Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Simon J. Foster. Tel: +44 114 222 4411. Fax: +44 114 272 8697. e-mail: s.foster{at}sheffield.ac.uk
Keywords: Bacillus subtilis, peptidoglycan hydrolase, cell wall, vegetative growth, sporulation
a Present address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
b Present address: School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK.
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Overview |
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In this review, we consider Bacillus subtilis as a genetically amenable, nonpathogenic model system for investigating the roles of autolysins in the cell-wall metabolism of an endospore-forming Gram-positive bacterium. The various phases of the B. subtilis life cycle afford excellent opportunities to study all the above proposed functions of autolysins except those solely involved in pathogenicity.
Although autolysins associated with B. subtilis have been known for many years (Rogers et al., 1984 ), the study of their physiological roles has been hampered by their great number and functional redundancy (Smith et al., 1996
). However, by analysis of multiply inactivated mutants, study of peptidoglycan fine structure and, most recently, by sequencing of the entire B. subtilis genome (Kunst et al., 1997
), it has become possible to define the roles played by individual autolysins in a number of important cellular processes. Here, we first seek to draw together enzymological and genome sequence information to define the autolysin complement of B. subtilis. We then review the known roles of autolysins at each stage in the life cycle, before considering how the activity of these potentially lethal enzymes is controlled in vivo. The lytic enzymes associated with prophages within the B. subtilis 168 chromosome are also briefly discussed. These are not autolysins sensu stricto because they are not host-encoded, but they are relevant because there appears to have been exchange of peptidoglycan hydrolase genes between B. subtilis and its phages. Lastly, we consider what opportunities arise from recent advances for a better understanding of the complex roles played by the autolysins of B. subtilis.
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B. subtilis autolysins and their substrate |
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The autolysin complement of B. subtilis |
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LytD glucosaminidase family
This contains only LytD itself, which is one of the major autolysins of vegetative growth. Autolytic activity resides within the C-terminal region (aa 569880; Margot et al., 1994 ; Rashid et al., 1995b
). The catalytic domain of LytD is also homologous to the glucosaminidase domain (aa 7761256) of the bifunctional autolysin, Atl, of Staphylococcus aureus (Foster, 1995
; Oshida et al., 1995
).
DL-Endopeptidase I family
Bacillus sphaericus produces two endopeptidases of diverse primary structure that cleave the peptide bond between D-Glu and A2pm (Hourdou et al., 1992 , 1993
). The 45 kDa zinc-requiring, sporulation-related endopeptidase I of B. sphaericus belongs to a large family of zinc-active-site peptidases with a variety of functions (Hourdou et al., 1993
). Its catalytic domain (aa 101396) has one significant homologue in B. subtilis, YqgT, which constitutes the DL-endopeptidase I family (Table 1
) and may also be sporulation specific.
DL-Endopeptidase II family
This family comprises homologues of endopeptidase II of B. sphaericus (Hourdou et al., 1992 ), which are also related to the C-terminal 120 aa (the presumed autolytic domain) of the p60 autolysin of Listeria monocytogenes (Wuenscher et al., 1993
). The experimentally determined bond specificity of B. sphaericus endopeptidase II (Hourdou et al., 1992
) and LytF (Margot et al., 1999
; Ohnishi et al., 1999
) of B. subtilis suggests that all these enzymes are
-D-glutamyl-L-diamino acid endopeptidases that hydrolyse the peptide bond between D-glutamate and A2pm. All the members of this family from B. subtilis, as well as endopeptidase II and p60, have an apparently catalytically essential cysteine residue within a conserved DCS(G/S) motif (Hourdou et al., 1992
).
Lysostaphin family
Searching the B. subtilis genome with the central catalytic portion (aa 236383; Baba & Schneewind, 1996 ) of lysostaphin from Staphylococcus simulans (Recsei et al., 1987
) revealed the lysostaphin family of possible peptidoglycan hydrolases, which has three members, YomI, YunA and SpoIIQ. Lysostaphin is an endopeptidase that hydrolyses the pentaglycine moieties that cross-link the branch peptides of S. aureus peptidoglycan (Schindler & Schuhardt, 1964
). The pentaglycine moiety has not been detected in B. subtilis peptidoglycan and so some of the lysostaphin homologues in Table 1
may act as antibiotics to kill staphylococci. Others may have different substrate specificity from lysostaphin or act on very rare unusual cross-links that have not yet been detected. One member of this family, YomI, a large protein with two apparently unrelated potential autolytic domains, also belongs to the Slt70 family.
