Degrees of O-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth

The progress of incorporation of radioactive glucosamine into the O-acetylated and non-O-acetylated sub-units of the insoluble peptidoglycan of growing Neisseria gonorrhoeae was examined. More than 80% of the final degree of cross-linking was achieved within 3 min; the remainder of the process took much longer. Rapid O-acetylation occupied up to 10 min, at which time only about 65% of the maximum value had been reached. There was thus evidence for maturation of peptidoglycan both in regard to cross-linking and to O-acetylation.     

Turnover of the cell wall peptidoglycan during growth of Neisseria gonorrhoeae and Escherichia coli. Relative stability of newly synthesized material

The peptidoglycan of a number of strains of Neisseria gonorrhoeae and Escherichia coli turned over during exponential growth as monitored by the loss of radioactivity (supplied as [14C]glucosamine) from SDS-insoluble material. However, no turnover of the peptide side chains of E. coli peptidoglycan was observed (monitored by diamino[3H]pimelic acid) even though turnover of glycan material was occurring. Turnover rates of 9 to 15% per generation were recorded for all the N. gonorrhoeae strains studied except for the autolytic variant RD5 which showed a higher rate of turnover (20 to 26% per generation). In contrast to previous interpretations, these rates of turnover were not affected by benzylpenicillin, unless sufficient antibiotic was present to affect culture turbidity, when lysis occurred. Examination of the fragments (monomer, dimer and their O-acetylated counterparts, and oligomers) produced by Chalaropsis B muramidase treatment of prelabelled peptidoglycan revealed that no fraction of the peptidoglycan was immune from turnover. However, peptidoglycan pulse-labelled for only 10 min did not show immediate turnover. The lapse of time before turnover commenced was strain dependent, with a maximum value of 1.5 generations. This work confirms that the peptidoglycan of N. gonorrhoeae undergoes a period of maturation and suggests that only mature peptidoglycan turns over.     

Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin

The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The beta-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by beta-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2.5 X MIC).     

Effects of beta-lactam antibiotics on peptidoglycan synthesis in growing Neisseria gonorrhoeae, including changes in the degree of O-acetylation

Low concentrations of beta-lactam antibiotics caused an increased uptake of radioactive glucosamine into the sodium dodecyl sulfate-insoluble peptidoglycan of growing Neisseria gonorrhoeae. There was no appreciable change in the (small) amount of sodium dodecyl sulfate-soluble polymer present in the cultures. The sodium dodecyl sulfate-insoluble product in control cells was only partially dissolved by egg-white lysozyme (about 40%), but could all be released by the Chalaropsis B muramidase. In cells exposed to beta-lactams the proportion of labeled peptidoglycan susceptible to lysozyme increased to 60%. Examination of the Chalaropsis B digests by thin-layer chromatography showed that they contained disaccharide-peptide monomers with and without O-acetylation and bis-disaccharide-peptide dimers with one or two O-acetyl groups, or with none. beta-Lactam antibiotics caused a decrease in the degree of O-acetylation but did not greatly affect the amount of peptidoglycan cross-linking. They also had the effect of enlarging the bacteria and conserving and thickening the septa that could be observed in thin sections under the electron microscope. The relationship between these results and the effects of beta-lactams on in vitro synthesis of peptidoglycan by ether-treated N. gonorrhoeae is discussed.     

Chemical composition and turnover of peptidoglycan in Neisseria gonorrhoeae.

The peptidoglycan of all four colonial types of a number of strains of Neisseria gonorrhoeae constituted 1 to 2% of the dry weight of the cell. The chemical composition of cell types examined was similar with molar ratios of 1:1:2:1:1 for muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid, respectively. Ninety-six percent of the mass of the peptidoglycan was composed of these compounds. A lipoprotein analogous to that observed in Escherichia coli was not detected. The chain length of the glycan varied from 80 to 110 disaccharide units. The peptide contained equimolar amounts of D- and L-alanine. The rate of turnover of peptidoglycan in strain RD5 was 50% per generation. Turnover proceeded without a lag and followed first-order kinetics.     

Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.     

