scholarly journals Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall

1974 ◽  
Vol 139 (3) ◽  
pp. 781-784 ◽  
Author(s):  
J. Barrie Ward ◽  
Harold R. Perkins
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Free Amino ◽  
Membrane Preparation ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Amino Group ◽  
Peptidoglycan Biosynthesis ◽  
Acetyl Group ◽  
Degraded Product

A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by 14C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.

Effect of cross-linking agents on insulin associated responses in adipocytes

1982 ◽  
Vol 60 (10) ◽  
pp. 987-1000 ◽  
Author(s):  
H. Joseph Goren ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Receptor ◽  
Glucose Transport ◽  
Free Amino ◽  
Glucose Oxidation ◽  
Insulin Binding ◽  
Cross Linking ◽  
Amino Group ◽  
Amino Groups ◽  
Disuccinimidyl Suberate ◽  
Reactive Groups

The effect of 10 bifunctional cross-linking agents and four monofunctional analogues was studied on isolated adipocytes. [125I]Insulin binding and degradation, basal and insulin-stimulated glucose oxidation, and 3-O-methyl glucose uptake were measured. Two cross-linkers, which possess succinimide ester residues (disuccinimidyl suberate and dithiobis(succinimidyl propionate)) and react selectively with amino groups, appeared to react relatively specifically with the insulin receptor. Both produced a slight stimulation of basal glucose transport and metabolism, a marked inhibition of insulin-stimulated glucose transport and metabolism, and a marked decrease in insulin binding. Pretreatment of cells with unlabelled insulin partially blocked the effect of disuccinimidyl suberate, and as has been previously shown, disuccinimidyl suberate cross-linked insulin to its receptor. A monofunctional analogue of these compounds was 100-fold less active in altering cellular metabolic activity. Bisimidates, such as dimethyl suberimidate, dimethyl adipimidate, and dimethyl dithiobispropionimidate, also react with free amino groups but are more hydrophilic. These agents produced similar effects on glucose oxidation as the succinimide esters, but had little or no effect on insulin binding. The effects of these agents are not blocked by insulin and they do not cross-link insulin to its receptor. Mixed bifunctional reagents containing either a succinimide ester or an imidate and a group which reacts with thiols produced effects similar to the cross-linkers containing two succinimide groups or bisimidates, respectively. The bifunctional arylating agents difluorodinitrobenzene and bis(fluoronitrophenyl)sulfone produce marked effects on insulin binding and glucose oxidation at micromolar concentrations, but the monofunctional analogue fluorodinitrobenzene is almost equally active suggesting that with these compounds chemical modifications and not cross-linking was important. With neither the mixed bifunctional reagents, nor the arylating agents, did insulin pretreatment alter the effect of cross-linker and none of these agents cross-linked [125I]insulin to its receptor. These data suggest that the insulin receptor possesses a free amino group in a hydrophobic environment in its active site. A reactive amino group in a hydrophilic environment as well as other reactive groups are also present in some component of the insulin receptor–effector complex. Chemical modification or cross-linking of these functional groups results in an inhibition or mimicking of insulin action. Further study will be required to identify the exact locus of these sites.

scholarly journals Applications of Chitin and Chitosan in Industry and Medical Science: A Review

2016 ◽  
Vol 16 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Shanta Pokhrel ◽  
Paras Nath Yadav ◽  
Rameshwar Adhikari
Keyword(s):  
Cell Wall ◽  
Free Amino ◽  
Science And Technology ◽  
Medical Science ◽  
Amino Group ◽  
Chitin And Chitosan

Chitin can be extracted from the exoskeletons of crustaceans, insects and mollusks and the cell wall of microorganisms. It can be converted into chitosan by deacetylation process. Chitosan shows more versatility than chitin due to its solubility and reactive free amino group (-NH2). This article helps to understand the importance and characteristics of chitin and chitosan by their various aspects such as properties and medical and industrial applications.Nepal Journal of Science and Technology Vol. 16, No.1 (2015) pp. 99-104

scholarly journals The configuration of 2,6-diamino-3-hydroxypimelic acid in microbial cell walls