Germination-specific lytic enzyme (GSLE) family
The GSLE family was uncovered by searching with the GSLE, SleB, of Bacillus cereus. SleB of B. cereus is a probable amidase that is released by germinating spores and has a role in germination (Makino et al., 1994 ). However, SleB of B. subtilis is likely to be a lytic transglycosylase (Boland et al., 2000
).
LD-Endopeptidase family
Searching with the L-alanoyl-D-glutamate endopeptidase AepA of the L. monocytogenes phage A500 (Loessner et al., 1995 ) revealed a single significant homologue (YcdD) in B. subtilis, which constitutes the LD-endopeptidase family.
Slt70 family
Two homologues of the lytic transglycosylase Slt70 of E. coli constitute the Slt70 family. Slt70 is unique among autolysins in that its X-ray crystal structure has been determined (Thunnissen et al., 1994 ). YomI and YjbJ, the Slt70 homologues in B. subtilis, are similar to Slt70 within its C-terminal domain (aa 451618), which has a similar structure to lysozyme (a muramidase) and is believed to be the catalytic part of the molecule. The N-terminal 450 aa of Slt70, which may function in substrate binding (Thunnissen et al., 1994
), is not significantly homologous to YomI or YjbJ, which may therefore recognize their substrates in quite different manners.
Enterococcal muramidase family
This contains YubE, which is homologous to the presumed catalytic N-terminal 200 aa of muramidase-2 from Enterococcus hirae (Joris et al., 1992 ). Searching with the homologous sequence of AlyS, a putative muramidase from Enterococcus faecalis (Joris et al., 1992
), also revealed YpbE. YpbE is weakly homologous to both the N-terminal (presumed catalytic) and C-terminal (proposed cell-wall-binding) domains of AlyS, and so was tentatively assigned to the enterococcal muramidase family as well. There is a weak similarity between the muramidase-2 and AlyS and some 100 aa within the catalytic domain of the major vegetative glucosaminidase LytD (Rashid et al., 1995b
), suggesting some similarity between these two groups of hexosaminidases.
LrgB family
LrgB, a 233 aa putative autolysin from S. aureus (Brunskill & Bayles, 1996 ), which shows no detectable sequence similarity to other identified families of enzymes, has three homologues in B. subtilis, which form the LrgB family.
XlyA amidase family
A family of seven potential peptidoglycan hydrolases was revealed by searching with the amidase XlyA of the B. subtilis prophage PBSX (Longchamp et al., 1994 ). This class of probable amidases is related to the amidase domain (aa 199775) of the bifunctional S. aureus autolysin Atl (Foster, 1995
; Oshida et al., 1995
) and the amidase domain (aa 31261) of the Ami amidase of L. monocytogenes (McLaughlan & Foster, 1998
; Braun et al., 1997
), but they show no detectable primary structure relationship to the amidases of the LytC class. As detailed below, several of these may be involved in cell lysis following prophage induction.
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Accuracy of assignment and general features of the autolysin complement |
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Many peptidoglycan hydrolases, from B. subtilis and other organisms, have noncatalytic domains, often containing tandem repeats, that are involved in binding to the cell wall (Joris et al., 1992 ; Wuenscher et al., 1993
; Lazarevic et al., 1992
; Ghuysen et al., 1994
; Baba & Schneewind, 1998
). The precise molecular nature of this recognition has yet to be elucidated, but it appears that anionic secondary polymers may be important (Lazarevic et al., 1992
). This modular design may have allowed interchange of catalytic and binding domains among autolysins and other wall-associated proteins during evolution. For instance, three members of the DL-endopeptidase II family (LytE, LytF and YojL) from B. subtilis are homologous within their N-terminal, probable wall-binding regions, to the putative wall-binding domain (aa 201666) of muramidase-2 from E. hirae (Joris et al., 1992
), although they have no detectable sequence similarity to the proposed catalytic domain of muramidase-2. YwtD, another member of the endopeptidase II family, strangely appears to have the endopeptidase II catalytic motif repeated three times, but only the central one has the catalytically important Cys. Maybe this domain is catalytic and the rest are involved only in substrate recognition. YocH, which is homologous to the amidase XlyA, also has repeats homologous to those of the endopeptidase II family but lacks the Cys at the proposed active centre of those enzymes.
