of Clostridium acetobutylicum
Dissertation
zur
Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Jan Reith, Dipl.-Biol.
Universität Konstanz
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
Konstanz, 2012
Tag der mündlichen Prüfung: 07.05.2012
Prüfungsvorsitzender: Prof. Dr. Wolfram Welte 1. Referent: Prof. Dr. Winfried Boos 2. Referent: PD Dr. Christoph Mayer
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-192901
Dedicated to my family – especially Christine and Thees.
TABLE OF CONTENTS
ZUSAMMENFASSUNG ... II SUMMARY ... IV
1. GENERAL INTRODUCTION ... 1
1.1. THE BACTERIAL CELL WALL ... 1
1.2. CHEMICAL COMPOSITION AND STRUCTURE OF PEPTIDOGLYCAN ... 4
1.3. PEPTIDOGLYCAN SYNTHESIS ... 8
1.4. CELL WALL GROWTH AND TURNOVER ... 12
1.5. RECYCLING OF PEPTIDOGLYCAN TURNOVER PRODUCTS ... 19
1.6. AIM OF THIS THESIS ... 26
2. LIST OF PUBLICATIONS ...27
3. CHAPTER 1 ...28
PEPTIDOGLYCAN TURNOVER AND RECYCLING IN GRAM-POSITIVE BACTERIA ... 28
3.1. ABSTRACT ... 28
3.2. INTRODUCTION ... 29
3.3. CELL WALL TURNOVER AND RECYCLING IN GRAM-NEGATIVE BACTERIA ... 31
3.4. DIFFERENCES IN THE CELL WALL STRUCTURE BETWEEN GRAM-NEGATIVE AND GRAM-POSITIVE BACTERIA AND THEIR IMPLICATIONS FOR TURNOVER AND RECYCLING ... 34
3.5. CELL WALL RECYCLING IN GRAM-POSITIVE BACTERIA ... 36
3.6. MUROPEPTIDE RECOVERY IN B. SUBTILIS AND C. ACETOBUTYLICUM ... 39
3.7. IMPACT OF CELL WALL RECYCLING ON BIOTECHNOLOGY ... 42
3.8. CONCLUDING REMARKS ... 43
4. CHAPTER 2 ...44
CHARACTERIZATION OF AN N-ACETYLMURAMIC ACID/N-ACETYLGLUCOSAMINE KINASE OF CLOSTRIDIUM ACETOBUTYLICUM ... 44
4.1. ABSTRACT ... 44
4.2. INTRODUCTION ... 45
4.3. MATERIALSANDMETHODS ... 47
4.4. RESULTS... 51
4.5. DISCUSSION ... 58
5. CHAPTER 3 ...61
CHARACTERIZATION OF A GLUCOSAMINE/GLUCOSAMINIDE N-ACETYLTRANSFERASE OF CLOSTRIDIUM ACETOBUTYLICUM ... 61
5.1. ABSTRACT ... 61
5.2. INTRODUCTION ... 62
5.3. MATERIALSANDMETHODS ... 64
5.4. RESULTS... 68
5.5. DISCUSSION ... 76
6. GENERAL REFERENCES ...79
7. DECLARATION OF AUTHOR CONTRIBUTION ...96
8. ACKNOWLEDGEMENTS ...97
ZUSAMMENFASSUNG
Die Zellwand ist ein essentielles Exoskelett nahezu aller Bakterien, das die Zellform aufrecht erhält und die Zelle gegen osmotische Lyse schützt. Hauptbestandteil der bakteriellen Zellwand ist Peptidoglykan, ein Heteropolymer bestehend aus Glykansträngen, die über kurze Peptidketten quervernetzt sind. Trotz seiner Starrheit ist Peptidoglykan eine hochdynamische Struktur, die im Laufe des Zellwachstums und der Zellteilung kontinuierlich dem Turnover und der Remodellierung unterliegt. Während des Turnoverprozesses freigesetzte Peptidoglykanfragmente werden von Gram-negativen Bakterien wie Escherichia coli effizient wiederverwertet (recycelt). Ob auch in Gram-positiven Bakterien Recycling stattfindet, ist allerding noch unbekannt. Für das Gram-positive Bakterium Bacillus subtilis wurde kürzlich ein Abbauweg für die Wiedergewinnung von Peptidoglykanfragmenten entdeckt, der die aufeinanderfolgende Umsetzung von N-Acetylglukosamin-N-Acetylmuraminsäure-Peptiden (Muropeptide) durch die sekretierten Enzyme N-Acetylglukosaminidase (NagZBs) und N-Acetylmuraminsäure-L-Alanin Amidase (AmiEBs) beinhaltet. Auch in Clostridium acetobutylicum, einem anaeroben Gram-positiven Lösungsmittelproduzenten, der eng mit Bacillus verwandt ist, sind Orthologe von Muropeptid recycelnden Enzymen aus B. subtilis vorhanden. Wir haben erkannt, dass Orthologe von NagZ (CA_C0182) und AmiE (CA_C0181) aus C. acetobutylicum anscheinend nicht wie in B. subtilis sekretiert werden, weshalb der Abbau von Muropeptiden möglicherweise anders verläuft.
Die vorliegende Promotionsarbeit untersucht den Peptidoglykanrecyclingweg von C.
acetobutylicum und fokussiert auf zwei Enzyme mit einer neuartigen Spezifität für die Aminozucker des bakteriellen Peptidoglykans, N-Acetylglukosamin (GlcNAc) und N-Acetylmuraminsäure (MurNAc). Wir identifizierten eine Kinase von C. acetobutylicum, benannt MurK (früher CA_C0183), die durch ein Gen kodiert wird, welches mit anderen vermutlichen Recyclinggenen in einem Cluster organisiert ist. MurK katalysiert jeweils die Phosphorylierung von GlcNAc und MurNAc an Position 6 zu GlcNAc-6-Phosphat und MurNAc-6-Phosphat. Die biochemische Charakterisierung von MurK offenbarte eine 1,5- fach höhere katalytische Aktivität für GlcNAc im Vergleich zu MurNAc. Anhand von Phosphorylierungsuntersuchungen mit radioaktivem ATP wurde gezeigt, dass strukturell verwandte Zellwandzucker, d.h. Epimere (N-Acetylgalaktosamin und N-Acetylmannosamin), nicht-N-acetylierte Derivate (Glukosamin und Muraminsäure) und eine Anhydroform von MurNAc (1,6-anhydroMurNAc) keine Substrate für dieses Enzym sind. MurK weist keine Gemeinsamkeiten in der Aminosäuresequenz mit den Aminozuckerkinasen AnmK und NagK
von E. coli auf, allerdings gibt es geringe Übereinstimmungen mit der menschlichen GlcNAc Kinase. Zudem wurde gezeigt, dass die MurK Kinase verwendet werden kann, um Zellwandzucker bis zu Mengen von 5 fmol nachzuweisen. Ein zweites kodiertes Enzym wurde innerhalb desselben Clusters stromabwärts von murK auf dem Genom von C. acetobutylicum als eine neuartige Acetyltransferase identifiziert und GlmA genannt (früher CA_C0184). Es katalysiert die Bildung von GlcNAc durch den Transfer einer Acetylgruppe von Acetyl-Coenzym A auf Glucosamin (GlcN) sowie endständige nichtreduzierte GlcN- Reste. Die katalytische Effizienz von GlmA für lineare Di- und Trisaccharide bestehend aus β-1,4-glykosidisch verknüpften GlcN-Resten ist im Vergleich zu GlcN um das Drei- bis Vierfache erhöht. Allerdings dient das Milchsäurederivat von Glukosamin, die Muraminsäure, nicht als Acetyl-Akzeptor von GlmA. Weitere Acetylierungsversuche mit Muropeptiden, freigesetzt aus dem Peptidoglykan von B. subtilis, zeigten, dass nicht-N-acetylierte GlcN- Reste erst acetyliert werden müssen, bevor die N-Acetylglucosaminidase NagZBs das endständige GlcNAc abspaltet. Peptidoglykan von E. coli hingegen, dem diese N-deacetylierten Modifizierungen fehlen, konnte nicht durch GlmA acetyliert werden.
