Biochemical analysis of a novel lipolytic enzyme YvdO from Bacillus subtilis 168.

The predicted amino acid sequence of Bacillus subtilis yvdO exhibits similarity to that of the proteins belonging to the patatin family of lipolytic enzymes. In the present study, YvdO was overproduced in Escherichia coli and purified, and its enzymatic properties were determined. YvdO hydrolyzed p-nitrophenyl fatty acid esters. The enzyme was specific to middle-chain fatty acids, and its optimum pH was approximately 7.5. It maintained 86% of its initial activity after incubation for 30 min at 80 degrees C, and its secondary structure was retained at up to 80 degrees C. Free myristic acid was detected as the product of the reaction with YvdO and 1-myristoly-2-lyso-sn-glycero-3-phosphocholine, while YvdO did not hydrolyze 1,2-dimyristoly-sn-glycero-3-phosphocholine. These results suggest that YvdO is a novel thermostable lipolytic enzyme that has the ability to hydrolyze lysophospholipids.

Bacillus subtilis spore coat protein LipC is a phospholipase B.

In Bacillus subtilis, the germination-related lipase LipC is located in the spore coat, and mutant spores are defective in L-alanine-stimulated germination. To determine the physiological role of LipC, the recombinant LipC expressed in Escherichia coli was purified and characterized. The enzyme hydrolyzes p-nitrophenyl ester substrates with various acyl-chain lengths. Thin-layer chromatography and gas chromatography-mass spectrometry analysis indicated that LipC cleaves the fatty acids at the sn-1 and sn-2 positions of phospholipids as phospholipase B, and that the enzyme shows no selectivity for the polar head groups of lipid molecules. When the amounts of free fatty acids in dormant wild-type and lipC mutant (YCSKd) spores were measured, the amount of free fatty acids in the YCSKd spores was about 35% less than in the wild-type spores. These results suggest the possibility that Bacillus subtilis LipC plays an important role in the degradation of the outer spore membrane during sporulation.

Physiological role of carbon dioxide in spore germination of Clostridium perfringens S40.

Germination of Clostridium perfringens is known to be triggered by nutrients such as l-alanine and inosine, and facilitated by CO2, however the role of CO2 has not been fully understood. During the studies of the germination-specific protease GSP, we found that CO2 could be replaced by bicarbonate or weakly acidic pH (pH 6.0-6.5). We also found that the spores obtained from the C. perfringens S40 overproducing GSP could germinate without CO2. Moreover, the spores could germinate in the absence of nutrients, when the spores were incubated with bicarbonate or under weakly acidic pH. GSP, which might consist of three homologous proteases, CspA, CspB, and CspC, is one of the key enzymes involved in the spore germination, and converts the pre-mature form of the spore cortex-lytic enzyme, SleC, to the mature form. Maturation of SleC in the spores obtained from the mother strain of C. perfringens S40 requires nutrients plus bicarbonate or weakly acidic pH. In contrast, mature SleC was found in the spores obtained from the cells overpoducing GSP, when the spores were treated by nutrients, bicarbonate or weakly acidic pH. Each nutrients, bicarbonate and weakly acidic pH can trigger the germination of the spores obtained from C. perfringens cells overproducing GSP.

Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40.

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.

A novel lipolytic enzyme, YcsK (LipC), located in the spore coat of Bacillus subtilis, is involved in spore germination.

The predicted amino acid sequence of Bacillus subtilis ycsK exhibits similarity to the GDSL family of lipolytic enzymes. Northern blot analysis showed that ycsK mRNA was first detected from 4 h after the onset of sporulation and that transcription of ycsK was dependent on SigK and GerE. The fluorescence of the YcsK-green fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the ycsK gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of ycsK spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in Escherichia coli showed lipolytic activity. We therefore propose that ycsK should be renamed lipC. This is the first study of a bacterial spore germination-related lipase.

Expression of germination-related enzymes, CspA, CspB, CspC, SleC, and SleM, of Clostridium perfringens S40 in the mother cell compartment of sporulating cells.

In Clostridium perfringens S40, spore germination-specific enzymes are synthesized during sporulation. Previous reports have demonstrated that two cortex-lytic enzymes, SleC and SleM, and a component of germination-specific protease, CspC, are located outside the cortex as an integral part of the dormant spore. In the present study, we examined the time and compartment of these enzymes' gene expression using reverse transcription-PCR (RT-PCR) and fluorescence microscopy on green fluorescence protein (GFP)-fused proteins. These results suggested that CspABC, SleC, and SleM are synthesized in the mother cell compartment of sporulating cells, probably at stages II approximately III of sporulation, and that the expression of cspABC genes is tricistronic.

Subcellular localization of a germiantion-specific cortex-lytic enzyme, SleB, of Bacilli during sporulation.

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB1-108-GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB1-108-GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic delta-lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB1-45-GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic delta-lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the delta-lactam structure of the cortex, not the formation of normal coat layer.

Hydrolysis of cortex peptidoglycan during bacterial spore germination.

Despite the most extreme dormancy and resistance properties among living systems, bacterial endospores retain an alert sensory mechanism to respond to the germinants and initiate germination. Although the molecular mechanism of the germination process is not completely described, current progress in the studies on the enzymes involved in the process gave us a somewhat clearer picture of the process of spore peptidoglycan (cortex) hydrolysis, a major biochemical event in germination. Germination-specific cortex-lytic enzymes require muramic acid d-lactam in their substrates. At least two types of enzymes are involved in the germination process: a spore cortex-lytic enzyme (SCLE) and a cortical fragment-lytic enzyme (CFLE). Except for their peptidoglycan-binding regions, the primary structures of SCLE and CFLE vary according species. Both enzymes differ in their hydrolytic bond-specificities and recognition of the substrates morphology. SCLE appears to initiate germination by uncrosslinking the intract cortex, and the CFLE further degrades the polysaccharide moiety of the SCLE-modified cortex. In vivo CFLE activity is likely regulated by its requirement for partially un-crosslinked cortex, while SCLE requires activation process. Clostridium perfringens SCLE is activated by a germination-specific serine protease during germination, but the activation mechanism of SCLE in Bacillus species is unknown. Cortex-lytic enzymes are expressed at the early stage of sporulation but the compartment of expression depends on proteins. However, all enzymes are located outside the cortex layer in dormant spores, suggesting that the hydrolysis process initiates at the exterior side of the cortex. The assembly of the germination apparatus is also discussed.