A few B. subtilis gene products show significant similarity only to apparently non-catalytic, probable wall-binding parts of known autolysins. Some or all of these proteins may be wall-associated but not autolysins. Several wall-associated non-autolysins appear to modulate autolysin activity, such as LytB, which enhances LytC activity (Lazarevic et al., 1992 ), and WapA, which modulates the effect of autolysin mutations on motility (Blackman et al., 1998
). Also, SpoIID, which is homologous to LytB and the wall-binding domain of LytC, may also have a role in autolysin activation (Kuroda et al., 1992
; Lazarevic et al., 1992
; Illing & Errington, 1991
).
Most wall-associated proteins, including autolysins, are expected to have high pI, since they are associated with the negatively charged cell wall. As can be seen in Table 1, most known autolysins are basic proteins, but we do not consider high estimated pI as a criterion for discounting a potential autolysin without further experimental evidence. Such a protein may still have access to the cell wall by being either membrane-associated or one of the so-called periplasmic proteins that are extracytoplasmic cell-associated proteins that remain bound to the cell wall when the cells are broken (Merchante et al., 1995
). Likewise, absence of a detectable signal sequence does not necessarily suggest that a particular enzyme is not secreted and so does not have access to the cell wall, as other mechanisms of secretion exist (Kuchler, 1993
). For instance, CwlC, the major amidase during late sporulation, has no signal sequence but has been shown to be present in the cell wall (Kuroda et al., 1993
; Smith & Foster, 1995
).
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Roles of autolysins |
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The mature glycan strands can have MurNAc at the nonreducing terminus, suggesting that glucosaminidase activity has taken place (Atrih et al., 1999 ). This activity can be provided by LytD (Margot et al., 1994
; Rashid et al., 1995b
), which is the only glucosaminidase in the B. subtilis genome that is currently identifiable by sequence similarity. However, a lytD mutant still exhibits the glucosaminidase product in its peptidoglycan and so at least one other enzyme is responsible (A. Atrih & S. J. Foster, unpublished). Peptide side-chains terminating in D-glutamate suggest the action of a D-glutamate-meso-diaminopimelate endopeptidase, which may be among the two identified DL-endopeptidase families (Table 1
). The existence in vegetative cell walls of MurNAc residues with complete cross-link peptides severed from the other glycan strand beyond the distal L-alanine indicates amidase activity. The presence of anhydromuropeptides strongly suggests the action of one or more lytic transglycosylase (Atrih et al., 1999
).
Cell separation, motility and competence.
Wild-type B. subtilis cells exist as short chains or, in stationary phase, primarily as single cells. Cell separation is probably important in facilitating dispersion of cells and allowing them to chemotact towards nutritionally favourable environments. Many autolysin-deficient mutants show abnormally long chains without changes in the dimensions of individual cells. This suggests involvement of autolysins in digesting peptidoglycan from the middle of the division septum to allow daughter cells to separate. Five gene products are known to have roles in cell separation. These are the amidase LytC, the glucosaminidase LytD, two members of the DL-endopeptidase II family (LytE and LytF) and the putative autolysin YwbG. There is considerable functional redundancy among these proteins: where multiple mutants have been constructed, inactivation of more autolysins generally causes the bacteria to form longer chains of cells (Blackman et al., 1998 ; Ishikawa et al., 1998a
; Margot et al., 1998
, 1999
; Ohnishi et al., 1999
; G. Horsburgh & S. J. Foster, unpublished). It is still unclear whether the functional redundancy of the autolysins involved in cell separation is complete, or whether certain steps in the process can be performed by some proteins but not others.