1969 ◽  
Vol 115 (4) ◽  
pp. 797-805 ◽  
Author(s):  
H R Perkins
Keyword(s):  
Cell Wall ◽  
Hydroxy Group ◽  
Cell Walls ◽  
Optical Rotation ◽  
Periodate Oxidation ◽  
Ring Closure ◽  
Diaminopimelic Acid ◽  
Amino Group ◽  
Amino Groups ◽  
Peptide Linkage

β-Hydroxydiaminopimelic acid, together with some diaminopimelic acid, occurs in the cell-wall mucopeptide of certain Actinomycetales. These components were converted into their di-DNP derivatives and separated by chromatography. Hence the relative proportions present in the cell walls of a number of species were measured. The problem of acid-induced inversion of configuration was studied. Of the diaminohydroxypimelic acids isomer B (see Scheme 2; amino groups meso, hydroxy group threo to its neighbouring amino group) always predominated but a small proportion of isomer D (amino groups l, hydroxy group erythro) also occurred. The configuration of the diaminohydroxypimelic acids was determined by periodate oxidation to glutamic γ-semialdehyde, which underwent spontaneous ring-closure. Reduction with sodium borohydride produced optically active proline, the configuration of which was determined by direct measurement of the optical rotation of DNP-proline. Un-cross-linked diaminohydroxypimelic acid in the cell wall was oxidized with periodate in the presence of ammonia. Since the remaining amino group was bound in peptide linkage, ring-closure was prevented and borohydride reduction of the aldehyde–ammonia presumed to be present resulted in the formation of ornithine. The quantity of ornithine was used as a measure of the degree of cross-linking.

Extent of Peptide Cross-Linking in the Peptidoglycan of Neisseria gonorrhoeae

1980 ◽  
Vol 28 (3) ◽  
pp. 867-875 ◽  
Author(s):  
R. S. Rosenthal ◽  
R. M. Wright ◽  
R. K. Sinha
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Free Amino ◽  
Group Analysis ◽  
Gel Filtration ◽  
Prototype Strain ◽  
Enzymatic Digestion ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Multiple Drugs ◽  
High Degree

The extent of peptide cross-linking in peptidoglycan (PG) isolated from various strains of Neisseria gonorrhoeae was examined. Purified PG, specifically labeled in the peptide moiety with [ 3 H]diaminopimelic acid (DAP) and labeled in the glycan with [ 14 C]glucosamine and [ 14 C]muramic acid, was digested completely with Chalaropsis B muramidase. Gel filtration of the digest on connected columns of Sephadex G-50 and G-25 revealed four well-defined peaks corresponding to soluble PG fragments and containing a constant ratio of 3 H to 14 C. On the basis of (i) K D values, (ii) amino acid composition, (iii) free amino group analysis of [ 3 H]DAP residues, (iv) borohydride reduction, (v) the β-elimination reaction, (vi) high-voltage electrophoresis, and (vii) paper chromatography in various solvents, the PG fragments were identified as un-cross-linked disaccharide peptide monomer, typical of chemotype I PG, and the corresponding peptide cross-linked dimers, trimers, and tetramers. The percent cross-linking of PG basically reflects the percentage of DAP residues that are involved in peptide cross-linking bonds. This value was estimated from the distribution of labeled fragments that resulted from the enzymatic digestion of PG and was confirmed by the analysis of free amino groups in [ 3 H]DAP of intact PG. Although there were subtle, strain- and medium-dependent differences in percent cross-linking, these values varied only over a relatively narrow range (36 to 44%). The percent cross-linking of PG in the prototype strain, RD 5 , grown in a standard gonococcal medium (LGCB + ) was 41.0 ± 2.0%. This is a relatively high degree of peptide cross-linking for a gram-negative bacterium. We also confirmed previous observations that the extent of PG cross-linking among isogenic gonococci was higher in strains, e.g., FA140 and FA136, carrying loci that govern increased resistance to multiple drugs.

scholarly journals Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan

1975 ◽  
Vol 146 (1) ◽  
pp. 253-267 ◽  
Author(s):  
Z Tynecka ◽  
J B Ward
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Cross Linking ◽  
Cross Links ◽  
Increased Sensitivity ◽  
Cross Bridges ◽  
Chain Lengths ◽  
Soluble Material ◽  
Subsequent Addition