Die hier präsentierten Ergebnisse zeigen, dass MurK, die GlcNAc/MurNAc Kinase, und GlmA, die Glukosamin/Glukosaminid N-Acetyltransferase, eine wesentliche Rolle in einem möglichen Recyclingweg spielen, der sich von den bekannten Abbauwegen aus B. subtilis und E. coli unterscheidet. Unser Modell für das Peptidoglykanrecycling in C. acetobutylicum beschreibt, dass die während des Zellwandturnovers freigesetzten de-N-acetylierten Muropeptide mit Hilfe des mutmaßlichen ABC Transporters AppABCDF in die Zelle aufgenommen werden. Die Wiedergewinnung von GlcN-Resten im Zytoplasma erfordert die N-Acetylierung am nichtreduzierten Ende. Das so entstandene endständige GlcNAc kann nun vom Muropeptid durch NagZ freigesetzt werden, während AmiE den Peptidanteil von MurNAc abspaltet. Beide Aminozucker können anschließend durch die MurK Kinase zu GlcNAc- und MurNAc-6-Phosphat phosphoryliert werden. Im weiteren Verlauf wird MurNAc-6-Phosphat durch die Etherase MurQ zu GlcNAc-6-Phosphat umgewandelt, welches nach erfolgter Deacetylierung entweder über die Glykolyse verstoffwechselt wird oder erneut für die Peptidoglykan-Biosynthese zur Verfügung steht.
SUMMARY
The cell wall is the essential exoskeleton of most bacteria that maintains cell shape and protects the cell against osmotic lysis. The major component of the bacterial cell wall is the peptidoglycan, a heteropolymer consisting of glycan strands cross-linked by short peptides.
Despite its rigidity, the peptidoglycan is a highly dynamic structure, which is continuously undergoing turnover and remodeling during cell growth and division. In Gram-negative bacteria like Escherichia coli, peptidoglycan fragments released during this turnover process are reutilized (recycled) efficiently. However, it is still unknown whether recycling also proceeds in Gram-positive bacteria. Recently, a pathway for the recovery of peptidoglycan fragments, which involves the sequential processing of N-acetylglucosamine-N- acetylmuramic acid-peptides (muropeptides) by the secreted enzymes N-acetylglucosaminidase (NagZBs) and N-acetylmuramic acid-L-alanine amidase (AmiEBs), has been proposed for the Gram-positive bacterium Bacillus subtilis. Orthologs of muropeptide recycling enzymes of B. subtilis are also present in Clostridium acetobutylicum, an anerobic Gram-positive solvent producer that is closely related to Bacillus. We recognized that orthologs of NagZ (CA_C0182) and AmiE (CA_C0181) of C. acetobutylicum are apparently not secreted as in B. subtilis, indicating that catabolism of muropeptides may proceed different.
This thesis presented here investigates the peptidoglycan recycling pathway of C. acetobutylicum and focuses on two enzymes showing a novel specificity for the amino sugars of the bacterial peptidoglycan, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). We identified a kinase of C. acetobutylicum, named MurK (formerly CA_C0183), which is encoded by a gene that is clustered with putative recycling genes.
MurK catalyzes the phosphorylation of both GlcNAc and MurNAc at the 6-position, yielding GlcNAc-6-phosphate and MurNAc-6-phosphate, respectively. The biochemical characterization of MurK revealed a 1.5-fold higher catalytic activity for GlcNAc than for MurNAc. Applying phosphorylation assays using radioactive ATP indicated that structurally related cell wall sugars, i.e., epimers (N-acetylgalactosamine and N-acetylmannosamine), non-N-acetylated derivatives (glucosamine and muramic acid) and an anhydro form of MurNAc (1,6-anhydroMurNAc) are no substrates for this enzyme. MurK displays no amino acid sequence similarity with the amino sugar kinases AnmK and NagK of E. coli but with human GlcNAc kinase. We showed that MurK kinase can be used for a sensitive detection of the cell wall sugars at amounts as low as 5 fmol. Within the same gene cluster downstream of
murK on the genome of C. acetobutylicum, we identified a second encoded enzyme, a novel acetyltransferase, named GlmA (formerly CA_C0184). It catalyzes the formation of GlcNAc by the transfer of an acetyl group from acetyl coenzyme A to glucosamine (GlcN) and terminal nonreducing GlcN residues, respectively. We determined a 3- to 4-fold higher catalytic efficiency of GlmA for linear di- and trisaccharides composed of β-1,4- glycosidically linked GlcN residues, compared to GlcN. However, the lactic acid derivative of GlcN, muramic acid, did not serve as an acetyl acceptor of GlmA. Acetylation assays with muropeptides derived from peptidoglycan of B. subtilis revealed that non-N-acetylated GlcN residues had to be N-acetylated by GlmA prior to the release of terminal GlcNAc by the N-acetylglucosaminidase NagZBs, whereas peptidoglycan of E. coli lacking these N-deacetylated modifications could not be acetylated by GlmA.
In conclusion, the results presented in this thesis indicate that MurK, the GlcNAc/MurNAc kinase, and GlmA, the glucosamine/glucosaminide N-acetyltransferase, both are involved in a putative recycling pathway in C. acetobutylicum, which proceeds distinct to the known pathways of B. subtilis and E. coli. Our model of the C. acetobutylicum peptidoglycan recycling proposes that de-N-acetylated muropeptides liberated during cell wall turnover are transported into the cell by the putative ABC transporter AppABCDF. In the cytoplasm, recovery of GlcN residues requires N-acetylation at the nonreducing end by GlmA. The generated terminal GlcNAc can then be released from muropeptides by NagZ and MurNAc from the peptide moiety by AmiE. Both amino sugars can now be used for phosphorylation by the MurK kinase, yielding GlcNAc- and MurNac-6-phosphate, respectively. Further processing involves the conversion of MurNAc-6-phosphate to GlcNAc-6-phosphate by the MurQ etherase, which enters, after deacetylation, either glycolysis or the peptidoglycan biosynthesis pathway.
1. GENERAL INTRODUCTION
Peptidoglycan is a giant bag-shaped macromolecule that surrounds the bacterial cell, forming the exoskeleton-like cell wall. Despite its stabilizing function, peptidoglycan is not static but always in a dynamic state. As the peptidoglycan sacculus is a covalently closed structure, its surface expansion requires a concerted interplay of peptidoglycan synthesizing and hydrolyzing enzymes to maintain cellular integrity while new material is inserted into the preexisting structure. Accordingly, bacterial growth and differentiation are accompanied by the breakdown of the cell wall and the release of turnover products that can be efficiently reutilized, as it has been shown for the Gram-negative bacterium Escherichia coli. The ability to recycle peptidoglycan provides a significant advantage for the survival of E. coli, since the bacterium is able to grow on cell wall-derived amino sugars as sole source of carbon and energy. Surprisingly, cell wall recycling in Gram-positive bacteria is less well studied, although the cell wall in these organisms is much thicker and recycling would generate an even greater benefit in terms preservation of resources. Indeed, Gram-positive organisms were shown to generate peptidoglycan turnover products in huge amount during cell growth but apparently use reutilization (recycling) pathways different from those of Gram-negative bacteria. Thus, a detailed understanding of the mechanisms of cell wall peptidoglycan contributes to clarify the fate of the turnover products in Gram-positive bacteria.
1.1. The bacterial cell wall
The integrity of the bacterial cell wall is of indispensable importance to cell viability. Its rigidity provides mechanical support to the cell and allows the bacterium to maintain its characteristic shape. By stabilizing the semipermeable, remarkably fragile plasma membrane, the primary function of the cell wall is to protect the underlying protoplast against environmental stresses from the outside and to prevent rupture or lysis caused by osmotic force from the inside. Since procaryotes usually live in hypotonic habitats with a lower solute concentration outside the cell, bacteria evolved to form such a bag-shaped sacculus.