Early discovered regulatory mutants deficient in autolysins were immotile and so it was suggested that these enzymes were required for motility (Pooley & Karamata, 1984a , b
). It is now clear that expression of the two major vegetative autolysins LytC and LytD, as well as the endopeptidase LytF, are largely coregulated with the genes required for flagellar motility and chemotaxis via the regulatory genes sigD and sinR (Lazarevic et al., 1992
; Rashid & Sekiguchi, 1996
; Margot et al., 1999
), strongly suggesting that these autolysins are involved in motility. Inactivation of the amidase LytC causes diminished swarming motility (Blackman et al., 1998
; Margot et al., 1994
; Rashid et al., 1995b
) and, under some conditions, the glucosaminidase LytD also has a role in this process (Blackman et al., 1998
; Rashid et al., 1995b
). The effect of these autolysin mutations on motility may be due at least partly to their effect on cell separation, which predominates in stationary phase, probably to facilitate motility. Uncoordinated chemotaxis of cells linked together in chains may produce less overall movement than when the cells can move separately, the pushmi-pullyu effect (Blackman et al., 1998
). The roles of other autolysins in motility, and the possibility that autolytic activity may be needed for extrusion of flagella through the cell wall (Dijkstra & Keck, 1996
) remain to be investigated. Likewise, the suggested involvement of autolysins in genetic competence, which was also inferred from pleiotropic mutants (Pooley & Karamata, 1984a
), has not yet been fully tested.
Cell expansion, cell-wall turnover and protein secretion.
Electron microscopy shows the wall as comprising three distinct layers (Graham & Beveridge, 1994 ). Pulse-labelling studies suggest an inside-to-outside flux of wall material, new wall being laid down along the cytoplasmic membrane and removed from the exterior by autolysins (Merad et al., 1989
; Pooley, 1976
). The inner zone of the wall may contain newly synthesized, unstressed peptidoglycan. As the cell elongates, this material passes outward and stretches, becoming the middle, stress-bearing zone. The outer zone may thus consist of old, partially hydrolysed peptidoglycan awaiting solubilization (Graham & Beveridge, 1994
). This inside-to-outside model of wall structural dynamics suggests that autolysins are necessary for hydrolysis of older peptidoglycan to allow newer peptidoglycan to expand and become stress bearing as the cell elongates, and to remove the oldest wall material from the outer surface (i.e. cell-wall turnover).
The inside-to-outside model for cell-wall growth suggests that breakage of covalent bonds within the peptidoglycan is essential for cell growth. Consistent with this, an early study found that exogenous addition of an autolysin-containing preparation could accelerate the growth rate (Fan & Beckman, 1971 ). However, since individual autolysin genes have been identified and inactivated, no single or multiple autolysin mutant has shown a serious defect in growth rate. In view of the functional redundancy observed with other autolysin functions, it may be that enzyme(s) capable of performing the essential hydrolytic events have already been inactivated, but suitable multiple mutants for detection of their roles in cell expansion have not yet been constructed. Alternatively, expansion of the cell may cause such great stress forces in the outer layers of the cell wall that critical covalent bonds are fractured without the need for enzyme-catalysed hydrolysis (Archibald et al., 1993
).
Although no link between autolytic activity and cell growth has been clearly established, the role of autolysins in cell-wall turnover has been convincingly proven. The major vegetative amidase LytC is important in this process. One study reported that its inactivation delayed the release of radiolabelled wall material in pulse-chase experiments without affecting the subsequent rate of turnover (Margot & Karamata, 1992 ), whilst another found that LytC inactivation caused cell-wall and septal thickening and a large decrease in the rate of turnover (Blackman et al., 1998
). Inactivation of lytD alone has no effect on turnover (Margot et al., 1994
), but absence of the glucosaminidase LytD further retards turnover in a lytC background. A minor autolysin controlled by
D, possibly the endopeptidase LytF, also functions in peptidoglycan turnover (Blackman et al., 1998
).
The cross-linked covalent network of peptidoglycan in bacterial cell walls can present a physical barrier to secretion of proteins, which may be lessened by the action of autolysins (Dijkstra & Keck, 1996 ). Preliminary results in our laboratory suggest that inactivation of lytC and/or lytD impedes secretion of proteins by B. subtilis (Blackman, 1998
). This effect may be at least partly due to the proposed requirement of cell-wall turnover for secretion of large proteins (Koch, 1995
).