Formation of the glycan chains in the synthesis of bacterial peptidoglycan

The main structural features of bacterial peptidoglycan are linear glycan chains interlinked by short peptides. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), all linkages between sugars being beta,1-->4. On the outside of the cytoplasmic membrane, two types of activities are involved in the polymerization of the peptidoglycan monomer unit: glycosyltransferases that catalyze the formation of the linear glycan chains and transpeptidases that catalyze the formation of the peptide cross-bridges. Contrary to the transpeptidation step, for which there is an abundant literature that has been regularly reviewed, the transglycosylation step has been studied to a far lesser extent. The aim of the present review is to summarize and evaluate the molecular and cellullar data concerning the formation of the glycan chains in the synthesis of peptidoglycan. Early work concerned the use of various in vivo and in vitro systems for the study of the polymerization steps, the attachment of newly made material to preexisting peptidoglycan, and the mechanism of action of antibiotics. The synthesis of the glycan chains is catalyzed by the N-terminal glycosyltransferase module of class A high-molecular-mass penicillin-binding proteins and by nonpenicillin-binding monofunctional glycosyltransferases. The multiplicity of these activities in a given organism presumably reflects a variety of in vivo functions. The topological localization of the incorporation of nascent peptidoglycan into the cell wall has revealed that bacteria have at least two peptidoglycan-synthesizing systems: one for septation, the other one for elongation or cell wall thickening. Owing to its location on the outside of the cytoplasmic membrane and its specificity, the transglycosylation step is an interesting target for antibacterials. Glycopeptides and moenomycins are the best studied antibiotics known to interfere with this step. Their mode of action and structure-activity relationships have been extensively studied. Attempts to synthesize other specific transglycosylation inhibitors have recently been made.     

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 1. The incorporation of peptidoglycan into the cell wall.

Ether-treated cells of Pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho-N-acetylglucosamine and uridinediphospho-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent-soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low-molecular-weight peptidoglycan fragments. Pulse-chase biosynthesis experiments show that the detergent-soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The DD-carboxypeptidase activity of P. aeruginosa is membrane-bound and does not hydrolyse C-terminal D-alanine residues from the L-lysine-containing nucleotide-precursor analogue. An LD-carboxypeptidase was also detected in P. aeruginosa.     

Growth pattern and cell division in Neisseria gonorrhoeae.

The gram-negative coccus Neisseria gonorrhoeae was found to grow regularly in at least two dimensions. Growth proceeded at a linear rate sequentially in each dimension. Growth in the second dimension (former width) was initiated slightly before the pole-division plane distance equalled the cell width. Penicillin treatment localized presumptive growth zones to the existing septum region. It was suggested that new growth zones were always formed perpendicular to the longitudinal axis created in the incipient daughter cells of a dividing coccus. Neither penicillin nor nalidixic acid induced filaments of N. gonorrhoeae. Such structures could nevertheless be formed in the rod-shaped species Neisseria elongata. N. gonorrhoeae divides by septation; however, complete septal structures with separated cytoplasms were rather infrequent. It is proposed that N. gonorrhoeae be regarded as a short rod which always extends parallel to the actual longitudinal axis and which never undergoes a rod-sphere-rod transition.     

Arrangement of peptidoglycan in the cell wall of Staphylococcus spp.

The arrangement of peptidoglycan in the cell wall of Staphylococcus was observed with the newly developed freeze-fracture technique, using n-octanol instead of water as the freezing medium. The replica of the trichloroacetic acid-extracted cell wall (TCA-wall) showed two areas. One of them has a concentric circular structure, a characteristic surface structure of the staphylococcal cell wall, and the other showed an irregular and rough surface. The chemical analysis of the wall revealed that the TCA-wall consisted of mostly peptidoglycan. By digesting the TCA-wall with lysozyme, the circular structures were greatly disturbed, and they disappeared after 60 min of treatment. From these observations it can be expected that the peptidoglycan is arranged in a concentric circular manner in the newly generated cell wall of Staphylococcus.     

AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system

Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.     

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'top-down' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.     

Genetic transformation of pilation and virulence into Neisseria gonorrhoeae T4.

Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.     

Synthesis of peptidoglycan in the form of soluble glycan chains by growing protoplasts (autoplasts) of Streptococcus faecalis.

Protoplasts (autoplasts) of Streptococcus faecalis were produced by the action of native autolytic N-acetylmuramidase in the absence of added peptidoglycan hydrolases and were grown in osmotically stabilized medium containing L-[3H]lysine and D-[14C]alanine. To reduce the level of muralytic hydrolysis of glycan chains during growth, heat-inactivated cell walls were added to the medium to bind autolytic enzyme, and tetracycline (1 mug/ml) was added to inhibit further enzyme synthesis. Under these conditions, protoplasts synthesized newly labeled peptidoglycan in the form of soluble, infrequently peptide cross-linked glycan chains which were released into the supernatant medium. These relatively large glycan chains were not transferred to exogenously added cell walls.     

Lipid intermediates in the biosynthesis of bacterial peptidoglycan.

This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.