Therefore, bacteria are able to accumulate high concentrations of nutrients inside the cell accompanied by the development of an exceedingly high internal osmotic pressure of about 2 to 5 atmospheres in Gram-negative bacteria and up to 50 atmospheres in Gram-positive bacteria (Archibald et al., 1993; Seltmann and Holst, 2002). Accordingly, the cell wall is made of a rigid, sufficiently porous material that has considerable tensile strength, thus
permitting the diffusion of metabolites to the plasma membrane as well as reversible cell expansion or shrinkage to tolerate changes in osmolarity of the environment. Peptidoglycan, also known as murein, is such a material, the major structural constituent of bacterial cell walls. Peptidoglycan is a heteropolymer that consists of long glycan strands of repeating disaccharide residues, cross-linked by short peptide bridges to form one large molecule of strong but elastic nature (Höltje, 1998; Park, 1996; Weidel and Pelzer, 1964). It is present in all bacteria with the exception of Mycoplasma, Planctomyces and a few other bacterial species that lack a cell wall (Moulder, 1993; Seltmann and Holst, 2002).
While in both Gram-negative and Gram-positive bacteria the chemical composition of peptidoglycan is very similar, its arrangement in the murein sacculus is different (Fig. 1). In Gram-negative bacteria, a thin peptidoglycan layer of a few nanometers, which accounts for less than 5% of the cell mass, is located in the periplasm between the inner and outer membrane (Fig. 2A). The peptidoglycan layer is covalently attached to the outer membrane by the murein lipoprotein called Lpp or Braun´s lipoprotein (Braun, 1975). About two thirds of the peptidoglycan in the Gram-negative bacterium E. coli is composed of only one layer, whereas the remainder has three layers (Labischinski et al., 1991). Unlike Gram-negative bacteria, peptidoglycan is highly abundant in Gram-positives, in which it makes up more than 20% of the cell mass.
Fig. 1. Most bacteria are protected against environmental stress by an exoskeleton-like structure called the bacterial cell wall. While the cell wall of Gram-positive bacteria is mainly comprised of a thick layer of peptidoglycan with covalently bound anionic polymers, Gram-negative bacteria possess a predominant monolayer of peptidoglycan within the periplasm, which is covered by an outer membrane. CAP, covalently attached protein; IMP, integral membrane protein; LP, lipoprotein; LPS, lipopolysaccharide; LTA, lipoteichoic acid; OMP, outer membrane protein; WTA, wall teichoic acid. Figure was adapted from (Silhavy et al., 2010).
Since Gram-positive bacteria typically lack an outer membrane, they are surrounded by multiple layers of peptidoglycan which can be up to 20-fold thicker than that of Gram- negative bacteria (Shockman and Barrett, 1983). Most Gram-positive bacteria protect the peptidoglycan sacculus, which is exposed to the external environment, by covering the surface with structures such as layers of proteins (S-layer) or polysaccharides (capsules) (Navarre and Schneewind, 1999). They may function, among others, as protective barriers or are involved in cell adhesion. In addition to peptidoglycan, teichoic acids are embedded in an approximately equal proportion within the Gram-positive cell wall (Foster and Popham, 2002). These anionic polymers consist of repeating residues of glycerol phosphate or ribitol phosphate and occur in two distinct forms. While wall teichoic acids are covalently attached to the peptidoglycan, the second major type that is anchored to the cytoplasmic membrane includes the lipoteichoic acids. Under phosphate-limiting growth conditions teichoic acids are almost entirely replaced by teichuronic acids, which are devoid of phosphate residues.
Glycopolymers like teichoic acids are not found within the cell wall of Gram-negative bacteria.
Fig. 2. Cryo-transmission electron microscopies of bacterial cell walls from Gram-negative and Gram- positive bacteria revealed distinct architectural differences. (A) The Gram-negative cell wall of E. coli includes an outer membrane (OM) that surrounds a thin peptidoglycan layer (PG) embedded in a periplasmic space between the plasma membrane (PM) and the outer membrane. Scale bar, 200 nm. (B) In contrast, Gram- positive bacteria like B. subtilis lack an outer membrane but possess a thick cell wall with varying mass distribution. The plasma membrane (PM) is enclosed by a low-density inner wall zone (IWZ), representing the Gram-positive periplasmic space, which is surrounded by a high-density outer wall zone (OWZ). Scale bare, 50 nm. Figure (A) was taken from (Matias et al., 2003) and (B) from (Matias and Beveridge, 2005).
Remarkably, a two-layered organization of the cell wall has been detected in Gram-positive bacteria using cryo-transmission electron microscopy (Fig. 2B). These studies revealed that Bacillus subtilis as well as Staphylococcus aureus possess a low-density inner cell wall zone overlying the outer surface of the plasma membrane, followed by an outer wall zone of higher density peptidoglycan with covalently linked teichoic acid (Matias and Beveridge, 2005, 2006). This inner cell wall zone contains soluble components but, in contrast to the outer wall zone, it is rid of the previously mentioned polymeric network. It is, therefore, suggested that the inner cell wall zone represents a periplasmic space analogous to that found in Gram- negative bacteria, as part of the Gram-positive cell wall (Matias and Beveridge, 2006).
The current knowledge of the cell wall peptidoglycan regarding structure, pathways and models presented in this thesis will focus predominantly on the rod-shaped Gram-negative bacterium E. coli and its Gram-positive counterpart B. subtilis, respectively.
1.2. Chemical composition and structure of peptidoglycan
The bacterial cell wall consists of peptidoglycan, a mesh-like polymer of glycan strands that are cross-linked to each other by short peptides (Fig. 3). The linear glycan strands are composed of alternating amino sugars of N-acetylglucosamine (GlcNAc) and its 3-O-lactyl ether derivative, N-acetylmuramic acid (MurNAc), which are linked by β-1,4 glycosidic bonds. Glycan strands of the mature peptidoglycan, especially the hexosamine residues, are frequently modified by O-acetylation, N-deacetylation, and/or N-glycosylation (Vollmer, 2008). While these secondary modifications are predominantly absent in Gram-negative bacteria, most of them have been detected in Gram-positive bacteria. They contribute to an increased protection against peptidoglycan hydrolyzing enzymes like lysozyme, as observed in the presence of high proportions of de-N-acetylated amino sugars, i.e., glucosamine and muramic acid residues in the peptidoglycan of some Bacillus species (Araki et al., 1971b;
Zipperle et al., 1984). De-N-acetylation of glucosamine and muramic acid is also known in B.
subtilis and occurs in 19 and 33% of the total vegetative cell peptidoglycan, respectively (Atrih et al., 1999). A distinct feature of the peptidoglycan of all Gram-negative bacteria is that the glycan strands do not contain a reducing end but have an 1,6-anhydro modification at the terminal MurNAc residue, which represents an intramolecular glycosidic linkage from C1 to C6 (Höltje et al., 1975). Only low amounts of 1,6-anhydroMurNAc residues are present in B. subtilis and in a few other Gram-positive representatives (Atrih et al., 1999). The length distribution of the glycan strands in the peptidoglycan is very broad among bacteria. An
average chain length of 21 disaccharide units was estimated for E. coli by a reversed-phase HPLC separation technique (Harz et al., 1990), whereas glycan strands in the peptidoglycan of B. subtilis are much longer, ranging between 54 and 96 disaccharide units (Ward, 1973). In contrast, atomic force microscopy (AFM) revealed an average chain length of isolated glycan strands of B. subtilis to be 1,300 disaccharide units (Hayhurst et al., 2008). The longest glycan strands identified in this way were as long as the cell at full length and contained up to 5,000 disaccharide units, corresponding to 5 µm.
Fig. 3. Schematic structure of the peptidoglycan of E. coli (Gram-negative) and B. subtilis (Gram-positive).
N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues are connected in an alternating sequence by β-1,4-glycosidic bonds forming linear glycan strands. Short stem peptides are linked to the D-lactyl group of each MurNAc. Adjacent stem peptides are directly cross-linked either between m-Dap and D-Ala (D,D- cross-linkage) or in some cases between two m-DAP (L,D-cross-linkage). The formation of the cross-linkage results in the concomitant removal of D-Ala at the terminal end of the pentapeptide (indicated by an arrow). In B.
subtilis, the free carboxyl groups of m-DAP are amidated (indicated by an asterisk). GlcNAc, N- acetylglucosamine (grey circle); MurNAc, N-acetylmuramic acid (grey square); 1,6-anhydroMurNAc, MurNAc possesses an intramolecular 1,6-glycosidic bond as the terminal residue of the glycan strands in E. coli (grey square with a cap). Figure was adapted from (Mayer, 2012).