Involvement of autolysins during differentiation
The differentiation processes of sporulation and subsequent germination require a number of rearrangements and modifications of cell-wall peptidoglycan that include at least four events in which autolysins are likely to be involved (Fig. 2b; Smith et al., 1996
). The first morphological change during sporulation is an asymmetric cell division (sporulation stage II) that produces daughter cells of unequal sizes. The smaller cell is the prespore, which develops into the endospore, and the larger compartment is the mother cell. The mother cell engulfs the prespore (sporulation stage III), which is thereafter known as the forespore. Two layers of peptidoglycan are laid down around the forespore (stage IV; Doi, 1989
). The forespore lays down the inner layer, which is the primordial cell wall; the outer layer, the cortex, is derived from the mother cell (Tipper & Linnett, 1976
). Proteinaceous coat layers develop around the forespore, which matures into a phase-bright, resistant endospore (stages V and VI). Finally, the mother cell lyses and releases the mature endospore (stage VII; Doi, 1989
). Despite its high level of resistance and quiescent state, the spore retains an alert sensory mechanism that can respond to specific germinants within minutes.
Asymmetric septum digestion.
The first stage at which autolysin activity appears necessary for sporulation is hydrolysis of the asymmetric septum to permit prespore engulfment. The autolysins involved have not been identified, but mutation of spoIID halts sporulation at stage IIb, when the septum is partially removed and remains as an annulus around the edge of the division plane (Illing & Errington, 1991 ). SpoIID is homologous to LytB, the modifier protein that enhances the activity of the major vegetative amidase LytC (Herbold & Glaser, 1975
; Lazarevic et al., 1992
; Kuroda et al., 1992
). Completion of asymmetric septum digestion may require activation of one or more autolysins by SpoIID. The lysostaphin homologue SpoIIQ also has a role in prespore engulfment, although apparently not in septum hydrolysis, because a spoIIQ-inactivated mutant is blocked after the septum has disappeared (Londoño-Vallejo et al., 1997
).
Cortex maturation.
The unique structure of cortical peptidoglycan, which is essential for maintaining the heat resistance and dormancy of the spore, indicates the action of autolysins in its maturation (Fig. 1b). About 50% of the disaccharide subunits in the cortex have the muramic acid
-lactam structure (Fig. 1b
), which is found only in bacterial endospores. The
-lactams are not randomly distributed, but occur predominantly at every alternate disaccharide. Two mechanisms have been proposed for
-lactam formation: amidase action followed by transacylation, or de-N-acetylation followed by transpeptidation (Tipper & Gauthier, 1972
). Inactivation of cwlD, which encodes a sporulation-specific putative amidase belonging to the LytC family (Table 1
), results in complete loss of the
-lactam structure (Atrih et al., 1996
; Popham et al., 1996a
). Thus amidase activity appears to be essential for producing this characteristic structure of cortical peptidoglycan.
Cortical peptidoglycan is very loosely cross-linked, at only 2·9 % of muramic acid residues (Atrih et al., 1996 ), compared to 2933% in vegetative cell walls (Atrih et al., 1999
). The anisotropic swelling theory of cortex expansion during forespore maturation suggests that the cortex is first synthesized with a relatively large number of peptide cross-links; selective enzymic cleavage of these reduces the cross-linking index and leads to radial expansion of the cortex causing inward mechanical pressure on the spore core. This pressure prevents the core taking up water and swelling, and thus the action of autolysins during cortex maturation may be essential in maintaining the spore in its dehydrated, quiescent state (Warth, 1978
). Alternatively, or in addition, DD-carboxypeptidase activity may prevent cross-link formation by removing C-terminal D-Ala residues from D-Ala-D-Ala-terminating peptide side-chains. This is supported by studies showing that mutations in the sporulation-specific penicillin-binding proteins DacB and DacF result in increased cross-linking (Atrih et al., 1996
; Popham et al., 1996b
, 1999
), showing that the cross-linking index of cortical peptidoglycan is at least partly controlled at the level of cross-link formation. The possible role of specific autolysin-catalysed cross-link hydrolysis in determining the final cross-linking index of mature cortex has yet to be fully investigated.
About 25% of the muramic acid residues in the cortex have single L-alanine side-chains (Fig. 1b; Atrih et al., 1996
; Popham et al., 1996b
). These may be formed from longer side-chains by the action of an L-alanoyl-D-glutamate endopeptidase. This suggests a role for YcdD, the only putative autolysin of B. subtilis predicted to have such activity (Table 1
). However, our recent data have shown that a ycdD mutant is still able to produce single L-alanine side-chains. Alternatively, the single L-alanine side-chains may result from sequential carboxypeptidase-catalysed steps.
