Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Abstract

Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB N ). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro. Members of the bacterial genera Bacillus and Clostridium form environmentally resistant spores in response to nutrient starvation. Subsequent exposure of spores to conditions that are amenable to vegetative growth and metabolism triggers a series of germination reactions that result ultimately in the emergence of new vegetative cells (1, 2). A major germination event concerns depolymerization of the thick layer of cortical peptidoglycan that surrounds the spore protoplast. The cortex serves to maintain the relatively dehydrated status of the spore core (27 to 55% water by weight versus 70 to 80% water in vegetative cells [3]), and its removal by lytic enzymes is essential for hydration of the core to levels that permit protein mobility and the resumption of metabolic activity (4). The cortex is composed of structurally distinct peptidoglycans comprising linear chains of alternating N-acetylglucosamine and N-acetylmuramic acid (NAM) residues, the latter characterized by enzymatically processed side chains that yield either single L-alanine residues (in ϳ25% of NAM residues) or the spore-specific muramic acid lactam (MAL; prevalent in ϳ50% of NAM residues) (5, 6). Both modifications result in reduced availability of peptide side chains for cross-linking of glycan chains in cortical peptidoglycan, conferring a peptidoglycan sacculus that is relatively loosely cross-linked compared to vegetative cell sacculi (7).The ...

Structural and functional analysis of SleL, a peptidoglycan lysin involved in germination of B acillus spores

“…Whereas the former hydrolyses (NAG) 2 units in a processive manner from the reducing end of chitin, which is a NAG polymer, SleL cleaves PG that contains NAM and MδL moieties in addition to NAG . Consideration of the products generated by SleL activity against spore sacculi partially digested with the lytic transglycosylase SleB—which include tetrasaccharide (MδL‐NAG‐NAM‐NAG) and anhydro‐trisaccharide (MδL‐NAG‐anhydroNAM) derivatives—indicates that NAG must occupy at least the +2 and −1 binding subsites. NAG may also occupy the +4 and −3 positions, although this will depend on the position that products are released from the active site.…”

“…Genes encoding SleL were amplified by PCR using genomic DNA from B. cereus ATCC 10876 (NCBI accession number EEK49693) and B. megaterium QM B1551 (NCBI accession number ADE67156). PCR fragments encoding entire open‐reading frames were cloned using a combined ligation‐independent cloning (LIC) and vector backbone exchange (VBEx) protocol to construct pNZ‐SleL derived plasmids for expression in Lactococcus lactis DML1, essentially as described previously . Intermediate pRE‐SleL plasmids were used as templates for site‐directed mutagenesis procedures, which were conducted using a QuikChange Lightning Site‐Directed Mutagenesis kit (Agilent Technologies, Wokingham, UK).…”

“…All SleL and variant proteins were expressed in L. lactis essentially as described previously, inducing with nisin (1 µg/L) when the optical density (A660 nm) of the cultures reached 0.6, with expression for 6 h before harvesting the cells. Cell pellets were resuspended in 16 mL of ice‐cold breakage buffer (20 m M NaH 2 PO 4 [pH 7.4], 500 m M NaCl, 1 m M PMSF, and 20 m M imidazole) and then the cells disrupted by four passes through a One‐Shot cell disrupter (Constant Systems, Northampton, United Kingdom) operating at 40 × 10 3 lb/in 2 .…”

“…Enzymatic activity of the various forms of SleL was assessed by incubating purified enzymes with B. subtilis spore PG sacculi (purified as described previously), suspended at an optical density (A600 nm) of 0.5 in 5 m M Tris‐HCl (pH 7.8) plus 1 m M DTT at 25°C. Sacculi were digested with recombinant B. megaterium SleB (0.5 µ M ) for 40 min, and then SleL proteins added to the suspensions in 96‐well plates at a final concentration of 1 µ M .…”

“…A third CLE common to Bacillus species, SleL, has been characterized as an N ‐acetylglucosaminidase, with catalytic activity against cortical fragments generated by SleB and CwlJ activity . Despite its characterisation as a cortical fragment lytic enzyme (CFLE), SleL can seemingly initiate cortex hydrolysis in B. megaterium sleB cwlJ double mutant spores, as long as YpeB, a non‐lytic protein with unknown function, is also present . However, in vitro analyses conducted with purified spore PG sacculi indicate that SleL is a CFLE; hence its role in initiating cortical hydrolysis together with YpeB in the absence of SleB and CwlJ in vivo is not clear.…”