In the peptidoglycan, the D-lactyl group of each MurNAc residue of the glycan strands is substituted by a stem peptide that contains alternating amino acids in L- and D-configuration, and one dibasic amino acid. The composition of the stem peptide varies but the common structure of most species, including E. coli and B. subtilis, is L-alanine-γ-D-glutamate-meso-
diaminopimelic acid-D-alanine-D-alanine of which the terminal L-alanine residue is attached to MurNAc (Quintela et al., 1995; Schleifer and Kandler, 1972; Vollmer et al., 2008a). The resulting D-L-D-L-sequence, beginning with the D-lactyl group of MurNAc and including the
L-center of m-DAP, prevents the formation of an α-helical peptide structure (Barnickel et al., 1979; Litzinger and Mayer, 2010). Thus, the peptides are much more flexible and can be stretched up to four-fold their relaxed length (Koch and Woeste, 1992). The third amino acid is the most variable component of the stem peptide but generally a dibasic amino acid is present, either meso-diaminopimelic acid (m-DAP) in Gram-negative bacteria but also in some bacilli together with mycobacteria, or L-lysine in most other Gram-positive bacteria. In B. subtilis, the free carboxyl group of m-DAP is amidated in vegetative wall peptidoglycan (Warth and Strominger, 1971). Neighboring glycan strands within the peptidoglycan are generally connected by D,D-peptide cross-linkages between the carboxyl group of D-alanine at position 4 of one stem peptide and the amino group of the diamino acid at position 3 of another stem peptide, thereby forming the mesh-like sacculus structure. Accordingly, the m-DAP-type stem peptides are usually directly cross-linked, whereas those of the L-lysine-type are cross-linked through an interpeptide bridge whose composition is species-specific. In S. aureus, for example, it is made up of 5 glycine residues.
Besides the main D,D-type of peptide cross-linkages, some bacterial species exhibit L,D- cross-linkages between two m-DAP residues on adjacent glycan strands at a lower frequency.
The degree of cross-linkages varies significantly between bacterial species, ranging from 40 to 60% of the stem peptides in Gram-negative bacteria and up to 90% in some Gram-positive bacteria; cf. table 1 of (Vollmer and Seligman, 2010). By comparison, the reported values for Gram-negative bacteria are much lower and in fact, almost half of the available stem peptides are apparently not cross-linked. These differences are probably caused by the presence of the rather bulky lactyl group at the MurNAc residue that forces the glycan strands to adopt a helical arrangement with presumably 4 disaccharide units per turn (Labischinski et al., 1985;
Leps et al., 1987). The consecutive stem peptides along the glycan strands are thought to be shifted by approximately 90 degrees to one another, protruding in all four directions.
Therefore, in a mostly monolayered peptidoglycan structure, as it occurs in Gram-negative bacteria, only every second stem peptide lies in the same plane and is likely to form a cross- linkage with another stem peptide protruding from a neighboring glycan strand. However, the recently solved NMR (nuclear magnetic resonance) structure of a small peptidoglycan fragment, a tetrasaccharide with two pentapeptides, consisting of L-alanine-γ-D-glutamate-L-
lysine-D-alanine-D-alanine, indicates a threefold symmetry of the glycan helix, which would prevent a planar arrangement of the cross-linked glycan strands (Meroueh et al., 2006).
Despite the detailed knowledge of the chemical composition of peptidoglycan, the three- dimensional arrangement of the glycan strands is still unknown. Currently, two models for the peptidoglycan architecture have been proposed. In the “horizontal model”, it is assumed that glycan strands are arranged in parallel to the cell membrane (Vollmer and Höltje, 2004).
Based on studies of isolated E. coli sacculi it was suggested that the more rigid glycan strands run perpendicular to the long axis of the cell, whereas the flexible peptides run in the direction to the long axis (Yao et al., 1999) (Fig. 4A). Thereby, the model describes a honeycomb pattern of hexagonal pores, the smallest of which is formed by two glycan strands and two cross-linked stem peptides, while in the stress-bearing layer the linear glycan strands follow a zigzag line (Demchick and Koch, 1996; Koch, 1998) (Fig. 4B). This classical concept of a layered peptidoglycan has been challenged by another model, the so-called “scaffold model”, in which the cross-linked glycan strands are extended perpendicularly to the cytoplasmic membrane (Dmitriev et al., 1999; Dmitriev et al., 2004; Dmitriev et al., 2003) (Fig. 4C).
However, a scaffold-like arrangement of the glycan strands is controversial because it is not in accordance to many experimental data, such as thickness, elasticity and amount of the covering peptidoglycan sacculus, as well as glycan chain length distribution and degree of cross-linkages (Vollmer and Höltje, 2004). Recently, the inner surface of the cell wall of B. subtilis has been visualized by AFM and revealed a number of helically coiled peptidoglycan cables of about 50 nm in width and cross striations of nearly 25 nm occurred within (Fig. 4D), arranged in parallel to the short axis around the cell but different to the known layered model of E. coli (Hayhurst et al., 2008). So far, further experiments are needed to confirm this cabling cell wall architecture in B. subtilis.
Fig. 4. Structural models of the peptidoglycan sacculus. (A) In the “horizontal model”, the glycan strands are oriented parallel to the cell membrane and along the short axis, whereas the peptide cross-linkages are arranged in the direction of the long axis of the cell. (B) According to the “horizontal model”, the smallest pore of a stressed cross-linked peptidoglycan is formed by two glycan strands (zigzag pattern of four disaccharide units) and two stem peptides (arrows and angels), as displayed inside the rectangle. It has been termed “tessera”. (C) In contrast, the “scaffold model” proposes an orientation of the glycan strands perpendicular to the cell membrane.
(D) Recently, atomic force microscopy revealed a novel model of cell wall architecture in B. subtilis. Thick cables of peptidoglycan with coiled structure running across the short axis of the cell. Figure (A) was taken from (Höltje, 1998), (B) from (Demchick and Koch, 1996), (C) from (Vollmer and Höltje, 2004) and (D) from (Hayhurst et al., 2008).
1.3. Peptidoglycan synthesis
The synthesis of peptidoglycan has been investigated extensively in Gram-positive and Gram- negative bacteria and occurs almost identically in three different stages (Fig. 5). The first stage concerns the formation of the nucleotide activated amino sugars uridindiphosphate-N- acetylglucosamine (UDP-GlcNAc) and uridindiphosphate-N-acetylmuramic acid- pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. In the second stage, the lipid- linked GlcNAc-MurNAc-pentapeptide (lipid II) is generated on the inner surface of the cytoplasmic membrane and then translocated across the cell membrane to the exterior. The third and final stage of polymerization terminates on the outer membrane surface in the periplasmic compartment with the incorporation of the precursors into the growing peptidoglycan structure, as reviewed by various authors (Barreteau et al., 2008; Bouhss et al.,
2008; Foster and Popham, 2002; Sauvage et al., 2008; van Heijenoort, 2001a, b, 2007;
Vollmer and Bertsche, 2008). In the following, the reaction pathways of E. coli are presented.
The assembly of the peptidoglycan precursors proceeds in the cytoplasm, where fructose- 6-phosphate is converted to UDP-GlcNAc, catalyzed by GlmS (aminotransferase) (Badet et al., 1987), GlmM (mutase) (Jolly et al., 2000; Mengin-Lecreulx and van Heijenoort, 1996) and GlmU (bifunctional transferase) (Mengin-Lecreulx and van Heijenoort, 1993, 1994) using glutamine (Gln) as nitrogen source for the amino group. Subsequently, UDP-MurNAc is formed in a two-step mechanism beginning with the transfer of an enolpyruvate from phosphoenolpyruvate (PEP) to UDP-GlcNAc by the transferase MurA (Marquardt et al., 1992) followed by its reduction to D-lactate, catalyzed by the reductase MurB (Pucci et al., 1992). Accordingly, the four ATP-dependent ligases MurC, MurD, MurE and MurF catalyze the formation of UDP-MurNAc-pentapeptide, the last soluble precursor of the peptidoglycan synthesis, by sequentially adding L-Ala, D-Glu, m-DAP and D-Ala-D-Ala dipeptide, respectively, to the D-lactyl group of UDP-MurNAc (Smith, 2006). Specific racemases convert the L-amino acids into the respective D-amino acids. In the following membrane- bound steps, MraY (Ikeda et al., 1991) catalyzes the transfer of the phospho-MurNAc- pentapeptide moiety of UDP-MurNAc-pentapeptide to the lipid carrier undecaprenyl- phosphate (C55-P or bactoprenol phosphate) on the inner side of the cytoplasmic membrane (Bouhss et al., 2008), thereby generating MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenyl (lipid I) and uridinmonophosphate (UMP). The subsequent addition of GlcNAc from UDP-GlcNAc to lipid I by MurG (Mengin-Lecreulx et al., 1991), which is also associated with the membrane, yields the final peptidoglycan precursor GlcNAc-MurNAc- (pentapeptide)-pyrophosphoryl-undecaprenyl, lipid II. The complete lipid II complex is then translocated across the hydrophobic environment of the cytoplasmic membrane. This process requires the activity of the lipid II flippase FtsW, an integral membrane protein that was recently identified as a transporter of the lipid-linked peptidoglycan precursors on the basis of fluorescence studies using E. coli membrane vesicles (Mohammadi et al., 2011; Van Dam et al., 2007). FtsW homologues, such as RodA and SpoVE, are also likely to be involved in the translocation of lipid II in B. subtilis. In the last stage of the peptidoglycan synthesis, which occurs on the exterior side of the cell surface, murein synthases catalyze the polymerization of the translocated disaccharide-pentapeptide units of lipid II by incorporation into the preexisting peptidoglycan.
Fig. 5. Synthesis of peptidoglycan in E. coli. The amino sugars of the glycan strands are indicated by: GlcNAc, N-acetylglucosamine (grey circle); MurNAc, N-acetylmuramic acid (grey square); 1,6-anhydroMurNAc, MurNAc with an intramolecular 1,6-glycosidic bond (grey square with a cap). The stem peptides L-Ala-γ-D-Glu- m-DAP-D-Ala-D-Ala are abbreviated with Ala-Glu-DAP-Ala-Ala. For details on the specificity and function of enzymes, see the text.
In E. coli, the membrane-bound murein synthases are either monofunctional transglycosylases, bifunctional transglycosylase/transpeptidases or monofunctional transpeptidases (Vollmer and Bertsche, 2008). In the transglycosylation reaction, the linear glycan strand, that contains repeating disaccharide-peptide units, is elongated by the transfer of the lipid-linked peptidoglycan strand on lipid II (Lovering et al., 2007; Yuan et al., 2007).
Accordingly, the reducing end of MurNAc on the nascent peptidoglycan strand and the GlcNAc moiety of lipid II are linked by a β-1,4-glycosidic bond. As a result, undecaprenyl- pyrophosphate is liberated and transported back to the cytoplasmic side of the inner membrane. It has to be regenerated by dephosphorylation yielding undecaprenyl-phosphate to become available again as lipid carrier for the synthesis of lipid I and lipid II (Cain et al., 1993; Ghachi et al., 2005; Tatar et al., 2007; Touzé et al., 2008). The newly synthesized
glycan strand will then be incorporated into the growing cell wall structure by the activity of transpeptidases, which catalyze the formation of cross-linkages between adjacent stem peptides. Because of the absence of energy sources such as ATP outside the cytoplasmic membrane, the hydrolysis of the D-Ala-D-Ala bond of one stem peptide results in the formation of an enzyme-substrate intermediate with concomitant release of the terminal
D-Ala, but also provides the energy required for the transpeptidation reaction. Therefore, the transpeptidation reaction usually ends with the transfer of the tetrapeptidyl group to the amino group of m-DAP of an acceptor stem peptide. This implies that a tight coordination between the activities of transpeptidases and transglycosylases regulates the formation of the cross- linked peptidoglycan sacculus.
Peptidoglycan synthases with transpeptidase activity are often referred to as penicillin binding proteins (PBPs) due to their ability to bind penicillin and other β-lactam antibiotics, which mimic the D-Ala-D-Ala structure of the stem peptide with their β-lactam rings. PBPs are classified as high-molecular weight (HMW) PBPs and low-molecular weight (LMW) PBPs (Sauvage et al., 2008; Scheffers and Pinho, 2005). All HMW PBPs present two domains on the periplasmic side, a C-terminal penicillin binding domain with transpeptidase activity and an N-terminal domain, which is anchored to the cytoplasmic membrane.
Depending on the structure and the catalytic activity of their N-terminal domain, HMW PBPs can be subdivided into two classes, A and B (Goffin and Ghuysen, 1998; Macheboeuf et al., 2006). HMW class A PBPs are bifunctional enzymes harboring a transglycosylase domain at the N-terminus, which allows them to catalyze the polymerization of the glycan strands and to form cross-linkages between the stem peptides. By contrast, HMW class B PBPs are solely transpeptidases, since they have a N-terminal domain of unknown function. Furthermore, many of the LMW PBPs are rather D,D-carboxypeptidases and D,D-endopeptidases, but not transpeptidases. These seemingly nonessential and enzymatically redundant
D,D-carboxypeptidases are most often involved in the peptidoglycan maturation by cleaving off the terminal D-Ala residue from the stem peptides, thereby regulating the extent of cross- linkages (Ghosh et al., 2008; Ghuysen, 1991; Massova and Mobashery, 1998).
The number of PBPs in bacteria is variable. Although E. coli possess 12 PBPs, only two of them, the class A PBP1A and PBP1B, are considered to be major bifunctional transpeptidase/transglycosylase enzymes, since the inactivation of both is lethal in contrast to that of at least 8 other PBPs with probably redundant activities present in this organism (Denome et al., 1999). However, the mutation of PBP1 of B. subtilis seems to cause slow
growth, abnormal cell morphology and the formation of asymmetric septa (Scheffers and Errington, 2004).
1.4. Cell wall growth and turnover
The task of maintaining and remodeling the cell wall during growth and division by inserting new glycan strands into the preexisting structure is accomplished by cell wall modifying enzymes, which include a number of hydrolases.
Specific hydrolases are essential for cell growth and exist for most connecting building blocks in the peptidoglycan. These enzymes can be classified into three types depending on the specific bond cleaved by the enzyme (Fig. 6). There are glycan strand-hydrolyzing enzymes such as muramidases (lysozyme-related and/or lytic transglycosylases) and glucosaminidases. Peptide bonds within the stem peptides of the peptidoglycan structure are hydrolyzed by endopeptidases and carboxypeptidases. Furthermore, (anhydro-)N-acetylmuramic acid-L-Ala amidases cleave the junction between the glycan strands and the linked stem peptides. Besides their important role in the growth of the cell wall, peptidoglycan hydrolases are also proposed to be involved in other cellular processes such as cell separation, turnover and recycling of cell wall components or sporulation (Scheffers and Pinho, 2005).
Several models for the growth of the peptidoglycan sacculus have been proposed for Gram-negative and Gram-positive bacteria, since the former have a thick multilayered cell wall compared to the predominantly monolayered structure of the latter. A safe enlargement of the sacculus can only be ensured during growth, when newly synthesized material is incorporated and cross-linked before the existing stress-bearing peptidoglycan is cleaved. This process is called the “make-before-break” strategy (Koch and Doyle, 1985). For Gram- positive bacteria, it has been shown that labeled peptidoglycan precursors are incorporated close to the inner membrane and are gradually displaced within the cell wall to the outside according to an “inside-to-outside” growth mechanism (Koch and Doyle, 1985). Therefore, new material is inserted in an unextended state, randomly along the cell cylinder, and underneath the stress-bearing peptidoglycan layers. This is followed by dissolution of the outermost layers, which are assumed to be more susceptible to the activity of peptidoglycan hydrolases at the cell surface, and their release into the growth medium. As a result of continuous peptidoglycan synthesis, newly synthesized material would be pushed outward and becomes stretched due to surface tension caused by the turgor pressure. In this model, the
cell wall is always steady and strong during growth as long as the covalently closed layers of the peptidoglycan are formed before the older and outer ones are degraded to prevent rupture of the cell wall.