The synthesis of peptidoglycan by an autolysin-deficient β-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with β-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.

scholarly journals The Cpx Envelope Stress Response Modifies Peptidoglycan Cross-Linking via the l,d-Transpeptidase LdtD and the Novel Protein YgaU

2014 ◽  
Vol 197 (3) ◽  
pp. 603-614 ◽  
Author(s):  
Margarita Bernal-Cabas ◽  
Juan Alfonso Ayala ◽  
Tracy L. Raivio
Keyword(s):  
Cell Wall ◽  
Stress Response ◽  
Protein Misfolding ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Content Type ◽  
Lytic Transglycosylase ◽  
Expression Of Genes ◽  
Envelope Stress ◽  
Cross Links

The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway inEscherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putativel,d-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized geneygaUin regulating cross-linking.

scholarly journals Sortase C-Mediated Anchoring of BasI to the Cell Wall Envelope of Bacillus anthracis

2007 ◽  
Vol 189 (17) ◽  
pp. 6425-6436 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Olaf Schneewind
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Active Site ◽  
Diaminopimelic Acid ◽  
Side Chain ◽  
Carboxyl Group ◽  
Amino Group ◽  
Active Site Cysteine ◽  
New Hosts ◽  
Host Death

ABSTRACT Vegetative forms of Bacillus anthracis replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of B. anthracis peptidoglycan.

scholarly journals The synthesis of peptidoglycan in an autolysin-deficient mutant of Bacillus licheniformis N.C.T.C. 6346 and the effect of β-lactam antibiotics, bacitracin and vancomycin

1974 ◽  
Vol 141 (1) ◽  
pp. 227-241 ◽  
Author(s):  
J. Barrie Ward
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Membrane Preparation ◽  
Deficient Mutant ◽  
Peptidoglycan Synthesis ◽  
Free Membrane ◽  
Negative Mutant

The synthesis of peptidoglycan by cell-free membrane and membrane+wall preparations from an autolysin-deficient, β-lactamase-negative mutant of Bacillus licheniformis N.C.T.C. 6346 was studied. The membrane preparation synthesized un-cross-linked polymer, the formation of which was not inhibited by β-lactam antibiotics. Release of d-alanine by the action of d-alanine carboxypeptidase was inhibited variably according to the antibiotic. This inhibition was reversed by neutral hydroxylamine but not by the action of β-lactamases or by washing. Bacitracin inhibited peptidoglycan synthesis, but not the d-alanine carboxypeptidase. Examination of peptidoglycan synthesized in the presence of excess of bacitracin showed that synthesis was not restricted to the addition of one disaccharide-pentapeptide unit at each synthetic site, an average of 2–3 disaccharide-pentapeptide units being added. Peptidoglycan synthesis was three- to four-fold more sensitive to vancomycin than was the release of d-alanine by the action of the carboxypeptidase. Incorporation of newly synthesized peptidoglycan into pre-existing cell wall was studied in membrane+wall preparations. This incorporation was catalysed by a benzylpenicillin- and cephaloridine-sensitive transpeptidase. The concentrations of these antibiotics giving 50% inhibition of incorporation were almost identical with those required to inhibit growth of the bacillus. Inhibition of the transpeptidase was reversed by treatment with β-lactamase or by washing.

scholarly journals Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a Useful Analytical Tool

2012 ◽  
Vol 79 (3) ◽  
pp. 768-773 ◽  
Author(s):  
Sebabrata Mahapatra ◽  
Charles Piechota ◽  
Filipa Gil ◽  
Yufang Ma ◽  
Hairong Huang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Mycobacterium Smegmatis ◽  
Analytical Tool ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Muramic Acid ◽  
Content Type ◽  
Mycobacterial Cell Wall

ABSTRACTSince the peptidoglycan isolated fromMycobacteriumspp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified thelysAgene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond betweenl-Ala andd-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of bothMycobacterium smegmatisandMycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides fromM. smegmatisare heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

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