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H R Perkins

Publications and source records attributed to H R Perkins.

At least 19 recordsLinked to original sources
PubMed1991

Cross-linking and O-acetylation of peptidoglycan in Staphylococcus aureus (strains H and MR-1) grown in the presence of sub-growth-inhibitory concentrations of beta-lactam antibiotics.

Staphylococcus aureus H was grown for 4 generation times with various sub-growth-inhibitory concentrations of beta-lactam antibiotics specific for particular penicillin-binding proteins (PBPs) - PBP2, clavulanic acid; PBP3, methicillin; PBP4, cefoxitin - and also with the non-specific benzylpenicillin. Isolated cell walls were digested with Chalaropsis muramidase and the resulting peptidoglycan fragments were fractionated by HPLC into disaccharide-peptide monomers and cross-linked dimers, trimers, tetramers and greater oligomers. The pattern of relative fragment concentrations with increasing amounts of drug was roughly the same regardless of the antibiotic used, monomers and dimers increasing while trimers and tetramers changed little and oligomers decreased rapidly. The patterns resembled closely those predicted by the 'random addition' model for multiple cross-link formation and not at all those predicted by the 'monomer addition' model. The O-acetylation of the peptidoglycan remained essentially unaffected under all these conditions. S. aureus MR-1, a constitutive producer of PBP2', gave similar results when treated with methicillin.

Acetylation↗
PubMed1990

Peptidoglycan cross-linking in Staphylococcus aureus. An apparent random polymerisation process.

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross-linked via its peptide chains. The material from wild-type (strain H) and mutants H28, H4B and MR-1 was freed from the teichoic-acid-linked component and then hydrolysed by Chalaropsis muramidase to yield disaccharide-repeating units of the glycan with attached peptides either non-cross-linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high-resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR-1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4-) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross-linking was observed, both in PBP4+ and PBP4- mutants. The implications for secondary cross-linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.

Binding Sites↗
PubMed1989

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.

Acetylation↗
PubMed1987

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).

Acetylation↗
PubMed1986

O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall.

Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.

Acetylation↗
PubMed1985

The peptidoglycan of Neisseria gonorrhoeae, with or without O-acetyl groups, contains anhydro-muramyl residues.

Muramidase digests of alkali-treated SDS-insoluble peptidoglycan from two strains of Neisseria gonorrhoeae were examined. Both strains contained disaccharide peptide monomers that had intramolecular 1,6-anhydro-muramyl ends. In contrast to strain 1L260, in which 50% of the monomer fraction is O-acetylated, the monomer fraction from strain RD5 was completely devoid of O-acetyl groups, as shown by HPLC. Penicillin decreased the O-acetylation of peptidoglycan but did not affect the proportion of anhydro-muramyl residues.

Acetylation↗
PubMed1985

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.

Bacterial Proteins↗
PubMed1984

Antibody to streptococcal cell wall peptidoglycan-polysaccharide polymers in seropositive and seronegative rheumatic disease.

An ELISA has been developed for serum antibodies to streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-GSP). A significantly increased prevalence of serum anti-PG-GSP antibody was found in juvenile chronic arthritis and both seropositive and seronegative rheumatoid arthritis (RA), compared with ankylosing spondylitis, systemic lupus erythematosus, myeloma and healthy controls. Anti-PG-GSP antibody was always of the IgG class and there was no correlation of anti-PG-GSP levels with C reactive protein, rheumatoid factor (RF) or anti-streptolysin O titres. There was no direct cross-reaction of RF with PG-GSP, nor did the presence of IgM-RF significantly interfere with the assay. Examination of paired serum and synovial fluid samples offered no evidence for local production of anti-PG-GSP antibody in synovial tissue. These data are compatible with an increased systemic immunization by bacterial fragments in RA.

Adult↗
PubMed1983

Peptidoglycan-degrading enzymes in ether-treated cells of Neisseria gonorrhoeae.

The incubation of peptidoglycan fragments with ether-treated cells of Neisseria gonorrhoeae resulted in breakdown products that showed the presence of previously undescribed lytic enzymes. The properties of an endopeptidase able to hydrolyse peptide-linked bis-disaccharide peptide dimer to monomer units were examined. An exo-N-acetyl-glucosaminidase was also shown to release free N-acetylglucosamine. The breakdown pattern of glycosidically-linked dimer indicated the existence of an endo-N-acetylglucosaminidase. The activities of the latter enzyme and of the endopeptidase were both sensitive to beta-lactam antibiotics.

Acetylglucosaminidase↗
PubMed1983

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.

Acetylation↗
PubMed1981

Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions.

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation.

Biological Transport↗
PubMed1981

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.

Acetylation↗
PubMed1980

The active centres in penicillin-sensitive enzymes.

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site no. 1. Interaction between the N-14 substituent of the bound molecule and binding site no. 2 induces changes in binding site no. 1. In turn, the catalytic site thus created increases the chemical reactivity of the beta-lactam amide bond. As the beta-lactam ring opens and acylates an enzyme serine residue, the interaction between the thiazolidine (or dihydrothiazine) ring and binding site no. 3 stabilizes the acyl-enzyme complex. Enzyme regeneration slowly proceeds either by direct elimination of the penicilloyl moiety or via C-5-C-6 splitting of the bound metabolite. The fragment arising from thiazolidine yields free N-formyl-D-penicillamine while the enzyme-linked N-acylglycyl fragment is immediately attacked by an exogenous nucleophile correctly positioned on the acceptor site. Similarly, the enzyme action on L-X-D-Ala-D-Ala terminated peptides is mediated via a binding site no. 1 that combines with D-Ala-D-Ala, a binding site no. 2 that interacts with the side chain of the preceding L-residue, an inducible catalytic site and an acceptor site. Enzymes are known that form a transitory L-X-D-Ala-enzyme complex where the acyl group is ester-linked to the same serine residue as that involved in the formation of the penicilloyl-enzyme complex (Waxman et al., this symposium). Other enzymes, however, may function as catalyst templates. Depending on the enzymes, the independence of the beta-lactam and L-X-D-Ala-D-Ala active centres is more or less pronounced.

Amino Acid Sequence↗