Mother-cell lysis.
Mother-cell lysis at the end of sporulation, which is required for release of the mature endospore, depends on two amidases of the LytC family, CwlC and LytC itself. CwlC and LytC have mutually compensatory roles in hydrolysis of mother-cell-wall peptidoglycan and so mother-cell lysis is blocked only when both are absent (Foster, 1992 ; Smith & Foster, 1995
). Recent results have shown that YqeE, which belongs to the XlyA amidase family, also has a role in mother-cell lysis (Nugroho et al., 1999
).
Germination.
The importance of the cortex in maintaining endospore dehydration, resistance and dormancy demands its selective hydrolysis as one of the necessary events of germination, to allow uptake of water, core expansion and outgrowth (Foster & Johnstone, 1990 ). Analysis of spore peptidoglycan and material released during germination shows that a subtle change, probably stereochemical, occurs at or near to the muramic
-lactam (Atrih et al., 1998
). Concomitantly, hydrolytic events, primarily lytic transglycosylase and, to a lesser extent, glucosaminidase, cause release of the cortical peptidoglycan as muropeptide fragments (Atrih et al., 1998
). Detectable peptidoglycan modification and hydrolysis are relatively late events in germination, being undetectable until 3 min after addition of germinant, by which time heat resistance has already begun to be lost and dipicolinate from the spore is released (Atrih et al., 1998
). It appears to be the muramic
-lactam that targets the cortex for digestion during germination, because the cwlD mutant, which lacks the
-lactam, produces resistant endospores but does not undergo cortex hydrolysis during germination and cannot outgrow (Atrih et al., 1996
, 1998
; Popham et al., 1996b
; Sekiguchi et al., 1995
). This specificity ensures that the primordial cell wall, which appears not to have the
-lactam, is not hydrolysed and remains to develop into the cell wall of the outgrowing spore. Little or no muramidase activity was observed during germination (Atrih et al., 1998
). Consistent with this, the germination-specific muramidase SleM found in Clostridium perfringens (Chen et al., 1997
) has no detectable homologue in B. subtilis.
GSLEs, which catalyse germination-like changes in permeabilized spores, have been isolated from germinating endospores in other species (reviewed by Atrih & Foster, 1999 ) and are weakly related to the XlyA amidase family. B. subtilis has three GSLE homologues, SleB, CwlJ and YkvT. The roles of SleB and CwlJ have been studied at the molecular level. Both have roles in the later stages of germination, and a sleB cwlJ double mutant is completely blocked in cortex hydrolysis and outgrowth (Ishikawa et al., 1998b
). The sleB mutant, which showed retarded germination, demonstrated no evidence of lytic transglycosylase activity during germination (Boland et al., 2000
). Thus, the GSLE SleB must be either a lytic transglycosylase or a protein with a different specificity whose action is necessary before the lytic transglycosylase can act. YjbJ, the only putative lytic transglycosylase that is not prophage-encoded, is not likely to be involved in germination because its inactivation caused no detectable germination deficiency (Blackman, 1998
) or lack of anhydromuropeptides during germination (A. Atrih & S. J. Foster, unpublished). Activity of sleB (but apparently not cwlJ) depends on ypeB, which encodes a protein of unknown function and is immediately downstream from sleB in the same apparent two-gene operon. ypeB is conserved in other endospore-forming bacteria (Boland et al., 2000
). The glucosaminidase activity observed during germination is due to an unidentified enzyme, as a lytD or cwlJ mutant still demonstrates generation of the products.
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Control of autolysins |
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The post-translational control of autolysin activity is more difficult to understand. B. subtilis cell walls contain a potentially lethal amount of autolytic activity throughout growth and must control this activity to prevent committing suicide by the action of their own enzymes. Although there has been much speculation about the mechanism of such control, there is still little hard evidence. Proposed mechanisms of autolysin control have included a number of possible inhomogeneities in the peptidoglycan substrate, particularly substrate conformation (Koch et al., 1985 ), covalent modification of the substrate (Clarke, 1993
), distribution of secondary polymers (Rogers et al., 1980
; Fischer et al., 1981
) and the wall ionic environment (Cheung & Freese, 1985
). Observations that cell-wall autolysis is enhanced by depolarizing agents has led to proposals that autolysins may be controlled by the proton-motive force (Jolliffe et al., 1981
; Kemper et al., 1993
). Any of these effects, or combinations of them, could modulate the susceptibility of different regions of the cell wall to a number of autolysins, thus explaining the existence of several enzymes during vegetative growth that have different bond specificities but are all, more or less, involved in the same diverse processes.
