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.

Necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens that results in significant economic losses, averaging damage of $0.05 per bird. The present study investigated the influence of a dietary supplement, Bacillus subtilis PB6, on performance, intestinal health, and gut integrity against C. perfringens-induced NE in broiler birds. Bacillus subtilis PB6 (ATCC-PTA 6737) is a natural strain isolated from healthy chicken gut that has been shown in in vitro to produce antimicrobial substances with broad activity against various strains of Campylobacter and Clostridium species. The animal study was conducted on broiler chickens (Cobb 400) for the period of 35 d using a completely randomized design. The experimental design included 3 treatments groups. Each treatment group contained 6 replicates, 3 male and 3 female, with 12 birds in each replicate. The 3 treatment groups were an uninfected control, an infected control, and an infected group supplemented with B. subtilis PB6 at 500 g/t of feed, containing 5 × 10(11) cfu/kg. Necrotic enteritis was induced in the broiler birds via oral inoculation of 30,000 oocysts of mixed strains of Eimeria species on d 14 followed by C. perfringens (10(8) cfu/mL) on d 19 through 21 of trial. The birds were analyzed for BW gain, mortality, feed conversion ratio (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. The infected control group showed markedly thickened mucosa, hemorrhages, intestinal lesions, and ballooning of intestine. The supplementation of B. subtilis PB6 reduced the FCR (P < 0.05) and intestinal C. perfringens counts significantly (P < 0.05) compared with the infected control group. It was also observed that B. subtilis PB6 improved villi length by 10.88 and 30.46% (P < 0.05) compared with uninfected and infected control groups, respectively. The group supplemented with B. subtilis PB6 significantly (P < 0.05) increased the villi length to crypt depth ratio by 49.11% compared with the infected group. In conclusion, the supplementation of B. subtilis PB6 not only controlled C. perfringens-induced NE, but also improved intestinal health in the broiler birds.

The role of the sleB gene of Bacillus subtilis, which encodes a putative sporecortex-lytic enzyme, and the downstream ypeB gene were investigated. Both SleB and YpeB were required for normal germination to occur. The corresponding mutants formed phase-bright, heat-resistant spores with no apparent defects in dormancy. However, mutant spore suspensions lost optical density slower than the wild-type and spores were phase-grey even 12 h after the triggering of germination. Since the loss of heat resistance and release of dipicolinic acid was similar to the wild-type, these mutants were blocked in the later stages of germination. The mutants were nevertheless capable of outgrowth on rich agar to form colonies, indicating that other spore components can compensate for their function sufficiently to allow outgrowth. The expression and regulation of the operon was examined using a lacZ transcriptional fusion. Expression of the operon began 2 h after the onset of sporulation and was under the control of RNA polymerase containing the forespore-specific sigma factor, σ G . The application of reverse phase HPLC revealed that the mutants do not have any structural defect in the dormant spore cortex and therefore these genes are not required for normal sporecortex synthesis. The analysis of peptidoglycan dynamics during germination showed, however, that the cortex was only partially hydrolysed in both mutants. This analysis also revealed that the likely hydrolytic bond specificity of SleB is likely to be that of a lytic transglycosylase. Keywords :Bacillus subtilis, endospores, germination, peptidoglycan, cortex hydrolysis INTRODUCTIONThe formation of resistant dormant endospores is a survival strategy used by members of the genera Bacillus and Clostridium during nutrient deprivation. The spore cortex maintains heat resistance and dormancy (Ellar, 1978). The cortex consists of a thick layer of peptidoglycan with a unique spore-specific structure (Atrih et al., 1996 ;Popham et al., 1996a). Hydrolysis of the cortex peptidoglycan is essential for later germination events and outgrowth Atrih et al., 1998 ;Popham et al., 1996b).A number of spore-cortex-lytic enzymes have been isolated from spores of different organisms. Two lytic enzymes have been isolated from Clostridium perfringens S40 spores -a 31 kDa enzyme encoded by the gene sleC (Miyata et al., 1995) and a 38 kDa enzyme encoded by the gene sleM (Chen et al., 1997). The Bacillus cereus IFO 13597 gene sleB encodes a 24 kDa enzyme (Makino et al., 1994 ; Moriyama et al., 1996b), and its homologue has been identified and inactivated in Bacillus subtilis. The resulting mutant germinates slower than the wild-type (Moriyama et al., 1996a).Foster & Johnstone (1987) isolated a germinationspecific lytic enzyme (GSLE) that was capable of cortex hydrolysis from the spores of Bacillus megaterium KM. The enzyme was activated in vivo during germination and has a high specificity for intact spore cortex. Western blot analysis revealed cross-reactivity with proteins from spore fractions of...

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.Spore germination is initiated by the interaction of the germinant molecule with a receptor in the spore. The nature of this receptor is not yet proven, but the available evidence suggests that the genes of the gerA family whose products are required for the response to specific germinants are likely to encode this receptor (16,20). The trigger reaction commits spores to undergo a series of successive events which result in the loss of spore dormancy and resistance properties. Spores of Bacillus cereus initiate germination in response to L-alanine or ribosides, of which inosine is the most effective (8). Inhibition of the alanine racemase activity associated with spores by Ocarbamyl D-serine is necessary to observe maximum rates of L-alanine-triggered germination, as D-alanine is a competitive inhibitor (8). The first measurable event after commitment is the loss of heat resistance (a rise in spore internal pH, a release of monovalent ions, and a release of dipicolinic acid (DPA) and calcium ions from spores are also early events), and later events include the activation of spore lytic enzymes (7, 17)...

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors.