Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli.

The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.     

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation.     

Streptomyces species with madurose (3-O-methyl-D-galactose) as a whole-cell sugar

Madurose, an actinomycete whole-cell sugar, was found in the strains of the genus Streptomyces: three strains of S. platensis, one strain each of S. platensis subsp. malvinus, and S. albus subsp. albus. The sugar was isolated from the hydrolysate of S. platensis IFO 14008 cells, and was identified as madurose (3-O-methyl-D-galactose) by chromatographic analyses, ¹H-NMR spectrometry, mass spectrometry as its alditol acetate, and demethylation with boron trichloride. Cell walls of the strain contained peptidoglycan and teichoic acids. ll-Diaminopimelic acid, glycine, glutamic acid, and alanine were present in the peptidoglycan fraction in molar ratios of 1.0:1.3:1.2:2.3. Madurose was detected in the teichoic acid fraction, which was composed of phosphorus, glycerol, galactose, and madurose in molar ratios of 9.3:8.5:2.9:1.0. Thus, madurose was found in the glycerol teichoic acid moiety of the cell walls of this strain.Abbreviations PC paper chromatography - TLC thin-layer chromatography - HPLC high performance liquid chromatography - GLC gas-liquid chromatography - TCA trichloroacetic acid     

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.     

Peptidoglycan synthesis and structure in Staphylococcus haemolyticus expressing increasing levels of resistance to glycopeptide antibiotics.

The structures of cytoplasmic peptidoglycan precursor and mature peptidoglycan of an isogenic series of Staphylococcus haemolyticus strains expressing increasing levels of resistance to the glycopeptide antibiotics teicoplanin and vancomycin (MICs, 8 to 32 and 4 to 16 microg/ml, respectively) were determined. High-performance liquid chromatography, mass spectrometry, amino acid analysis, digestion by R39 D,D-carboxypeptidase, and N-terminal amino acid sequencing were utilized. UDP-muramyl-tetrapeptide-D-lactate constituted 1.7% of total cytoplasmic peptidoglycan precursors in the most resistant strain. It is not clear if this amount of depsipeptide precursor can account for the levels of resistance achieved by this strain. Detailed structural analysis of mature peptidoglycan, examined for the first time for this species, revealed that the peptidoglycan of these strains, like that of other staphylococci, is highly cross-linked and is composed of a lysine muropeptide acceptor containing a substitution at its epsilon-amino position of a glycine-containing cross bridge to the D-Ala 4 of the donor, with disaccharide-pentapeptide frequently serving as an acceptor for transpeptidation. The predominant cross bridges were found to be COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2. Liquid chromatography-mass spectrometry analysis of the peptidoglycan of resistant strains revealed polymeric muropeptides bearing cross bridges containing an additional serine in place of glycine (probable structures, COOH-Gly-Ser-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Ser-Gly-NH2). Muropeptides bearing an additional serine in their cross bridges are estimated to account for 13.6% of peptidoglycan analyzed from resistant strains of S. haemolyticus. A soluble glycopeptide target (L-Ala-gamma-D-iso-glutamyl-L-Lys-D-Ala-D-Ala) was able to more effectively compete for vancomycin when assayed in the presence of resistant cells than when assayed in the presence of susceptible cells, suggesting that some of the resistance was directed towards the cooperativity of glycopeptide binding to its target. These results are consistent with a hypothesis that alterations at the level of the cross bridge might interfere with the binding of glycopeptide dimers and therefore with the cooperative binding of the antibiotic to its target in situ. Glycopeptide resistance in S. haemolyticus may be multifactorial.     

Peptidoglycan synthesis and turnover in cell division mutants of Agmenellum.

The synthesis and turnover of peptidoglycan in Agmenellum quadruplicatum was investigated using D-[U-14C]alanine followed by proteolytic digestion. The rate of turnover of alanine in the peptide portion of the peptidoglycan was measured in strain BG-1 and in two division mutants of this strain: one was blocked in cell separation; and the other was a low-temperature, conditional cell division mutant. The peptide portion of peptidoglycan turned over in all three strains tested, but no correlation was observed between septum formation or cell separation and the rate of turnover. Peptidoglycan synthesis was measured during induced division in snake forms of strain SN-29. A stimulation of peptidoglycan synthesis was observed during the period of cross-wall formation, even in the absence of new protein synthesis. Thus in A. quadruplicatum, cross-wall synthesis is accompanied by a stimulation of peptidoglycan synthesis.     

Differential methicillin susceptibilities of peptidoglycan syntheses in methicillin-resistant Staphylococcus aureus.

The mechanism of staphylococcal resistance to methicillin is unknown. Peptidoglycan synthesis was studied in a methicillin-resistant and a derived methicillin-sensitive Staphylococcus aureus strain. Although the methicillin minimum inhibitory concentration for growth of the methicillin-resistant strain was 1,600 micrograms/ml, peptidoglycan synthesis by the organism incubated in a wall synthesis solution was inhibited about 90% by 5 micrograms of methicillin per ml. In contrast, high concentrations of methicillin added to actively growing cultures of the methicillin-resistant strain had little effect on growth or peptidoglycan synthesis. Peptidoglycan synthesis in chloramphenicol-treated cultures was more susceptible to methicillin than it was in actively growing cultures of the methicillin-resistant strain. It is proposed that in this strain cell wall thickening peptidoglycan synthesis which predominates in cell wall synthesis solution and chloramphenicol-treated cultures is methicillin sensitive, whereas peptidoglycan synthesis involved in cell division, primarily in the region of the septum, which predominates in actively growing cultures is methicillin resistant. Both cell wall thickening and septal peptidoglycan syntheses are methicillin sensitive in the methicillin-sensitive strain.     

Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum.

The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls.     

Incorporation of LL-diaminopimelic acid into peptidoglycan of Escherichia coli mutants lacking diaminopimelate epimerase encoded by dapF.

Recently a dapF mutant of Escherichia coli lacking the diaminopimelate epimerase was found to have an unusual large LL-diaminopimelic acid (LL-DAP) pool as compared with that of meso-DAP (C. Richaud, W. Higgins, D. Mengin-Lecreulx, and P. Stragier, J. Bacteriol. 169:1454-1459, 1987). In this report, the consequences of high cellular LL-DAP/meso-DAP ratios on the structure and metabolism of peptidoglycan were investigated. For this purpose new efficient high-pressure liquid chromatography techniques for the separation of the DAP isomers were developed. Sacculi from dapF mutants contained a high proportion of LL-DAP that varied greatly with growth conditions. The same was observed with the two DAP-containing precursors, UDP-N-acetylmuramyl-tripeptide and UDP-N-acetylmuramyl-pentapeptide. The limiting steps for the incorporation of LL-DAP into peptidoglycan were found to be its addition to UDP-N-acetylmuramyl-L-alanyl-D-glutamate and the formation of the D-alanyl-DAP cross-bridges. The Km value of the DAP-adding enzyme for LL-DAP was 3.6 x 10(-2) M as compared with 1.1 x 10(-5) M for meso-DAP. When isolated sacculi were treated with Chalaropsis N-acetylmuramidase and the resulting soluble products were analyzed by high-pressure liquid chromatography, the proportion of the main peptidoglycan dimer was lower in the dapF mutant than in the parental strain. Moreover, the proportion of LL-DAP was higher in the main monomer than in the main dimer, where it was almost exclusively located in the donor unit. There are thus very few D-alanyl-LL-DAP cross-bridges, if any. We also observed that large amounts of LL-DAP and N-succinyl-LL-DAP were excreted in the growth medium by the dapF mutant.     

Hydrolysis of a Staphylococcus aureus cell wall peptidoglycan by 209 P lysoamidase

Hydrolysis of Staphylococcus aureus 209 P cell wall peptidoglycan was accompanied by the liberation of 1.3 mol of C-terminal and 1.2 mol of N-terminal glycine per mole of Glu as well as of 0.5 mol of N-terminal and 0.3 mol of C-terminal alanine. Gel chromatography on Sephadex G-25, ion-exchange chromatography on QAE-Sephadex A-50 and paper electrophoresis of S. aureus peptidoglycan hydrolysates gave seven homogeneous fractions; these fractions were structurally defined. Lysoamidase hydrolyzed bonds Mur-Ala, Gly-Gly and Mur-GlcN in the peptidoglycan molecule. Hydrolysis of glycan chains was accompanied by the formation of large fragments, (GlcN-Mur)9 and (GlcN-Mur)28. The lytic effect of lysoamidase on S. aureus peptidoglycan is coupled with bacteriolytic enzymes of lysoamidase: acetmuramyl amidase, glycyl--glycine endopeptidase and acetyl--muramidase.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Isogenic variants of Escherichia coli with altered morphology have peptidoglycan with identical muropeptide composition.

The peptidoglycan compositions of three isogenic morphological mutants of Escherichia coli were determined by high-pressure liquid chromatography analysis. The muropeptide compositions of the peptidoglycan of these mutants were the same, indicating that the shape of E. coli is not (solely) determined by the chemical composition of the peptidoglycan. Furthermore, it appeared that the muropeptide composition of the peptidoglycan was not affected by growth temperature.     

Unstable Escherichia coli L forms revisited: growth requires peptidoglycan synthesis

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the beta-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the beta-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli-and possibly all L forms-have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.     

Interaction between two major outer membrane proteins of Escherichia coli: the matrix protein and the lipoprotein.

The affinity to the matrix protein, one of the major outer membrane proteins of Escherichia coli, for the peptidoglycan was examined of extracting the cell envelope complex at 55 degrees C and 2% sodium dodecyl sulfate containing different amounts of NaCl. It was found that the matrix protein was extracted from the peptidoglycan of a mutant strain (lpo) that lacks another major membrane protein, the lipoprotein, at a lower NaCl concentration than was the matrix protein of the wild-type cell (lpo+). When the envelope fraction of the wild-type strain was treated with trypsin, which is known to cleave the bound-form lipoprotein from the peptidoglycan, the affinity of the matrix protein for the peptidoglycan decreased to the same level as that of the affinity of the matrix protein for the peptidoglycan of the mutant strain. It was further shown that the free-form lipoprotein was also retained in the matrix protein-peptidoglycan complex, although the extent of retention of the free form of the lipoprotein was less than that of the matrix protein. These results indicate that both the free and the bound forms of the lipoprotein are closely associated with the matrix protein and that the bound form of the lipoprotein plays and important role in the association between the matrix protein and the peptidoglycan.     