Fig. 6. Peptidoglycan hydrolyzing enzymes in E. coli or B. subtilis. The arrows indicate the cleavage sites of the different hydrolases. Glycosidic bonds in the peptidoglycan are hydrolyzed by glucosaminidases and muramidases, yielding GlcNAc (grey circle) and disaccharides containing among MurNAc (grey square) or 1,6-anhydroMurNAc (grey square with cap), respectively. Amidases, (1,6-anhydro-)N-acetylmuramic acid-L-Ala amidases, hydrolyze the amide bonds between the lactyl group of (1,6-anhydro-)MurNAc and the N-terminal L-alanine of the stem peptides, thereby releasing oligopeptides. In contrast to endopeptidases, which cleave specific peptide bonds within the stem peptides, carboxypeptidases remove only C-terminal D- or L-amino acids.
The stem peptides L-Ala-γ-D-Glu-m-DAP-D-Ala-D-Ala are abbreviated with Ala-Glu-DAP-Ala-Ala. Figure was adapted from (Mayer, 2012).
Based on the “inside-to-outside” growth mechanism of Gram-positive bacteria, even the predominantly monolayered peptidoglycan sacculus of Gram-negative bacteria follows the
“make-before-brake” strategy. Accordingly, the “three-for-one” model has been proposed for the peptidoglycan synthesis in E. coli (Höltje, 1998) (Fig. 7A). This model suggests that three new glycan strands are synthesized and cross-linked to each neighbor, whereby a triplet is formed. This is achieved by transglycosylation and transpeptidation catalyzed by a dimer of a bifunctional PBP (PBP1A or PBP1B) as well as a monofunctional PBP (PBP1C) (Bertsche et al., 2005; Born et al., 2006; Schiffer and Höltje, 1999). Now, the triplet is covalently attached in a relaxed conformation below an existing glycan strand, the so-called docking strand, in the sacculus. A dimer of a transpeptidase (PBP2 or PBP3) cross-links the glycan strands on the outside of the triplet to both sides of the docking strand (Spratt, 1975). Upon simultaneous removal of the docking strand by degradation to monomeric disaccharide peptide subunits by
the concerted action of a dimer of an endopeptidase (PBP4 or PBP7) (Korat et al., 1991;
Romeis and Höltje, 1994a) and a lytic transglycosylase, the triplet of glycan strands is pulled into the stress-bearing sacculus by the force of the internal pressure of the cell.
Fig. 7. Proposed model for the peptidoglycan synthesis in Gram-negative bacteria. (A) The “three-for-one”
growth model. A triplet of three newly synthesized, cross-linked glycan strands is covalently attached in an unextended state to the free amino groups of m-DAP (A2pm) present in the stem peptides on both sides of an
“old” docking strand. Specific cleavage of the preexisting cross-linkages results in the removal of the docking strand followed by the insertion of the triplet into the stress-bearing peptidoglycan sacculus due to surface tension. (B) According to the “three-for-one” model, a multienzyme complex consisting of peptidoglycan synthases and hydrolases slides along the docking strand to synthesize the peptidoglycan triplet. While a monofunctional transglycosylase (TG) polymerizes the middle strand, two bifunctional transpeptidase/transglycosylases (TP/TG) polymerize the two outer strands and cross-link them to the middle strand. After its attachment to the existing peptidoglycan by two monofunctional transpeptidases (TP), the docking strand is removed by a lytic transglycosylase (TG) and two endopeptidases (EP) and/or two amidases (AM). Figure (A) was taken from (Vollmer and Höltje, 2001) and (B) from (Höltje, 1998).
Interestingly, the localization of the enzymes reflects the “inside-to-outside” growth mechanism of the “three-for-one” model in E. coli. The polymerizing enzymes (PBP1A, 1B, 1C, 2, 3) that form the new peptidoglycan layers are bound to the cytoplasmic membrane, whereas most lytic transglycosylases that act on the outer layers are lipoproteins anchored to the outer membrane. In this regard, the “three-for-one” model predicts that an interaction of peptidoglycan synthases (i.e., transpeptidases and transglycosylases) and peptidoglycan hydrolases with each other must be coordinated by the formation of a multienzyme complex to keep the activities of both in a balanced ratio (Höltje, 1996, 1998) (Fig. 7B). In agreement with the “make-before-break” strategy, peptidoglycan hydrolases in the multienzyme complex should be arranged behind the peptidoglycan synthases, thus ensuring that the insertion of new material precedes the hydrolysis of preexisting peptidoglycan. This allows a safe enlargement of the stress-bearing peptidoglycan sacculus. An evidence for the existence of this multienzyme complex in E. coli was presented by the detection of specific protein-protein
interactions between different PBPs, peptidoglycan hydrolases and PBPs, as well as PPBs and structural proteins (Bhavsar and Brown, 2006; Romeis and Höltje, 1994b; Vollmer and Bertsche, 2008; Vollmer et al., 1999; von Rechenberg et al., 1996). The results of these studies suggest the presence of two multienzyme complexes, which were predicted to be involved in cell elongation and cell division, respectively. So far, a clear evidence for a multienzyme complex in Gram-positive bacteria has not been demonstrated.
Peptidoglycan synthesis might be directed in most rod-shaped bacteria, including E. coli and B. subtilis, by interaction of the intracellular cytoskeleton acting as a scaffold that recruits and assembles the multienzyme complex required for cell wall growth. During cell elongation, the actin-like cytoskeletal elements MreB in Gram-negative bacteria and its paralogs in Gram-positive bacteria are essential for peptidoglycan synthesis along the cylindrical sidewall to maintain the rod shape of the cell. They appear to form helical filaments on the inner side of the cytoplasmic membrane extending from one cell pole to the other (Jones et al., 2001). As visualized by fluorescence microscopy in B. subtilis, peptidoglycan insertion into the existing sacculus was observed to occur in a pattern similar to that formed by Mbl (Daniel and Errington, 2003). In contrast, total internal reflection fluorescence microscopy (TIRFM) is a more sensitive method for imaging events near the cell surface. Studies that based on this technique revealed that during exponential growth, MreB isoforms in B. subtilis (MreB, Mbl and MreBH) did not form helical structures but assembled into discrete patches that moved bidirectionally along peripheral tracks perpendicular to the cell axis (Domínguez-Escobar et al., 2011). In the absence of Mbl, peptidoglycan synthesis is localized at mid-cell. Accompanied by the accumulation of the bacterial tubulin-homolog FtsZ (Bi and Lutkenhaus, 1991), a contractile ring-like structure is polymerized that presumably redirects the multienzyme complex to the division site. This switch from cell elongation to cell division depends on remodeling of the multienzyme complex as illustrated for E. coli (Vollmer and Bertsche, 2008) (Fig. 8).
Cell growth and division of bacteria is accompanied by a massive release of peptidoglycan fragments, so-called muropeptides, from the stressed, outermost layers of the cell wall. This process that is termed cell wall turnover appears as an intrinsic consequence of a growth mechanism, which couples the degradation of the cell wall with the simultaneous insertion of newly synthesized material. Therefore, cell wall turnover occurs as a result of the need for the cell to expand its surface. It has been studied extensively in Gram-positive bacteria (Doyle et al., 1988).
Fig. 8. Model for the peptidoglycan synthesis complexes controlled by the cytoskeletal elements MreB and FtsZ in E. coli. Indicated are the specific composition and cellular localization of the subcomplexes. During cell elongation, synthesis (MraY and MurG) of peptidoglycan precursors is followed by their incorporation (PBPs) at the filament of MreB. Amidases (AMI), lytic transglycosylases (LT) and endopeptidases (EP) are responsible for the hydrolysis of older peptidoglycan layers. Formation of the FtsZ-ring at mid-cell initiates the assembly of the multienzyme complexes for cell division to generate a septum that matures to two new cell poles. Figure was taken from (Vollmer and Bertsche, 2008).
During vegetative growth, B. subtilis and other Gram-positive species turn over up to 50% of the existing cell wall material in one generation (Boothby et al., 1973; Chaloupka et al., 1962a; Chaloupka et al., 1962b; Mauck et al., 1971; Mauck and Glaser, 1970; Rogers, 1967).
