. In this system, loss of heat resistance was associated with commitment, whereas selective cortex hydrolysis, release of pyridine-2,6-dicarboxylic acid, Zn2+ and soluble peptidoglycan, as well as loss of refractility, were identified as post-commitment events. The commitment event was reversibly inhibited by several proteinase inhibitors and a membrane bulking agent. A model of spore germination based on these results is presented.

Collapse of cortex expansion during germination of Bacillus megaterium spores.

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast-plus-cortex, C/(P+C), of the CdCl2-germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose-induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.

Role of uricase in the triggering of germination of Bacillus fastidiosus spores.

The likelihood that uric acid was the only compound capable of triggering germination of Bacillus fastidiosus spores was reinforced by the finding that ureidoglycollic acid, urea, NH4Cl, 2,8-dihydroxypurine and a combination of L-alanine and O-carbamoyl-D-serine were ineffective as germinants. Uric acid-triggered germination of B. fastidiosus was prevented by a range of inhibitors that also inhibited uricase activity in dormant spore extracts. O2 uptake during germination started immediately after addition of uric acid, possibly as a consequence of the oxidation of uric acid by the enzyme uricase. Germination showed a dependence on uric acid concentration, with a relatively high Km (4-5 mM). During the first 10 min of germination of heat-activated spores there was no detectable change in the number of spore-cortex reducing groups, indicating that selective cortex hydrolysis is not involved in the trigger mechanism of germination of B. fastidiosus. On the basis of the results, a model is proposed in which re-initiation of uricase activity is the mechanism by which B. fastidiosus spores are triggered to emerge from the dormant state.

Inhibition of cortex hydrolysis during spore germination by CdCl2.

When the spores of Bacillus megaterium QM B1551 (ATCC 12872) were incubated with 5 mM CdCl2 at 30 C, they underwent the early germination events, such as loss of heat resistance and release of calcium dipicolinate, in the same way as when they were germinated by glucose + KNO3. However, germination by CdCl2 caused no increase in the reducing groups in the cortex and no excretion of glucosamine-containing materials due to the hydrolysis of the cortex peptidoglycan. Addition of CdCl2 at any time during germination by glucose + KNO3 inhibited the release of glucosamine-containing materials from the spores, whereas removal of cadmium from the CdCl2-germinated spores by treatment with cysteine restored the hydrolysis of peptidoglycan. These results suggested that CdCl2 caused the early events of spore germination but prevented the spores from undergoing the events following germination by inhibiting the enzymatic lysis of the cortex peptidoglycan. The conclusion from the study is that cortex degradation is not always required for the initiation of germination.

The role of cortex hydrolysis in the triggering of germination of Bacillus megaterium KM endospores

The appearance of new cortical reducing groups has been measured in relation to other germination events in Bacillus megaterium KM in order to test the hypothesis that spore germination is a result of selective cortex hydrolysis. New reducing groups were detectable from 2 min after the additin of germinants at 30°C whereas spore metabolism as measured by net ATP synthesis and irreversible tritium incorporation from tritiated water started at least 1 min later. Since triggering of B. megaterium KM spore germination takes place in the absence of both general spore metabolism and germinant metabolism, cortex hydrolysis may be concluded to represent the first detectable enzyme catalysed event during the triggering of spore germination. The new reducing groups were identified as the sodium borotritide reduction products of muramic acid and muramic acid δ-lactam which arise as the result of glycan chain hydrolysis and the uncrosslinking of cortex peptidoglycan, respectively. An average peptidoglycan chain length of 310 disaccharides was calculated from the ratio of reducing to non-reducing muramic acid in isolated pure cortex. These results provide support for a model of spore germination in which cortex hydrolysis is the first detectable metabolic event.