The role of substrate conformation in determining autolysin activity is demonstrated by the GSLEs, which hydrolyse stressed, intact cortex, but not the same material from mechanically broken spores. One of the clearest examples of the role of covalent modification of peptidoglycan in determining autolysin activity is the muramic -lactam of cortical peptidoglycan, which appears to be the feature of the cortex that makes it sensitive to the germination autolysins, allowing its selective removal without digestion of the primordial cell wall (Atrih et al., 1996
; Popham et al., 1996a
). However, the
-lactam cannot be the only feature of cortex that targets it for specific autolytic attack, because a 41 kDa sporulation-specific autolysin, which hydrolyses cortex but not vegetative cell walls, is active even against cortex from the cwlD mutant (Smith & Foster, 1997
).
Differential control of different teichoic acid genes with differing cell division rates suggests that the teichoic acid (one of the major anionic polymers) may have a different structure in the septa than in the cylindrical parts of the wall (Mauël et al., 1995 ). For instance, an uneven distribution of negative charge over the cell wall was observed by preferential binding of positively charged cytochrome c to the cylindrical regions rather than the poles (Wecke et al., 1997
). If the primarily positively charged major autolysins were distributed similarly, this would explain the observed higher rate of wall turnover at the cylinders than the poles (Clarke-Sturman et al., 1989
). Accelerated cell autolysis in a mutant with more negatively charged secondary polymers is consistent with this theory (Wecke et al., 1997
). The observed de-N-acetylation of certain glucosamine residues in vegetative peptidoglycan also offers a mechanism for autolysin control (Atrih et al., 1999
). Differential binding of autolysins, via their varied cell-wall-binding domains, to different parts of the cell surface may allow exquisite control of their sites and extent of action. Specific binding of the major S. aureus autolysin Atl around the plane of cell division has already been observed (Sugai et al., 1995
). It seems unlikely that the major autolysins, which appear to act at numerous sites around the cell wall, are highly localized, but as yet uncharacterized minor enzymes with specialized functions may well be. Proteinprotein interaction may also mediate the localization and control of autolysin activity. For instance, the modifier protein LytB, which enhances the in vitro activity of the major vegetative amidase LytC (Lazarevic et al., 1992
), may be one of a number of factors involved in controlling autolytic activity in vivo.
Although the nature of the control mechanisms that cease upon cell death remain largely unknown, the major autolysins involved in post-mortem cell autolysis have been identified. The amidase LytC and the putative endopeptidase LytE have roles in cell-wall lysis after exposure of the culture to the metabolic poison sodium azide. No role for the major vegetative glucosaminidase LytD during cell lysis has been found (Blackman et al., 1998 ; Margot et al., 1994
, 1998
; Margot & Karamata, 1992
). Both LytC and LytD have mutually compensatory roles in lysis after exposure to the ß-lactam antibiotic cloxacillin, although neither is required for cloxacillin-induced cell death (Blackman et al., 1998
).
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Prophage lytic enzymes |
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YomI, which has both Slt 70- and lysostaphin-like domains, is found within SPß and so may function in SPß lysis in addition to YomC (BlyA). The gene encoding YddH, a member of the DL-endopeptidase II family, resides within the presumptive prophage 2 and so may be or have been involved in its lysis of the cell. ydhD and yocH appear to be phage-related amidase genes distant on the chromosome from identified prophages, which may encode autolysins that are controlled by the host. yqeE, which is near to but outside skin, could be another such gene. The existence of such genes implies exchange of peptidoglycan hydrolase genes between the host and its phages, a phenomenon that has also been described in pneumococci (Sheenan et al., 1997 ). xlyB, a putative amidase gene of the XlyB family, lies near to but outside PBSX. Induction of PBSX produces two lytic enzymes, a 32 kDa enzyme that corresponds to XlyA and another of 34 kDa (Longchamp et al., 1994
), very similar in size to the 33·7 kDa XlyB. This confirms that part of the PBSX genome is, to some extent, distributed around the chromosome (Longchamp et al., 1994
).