The liberated degradation products are released into the growth medium. It has also been observed that the process of cell wall turnover is not restricted solely to peptidoglycan but also refers to the loss of peptidoglycan-attached polymers, such as teichoic acids and capsular polysaccharides at the cell surface (Hancock and Cox, 1991; Mauck et al., 1971; Wong et al., 1974). Peptidoglycan turnover in B. subtilis proceeds rapidly along the cell cylinder, since the cell poles are metabolically inert (Mobley et al., 1984).
Lysis of the peptidoglycan proceeds by the induction of the autolytic system with its hydrolyzing enzymes (autolysins), which contribute to the cleavage of the covalent bonds in the intact sacculus. For B. subtilis, more than 35 candidate peptidoglycan hydrolases have been identified on the basis of amino acid sequence similarities, and classified as N-acetylglucosaminidases (digesting GlcNAc-MurNAc linkage), N-acetylmuramic acid-L-Ala amidases (digesting MurNAc-L-Ala linkage), D,L-endopeptidases (digesting D-Glu-m-DAP
linkage) and L,D-endopeptidases (digesting L-Ala-D-Glu linkage) (Kunst et al., 1997; Smith et al., 2000). Among the best characterized autolysins, the amidase LytC (Kuroda and Sekiguchi, 1991) and the glucosaminidases LytD (Margot et al., 1994; Rashid et al., 1995) and LytG, an exo-N-acetylglucosaminidase, (Horsburgh et al., 2003) are assumed to be responsible for more than 95% of the autolytic activity in B. subtilis (Blackman et al., 1998).
Recently, two bifunctional autolysins, B. subtilis CwlT (YddH) (Fukushima et al., 2008) and CwlP (YomI) (Sudiarta et al., 2010a), have been associated with cell wall turnover. The C-terminal domain of both enzymes exhibits D,L- and D,D-endopeptidase activity, respectively, whereas the N-terminal domain is an N-acetylmuramidase that hydrolyzes the linkage of MurNAc-GlcNAc, thereby releasing tetra-, hexa- and octasaccharides from purified glycan strands of B. subtilis peptidoglycan. Glycan strand-cleaving enzymes such as muramidases are thought to be responsible for turnover of peptidoglycan in Gram-positive bacteria (Atrih et al., 1999). Lytic transglycosylases act like muramidases because they also target the β-1,4-glycosidic linkage between the MurNAc and GlcNAc residue of peptidoglycan, but they catalyze an intramolecular transglycosylation reaction by formation of a 1,6-anhydro ring at the terminal MurNAc residue, thereby producing anhydromuropeptides (Höltje et al., 1975; Scheurwater et al., 2008) (Fig. 9). Analysis of the peptidoglycan structure of B. subtilis revealed that anhydromuropeptides represent only 0.4% of the total cell wall degradation products, in contrast to the large amounts in E. coli (80%) (Kitano et al., 1986), and thus lytic transglycosylases are thought to have only a minor impact on the peptidoglycan turnover in Gram-positive bacteria (Atrih et al., 1999). Therefore, CwlQ (YjbJ) was identified as another bifunctional cell wall hydrolase, but the first one in B. subtilis that exhibits a strong lytic transglycosylase activity besides muramidase activity (Sudiarta et al., 2010b).
Cell wall turnover in the Gram-negative bacterium E. coli has been underestimated for a long time, since only a minor portion of about 8% of released muropeptides was isolated from the culture supernatants (Goodell and Schwarz, 1985). But E. coli also degrades almost half of its peptidoglycan in each generation. The majority of the turnover products have been found to accumulate in the periplasm as a result of turnover, from where they are continuously transported into the cytoplasm and reutilized (i.e., recycled) efficiently (Goodell, 1985). The main turnover products are anhydromuropeptides such as GlcNAc-anhydroMurNAc-L-Ala-γ-
D-Glu-m-DAP(-D-Ala) (Höltje, 1998). Their monomeric structure indicates that they are in principle products of lytic transglycosylases and endopeptidases (PBP4, 7 and MepA) (Keck et al., 1990; Korat et al., 1991; Romeis and Höltje, 1994a), released as a result of the vegetative growth process. Lytic transglycosylases represent the major class of muramidases
in Gram-negative bacteria. At least one soluble lytic transglycosylase (Slt70) and seven membrane-bound lytic transglycosylases (MltA, B, C, D, E (EmtA), F), which are lipoproteins anchored to the inner leaflet of the outer membrane, have been described in E. coli (Ehlert et al., 1995; Höltje et al., 1975; Kraft et al., 1998; Lommatzsch et al., 1997;
Vollmer and Bertsche, 2008; Vollmer et al., 2008b). Most lytic transglycosylases appear to act as exo-acting enzymes (Slt70 and MltA, B), removing disaccharide-peptide repeats from the 1,6-anhydroMurNAc glycan strand end (Vollmer and Bertsche, 2008). The only known exception in E. coli is MltE (EmtA), which is proposed to be an endo-specific lytic transglycosylase (Kraft et al., 1998). However, Slt70 found in the periplasm of E. coli is predicted to be the principle lytic transglycosylase responsible for the turnover of peptidoglycan.
Fig. 9. Cleavage of the β-1,4-glycosidic linkage in glycan strands of peptidoglycan by glucosaminidases (1) or muramidases such as lysozyme-like transglycosylases (2) or lytic transglycosylases (3). Reaction of the lytic transglycosylases is accompanied by an intramolecular transglycosylation at the MurNAc residue, resulting in the formation of a 1,6-anhydro bond. R, stem peptide attached to the lactyl residue of MurNAc. Figure was taken from (Vollmer et al., 2008b).
Furthermore, E. coli possesses five MurNAc-L-Ala amidases, i.e., three periplasmic enzymes (AmiA, B, C) (Heidrich et al., 2001), one outer membrane-anchored enzyme (AmiD) (Uehara and Park, 2007), and one cytoplasmic enzyme (AmpD) (Höltje et al., 1994). All amidases hydrolyze the amide bonds between the glycan strands and the stem peptides with concomitant turnover of the peptide moiety in form of tetra- or tripeptides besides the anhydrodisaccharides from the sacculus. As a result of the combined lytic activities, more than 80 different types of muropeptides were identified in peptidoglycan preparations from E. coli (Glauner et al., 1988).
1.5. Recycling of peptidoglycan turnover products
As mentioned above, peptidoglycan undergoes extensive degradation and resynthesis during bacterial growth, thereby resulting in a massive turnover of almost 50% each generation (Doyle et al., 1988). The muropeptides released from the endogenous cell wall are subject to an efficient recycling pathway in E. coli (Goodell, 1985; Goodell and Schwarz, 1985). Indeed, peptidoglycan recycling is not crucial to cell survival under laboratory conditions, but provides the formation of new precursors for peptidoglycan biosynthesis. The pathways for recycling of peptidoglycan turnover products have been investigated in great detail in the Gram-negative bacterium E. coli and hence almost a dozen genes encoding enzymes involved in this process have been identified (Park and Uehara, 2008) (Fig. 10).
The major turnover products from peptidoglycan of E. coli, being 1,6-anhydro- disaccharide-tetra- and tripeptides (Höltje, 1998), are transported into the cytoplasm by AmpG, a secondary transporter that recognizes the GlcNAc-anhydroMurNAc disaccharide carrying a stem peptide as well as the free disaccharide (Cheng and Park, 2002; Jacobs et al., 1994). The peptide moiety of the anhydromuropeptides is liberated by the cytoplasmic amidase AmpD that acts solely on anhydroMurNAc-L-Ala bonds and therefore does not hydrolyze peptidoglycan precursors such as UDP-MurNAc-pentapeptide, which is also present in the cytoplasm (Höltje et al., 1994; Jacobs et al., 1995). The tetrapeptide L-Ala-γ-D- Glu-m-DAP-L-Ala will be processed further by the L,D-carboxypeptidase LdcA, which removes the C-terminal L-Ala (Templin et al., 1999). The resulting tripeptide L-Ala-γ-D-Glu- m-DAP is reutilized by the muropeptide ligase Mpl, linking it to UDP-MurNAc of the peptidoglycan synthesis pathway (Mengin-Lecreulx et al., 1996). Therefore, LdcA from E. coli is essential for survival, since in its absence tetrapeptides accumulate and are used by Mpl to form UDP-MurNAc-tetrapeptide, which will be incorporated into the peptidoglycan sacculus instead of UDP-MurNAc-pentapeptide (Templin et al., 1999). Only the latter is required for cross-linking the glycan strands of peptidoglycan and the loss of it leads to a weakened cell wall sacculus. Accordingly, ldcA mutants appear as thick, oval cells that usually lyze during the late exponential phase of growth. Interestingly, a mutation in the ldcA gene constitutes an exception to those in the genes known to be involved in recycling of peptidoglycan because it is the only one that shows an obvious phenotype. Besides the intact anhydromuropeptides, tri- or tetrapeptides also occur as turnover products, derived from the peptidoglycan during maturation by the action of the periplasmic amidases AmiA, B and/or C that hydrolyze the MurNAc-L-Ala bond (Heidrich et al., 2001; Priyadarshini et al., 2006).