Myxospore Formation in Myxococcus xanthus: Chemical Changes in the Cell Wall During Cellular Morphogenesis

Vegetative cells of Myxococcus xanthus (strain FB) were induced to form myxospores by the glycerol induction technique. Several structural changes took place in the peptidoglycan during myxospore formation. The percent of the peptidoglycan comprised of monomer (disaccharide peptide) decreased from about 20% to approximately 7%. The proportion of the total diaminopimelic acid possessing a free amino group decreased about 11%. A carbohydrate containing only glucose was found to be bound, possibly covalently, to the vegetative cell and myxospore peptidoglycan. The amount of carbohydrate relative to peptidoglycan decreased by two-thirds during myxospore formation. None of the above changes in the peptidoglycan were observed in a mutant (strain GNI) of M. xanthus which was unable to convert to myxospores when incubated in the glycerol induction medium, or in the parental wild type (FB) when it was incubated in induction medium lacking the myxospore inducer, glycerol.     

Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli.

In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine. The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used. Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid. Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi. For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed. The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values. Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis. The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides. HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi.     

N-acetylputrescine as a characteristic constituent of cyanelle peptidoglycan in glaucocystophyte algae.

Cyanelle peptidoglycan from the glaucocystophyte algae Glaucocystis nostochinearum and Cyanoptyche gloeocystis was investigated by high-performance liquid chromatography of muropeptides, supported by matrix-assisted laser desorption-ionization mass spectrometry. The peptidoglycans of both species are modified with N-acetylputrescine, as has been demonstrated for cyanelle peptidoglycan of Cyanophora paradoxa.     

Purification and properties of bacteriolytic enzymes from Bacillus licheniformis YS-1005 against Streptococcus mutans

To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37 degrees C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product.     

Effects of peptidoglycans from periodontal pathogens on selected biological activities of CD-1 murine peritoneal macrophages

Resident CD-1 murine peritoneal macrophages were exposed to various concentrations of purified peptidoglycan isolated from members of the genera Bacteroides, Eikenella, and Actinomyces. Macrophage viability, the release of lysozyme, acid phosphatase, and prostaglandins E1 and E2 were assayed as a function of peptidoglycan concentration and time. Macrophages responded as a function of peptidoglycan concentration with increased release of acid phosphatase and prostaglandins; all cells remained greater than 90% viable during the course of the experiments. However, concentrations of peptidoglycan greater than 50 micrograms/mL were toxic to the macrophages, while the peptidoglycan from B. capillus strain 925.08 and Actinomyces viscosus strain T14AV consumed complement by both the classical and the alternate pathways. Cellular lysozyme activity and phagocytosis of Saccharomyces cerevisiae were significantly reduced in the presence of peptidoglycan. When viewed by scanning electron microscopy, the activated macrophages were rounded, lacked distinct pseudopod extensions, and possessed an increased number of microvilli and plasma membrane associated vesicles. These morphological alterations occurred as early as 3 h. Transmission electron microscopy revealed the purified peptidoglycan to have been taken up into numerous phagosomes; however, even after 24 h incubation, it was only partially degraded.     

The Listeria monocytogenes serotype 4b autolysin IspC has N-acetylglucosaminidase activity

IspC is a novel peptidoglycan (PG) hydrolase that is conserved in Listeria monocytogenes serotype 4b strains and is involved in virulence. The aim of this study was to establish the hydrolytic bond specificity of IspC. Purified L. monocytogenes peptidoglycan was digested by recombinant IspC and the resulting muropeptides were separated by reverse phase high-performance liquid chromatography. The structure of each muropeptide was determined using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry in combination with MALDI-post-source decay mass spectrometry. The structure of muropeptides resulting from IspC-mediated hydrolysis indicated that IspC has N-acetylglucosaminidase activity. These muropeptides also had a high proportion of N-acetylated glucosamine residues. To determine whether IspC is more effective at hydrolysing N-acetylated peptidoglycan than de-N-acetylated peptidoglycan, a peptidoglycan deacetylase (PgdA) in-frame deletion mutant was created. This mutant was shown to have fully N-acetylated peptidoglycan and was more susceptible to hydrolysis by IspC when compared with the partially de-N-acetylated wild-type peptidoglycan. This indicates that IspC is more efficient when hydrolysing a fully N-acetylated peptidoglycan substrate. The finding that IspC acts as an N-acetylglucosaminidase is consistent with its categorization, based on amino acid sequence, as a member of the GH73 family. As with other members of this family, de-N-acetylation seems to be an important mechanism for regulating the activity of IspC.