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Conclusions and future prospects |
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As more microbial genome sequences become available, it becomes clear that apparent redundancy of gene function is common among genes involved in a wide range of physiological processes. Significant differences in the phenotypes of genetically very similar autolysin-deficient mutants observed in different laboratories underlines the conclusion that the precise roles of individual autolysins in particular processes is highly sensitive to growth conditions and genetic background. These observations are consistent with functional redundancy arising from a need to adapt to a wide range of growth conditions in the environment.
Among the autolysins of B. subtilis, functional redundancy is evident throughout the life cycle, but is more pronounced during vegetative growth than sporulation. During vegetative growth there are two orders of functional redundancy: individual processes can be performed by any one of a number of enzymes, and individual enzymes can be involved in a number of processes. During differentiation, on the other hand, functional redundancy is less extreme. Perhaps the autolytic processes of differentiation are less susceptible to, or better isolated from, the environment than those of vegetative growth.
The autolysins of B. subtilis are remarkable not only in their number but also in their variety. We have identified no fewer than 11 distinct families of autolysins and possible autolysins, and biochemical studies show that there are at least six different types of hydrolytic bond specificity involved in cell-wall metabolism. Indeed, only one of the proposed hydrolytic bond specificities (D-alanine-meso-diaminopimelate endopeptidase, see Fig. 1) has not been observed. During vegetative growth it is surprising that the process of cell separation, which involves the specific removal of part of the septal peptidoglycan, can be performed by enzymes with at least three different hydrolytic bond specificities. It appears that the total level of autolysin activity is what governs the probability of daughter-cell separation. The compensatory nature of the autolysins involved in this process as well as others can be explained by the effect of lack of a particular class of enzymes on substrate availability. Thus, having one enzyme missing may provide more substrate for another resident enzyme, which will therefore compensate for the absence of the first and so avoid any potential problem for the cell. Weak, though significant, primary-structure similarities among a number of different classes suggest that the three-dimensional structures and catalytic mechanisms of some of the families are interrelated, although such theories remain to be substantiated by determination of the three-dimensional structure of bacillary autolysins.
It is intriguing that autolysins essential for vegetative growth of B. subtilis have not been identified. It is difficult to conceive how cell growth could be achieved without selected disconnection of covalent bonds within the peptidoglycan to permit cell-wall expansion, but the autolysins involved in this essential process have so far proved elusive. During cell elongation, maybe minor autolysins hydrolyse relatively few bonds, but create sites suitable for the action of the major autolysins. During cell growth, the former activity may correspond to the essential autolytic events essential for cell growth and the latter to the inessential wall-turnover-related autolysin activity. It is possible that enzyme activity is not involved or is inessential, and that the necessary breakage of bonds can occur by mechanical stress from the expanding cytoplasm (Archibald et al., 1993 ). More likely, however, is that the autolysins required in this carefully regulated process have not yet been identified.
The control of autolysin activity remains a mystery, and is one that seems difficult to solve by in vitro studies with isolated cell walls, when most autolysins have apparently run-away activity. One way forward is to isolate mutants that interfere with autolytic activity, thus defining what properties of the cell-wall environment are important. In this manner, study of a strain with cortical peptidoglycan lacking the muramic -lactam structure has already identified the
-lactam as necessary for targeting the cortex for hydrolysis during germination (Atrih et al., 1996
, 1998
; Popham et al., 1996a
).
We propose that the molecular origin of autolysin specificity, post-translational control of autolysin activity and characterization of the roles, if any, of autolysins in cell expansion are the major current questions that remain to be answered in the study of these important enzymes. A combined approach, using multiple gene inactivations, morphological and physiological studies, together with analysis of peptidoglycan fine structure, has already begun to unravel the molecular functions of the plethora of autolysins in B. subtilis. We suggest that continued application of similar technology may reveal every autolysin involved in each autolysin-requiring process. Together with parallel studies of the mechanism of peptidoglycan synthesis, a thorough understanding of the molecular origin of cell-wall dynamics in B. subtilis now seems feasible.
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ACKNOWLEDGEMENTS |
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