Fig. 10. Schematic of peptidoglycan turnover and recycling pathways of E. coli. During cell wall turnover, lytic transglycosylases (LT), endopeptidases (EP) and amidases release peptidoglycan fragments from the endogenous cell wall. While anhydromuropeptides are taken up into the cell by the AmpG permease, cell wall- derived amino sugars, including GlcNAc (grey circle), MurNAc (grey square) and 1,6-anhydroMurNAc (grey square with cap) are transported by the specific phosphotransferase systems (PTS) MurP and NagE, respectively, and the uptake of the liberated peptide moiety of peptidoglycan requires the oligopeptide transport system MppA/Opp. All of the turnover products are recycled within the cytoplasm, thereby returning them either to the glycolysis pathway or the peptidoglycan synthesis pathway. For a detailed description of enzymes and substrates involved in the pathways, see text. The stem peptides L-Ala-γ-D-Glu-m-DAP-D-Ala-D-Ala are abbreviated with Ala-Glu-DAP-Ala-Ala. OM, outer membrane, IM, inner membrane. Figure was taken from (Jaeger and Mayer, 2008a).
The free tripeptides are taken up by the oligopeptide ABC-transporter Opp that recruits the periplasmic binding protein MppA (Park, 1993; Park et al., 1998). To a lesser extent, the
L-Ala-γ-D-Glu-m-DAP tripeptide can also be degraded to single amino acids. This pathway involves in turn MpaA, a D,L-carboxypeptidase (Uehara and Park, 2003), which releases m-DAP, and the epimerase YcjG, which converts the resulting L-Ala-γ-D-Glu dipeptide to
L-Ala-L-Glu (Schmidt et al., 2001). This is then hydrolyzed by the peptidase PepD (Schroeder et al., 1994), yielding L-Ala and L-Glu.
The amino sugars of the imported turnover products were also shown to be recycled.
NagZ, a β-N-acetylglucosaminidase, hydrolyzes the β-1,4-glycosidic bond of the GlcNAc- anhydroMurNAc disaccharide to release GlcNAc and anhydroMurNAc in the cytoplasm (Vötsch and Templin, 2000), and is also active on substrates containing attached peptides. To make the free amino sugars available for metabolic reutilization, the cytoplasmic GlcNAc and
anhydroMurNAc must be converted to GlcNAc-6-phosphate. While GlcNAc is directly phosphorylated to GlcNAc-6-phosphate by NagK (Uehara and Park, 2004), an N-acetylglucosamine kinase, anhydroMurNAc is first phosphorylated by a specific anhydro-N-acetylmuramic acid kinase, AnmK (Uehara et al., 2005). In addition to phosphorylation, AnmK catalyzes the simultaneous cleavage of the 1,6-anhydro ring of anhydroMurNAc, thereby generating MurNAc-6-phosphate. The latter is then converted to GlcNAc-6-phosphate by the MurQ etherase of E. coli, which hydrolyzes the ether bond between D-lactate and the GlcNAc moiety of MurNAc-6-phosphate (Jaeger et al., 2005;
Uehara et al., 2006). Alternatively, both GlcNAc and MurNAc can be taken up directly into the cell with concomitant phosphorylation at the 6-position by the specific phosphotransferase system (PTS) transporters NagE and MurP, respectively (Dahl et al., 2004; Plumbridge, 2009). Besides recycling anhydromuropeptides, MurP and MurQ are also essential for E. coli to grow on external MurNAc as sole source of carbon (Jaeger and Mayer, 2008b). The following deacetylation of GlcNAc-6-phosphate by NagA yields glucosamine-6-phosphate (GlcN-6-phosphate) (White and Pasternak, 1967), which can either be shuttled into the glycolysis pathway upon deamination to fructose-6-phosphate by NagB (White, 1968), or be converted to GlcN-1-phosphate by GlmM, a phosphoglucosamine mutase. (Mengin-Lecreulx and van Heijenoort, 1996). Thereafter, the bifunctional GlmU transferase catalyzes both the reacetylation of GlcN-1-phosphate and also the activation of the resulting product GlcNAc-1- phosphate in the presence of UTP to form UDP-GlcNAc, the first dedicated precursor for the peptidoglycan synthesis pathway (Mengin-Lecreulx and van Heijenoort, 1993, 1994).
Interestingly, although the process of peptidoglycan turnover has been reported for many Gram-positive bacteria (Doyle et al., 1988), it has never been demonstrated whether turnover products are also recycled in these organisms. Since large amounts of peptidoglycan fragments were isolated from the growth medium, presumably due to the lack of an outer membrane that usually holds in the components, it was assumed that Gram-positive bacteria do not recycle turnover products as they are broken down from peptidoglycan (Mauck et al., 1971; Mauck and Glaser, 1970). In Gram-positive bacteria like B. subtilis, specific orthologs of recycling enzymes that are well-conserved in Gram-negative bacteria are apparently not present. Indeed, an AmpG ortholog, which is required for the uptake of anhydromuropeptides, and orthologs of recycling enzymes of E. coli, such as the AmpD amidase and the AnmK kinase, to process turnover products have not yet been identified in B. subtilis (Park and Uehara, 2008). However, B. subtilis possesses orthologs for the recovery of MurNAc, including the MurNAc-specific phosphotransferase system MurPBs (YbbF) and the MurNAc-
6-phosphate etherase MurQBs (YbbI), as well an ortholog of the GlcNAc-specific phosphotransferase system NagE. Interestingly, MurQBs and MurPBs are encoded in a putative operon in B. subtilis next to two orthologous genes encoding for the β-N-acetylglucosaminidase NagZBs (YbbD) and the N-acetylmuramic acid-L-Ala amidase AmiEBs (YbbE) (Litzinger et al., 2010a) (Fig. 11). AmiEBs is an ortholog of E. coli AmpD.
But in contrast to NagZ and AmpD of E. coli, both enzymes are secreted to the exterior due to an N-terminal signal sequence, while they are noncovalently bound to the cell wall of B. subtilis (Litzinger et al., 2010a). According to the data from sequence homologies and studies on the function of NagZBs and AmiEBs, a putative cell wall recycling pathway of B. subtilis was recently proposed, as described below (Litzinger et al., 2010a) (Fig. 11).
Fig. 11. Model of the proposed muropeptide recovery pathway of B. subtilis and organization of the corresponding gene cluster. In B. subtilis, muropeptides like GlcNAc-MurNAc-peptides (GlcNAc, grey circle;
MurNAc, grey square; stem peptide L-Ala-γ-D-Glu-m-DAP-D-Ala is abbreviated with Ala-Glu-DAP-Ala) released from the endogenous cell wall during growth are further processed in the extracellular compartment by NagZ (YbbD) and AmiE (YbbE), as indicated. The recovery of MurNAc requires the specific phosphotransferase system (PTS) MurP (YbbF) and the following conversion by MurQ (YbbI). YbbH (MurR) is a putative MurNAc-6-phosphate-specific transcriptional regulator, and the function of YbbC is so far unknown.
For more information on the pathway, see text. CM, cytoplasmic membrane; EIIA, enzyme IIA; HPr, histidine- containing phosphocarrier protein; EI, enzyme I; PEP, phosphoenolpyruvate. Figure was adapted from (Litzinger et al., 2010a).
