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Preparation of arthritogenic hydrosoluble peptidoglycans from both arthritogenic and non-arthritogenic bacterial cell walls.

Cell wall lytic enzyme (Kyowa lytic no. 2 enzyme) liberated arthritogenic hydrosoluble peptidoglycans from both arthritogenic and non-arthritogenic bacterial cell walls. From these cell walls, mutanolysin (peptidoglycan-degrading enzyme) also liberated hydrosoluble peptidoglycans which, however, lacked arthritogenicity. Based on the chemical composition of these peptidoglycans, it was suggested that their arthritis-inducing ability depends on a relatively long chain of glycan units that consists of repeated units of N-acetylglucosaminyl-N-acetylmuramic acid. However, the glycan chain lengths on these peptidoglycans appeared to be related to their adjuvancy rather than to an antigen(s) responsible for development of arthritis in rats.

Animals

Nitrate modulates pectin metabolism and cell wall mechanics during cell expansion in Arabidopsis.

Nitrate is a key nutrient and one of the most important nitrogen sources for land plants. Besides its nutritional role, nitrate is a signal molecule that regulates plant gene expression, metabolism, physiology, growth, and development. In cotyledons and true leaves, nitrate promotes growth by inducing cell expansion. Plant cell expansion requires changes in the cell wall. However, there is scant information on the influence of nitrate on cell wall metabolism and properties during cell expansion and growth. Here, we demonstrate that nitrate availability modulates pectin metabolism, a major polysaccharide of the primary cell wall. Using colorimetric assays, immunohistochemistry, and confocal microscopy, we show that nitrate enhances methylesterified pectin during cotyledon cell expansion. This is achieved by increasing galacturonic acid (GalA) deposition as homogalacturonan (HG) and by decreasing global PME activity. We further show that this regulation is dependent on nitrate signaling pathway components, including NRT1.1 and NLP7. Pectin methylesterification state impacts the mechanical properties of the cell wall. We characterized cell wall elasticity changes during nitrate-induced expansion using atomic force microscopy (AFM) and automatic confocal microextensometry (ACME). We found that nitrate induces cell wall softening at both cellular and whole-tissue levels during this expansion process. Our results indicate pectin metabolism plays an important role in nitrate-induced cell expansion and cotyledon growth in Arabidopsis. We provide insights into the interplay between nitrate signaling, cell wall metabolism, and biomechanical properties for cell expansion. Our results contribute to our understanding of how plants sense and respond to environmental cues for growth.

Pectins

Relation between cell wall turnover and cell growth in Bacillus subtilis.

The kinetics of cell wall turnover in Bacillus subtilis have been examined in detail. After pulse labeling of the peptidoglycan with N-acetylglucosamine, the newly formed peptidoglycan is stable for approximately three-quarters of a generation and is then degraded by a process that follows first-order kinetics. Deprivation of an auxotroph of amino acids required for protein synthesis results in a cessation of turnover. If a period of amino acid starvation occurs during the lag phase of turnover, then the initiation of turnover is delayed for a period of time equivalent to the starvation period. During amino acid starvation, new cell wall peptidoglycan is synthesized and added to preexisting cell wall. This peptidoglycan after resumption of growth is also subject to degradation (turnover). It is suggested that cell wall turnover is dependent on cell growth and elongation. Several possible control mechanisms for cell wall autolytic enzymes are discussed in light of these observations.

Acetylglucosamine

Immunochemical characterization of cell wall protein antigen purified from the cell wall autolysate of Clostridium botulinum type A.

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components in immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.

Antigens, Bacterial

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Mycobacterium tuberculosis

Stimulation of human B lymphocytes by Listeria cell wall fraction.

Cell wall fraction of Listeria monocytogenes (LCWF), a B cell mitogen for mouse spleen cells, is also mitogenic for human adult and cord peripheral blood lymphocytes. Purified B-cell suspensions responded to LCWF in vitro proliferation, to a similar extent as the unfractionated suspensions. Furthermore, LCWF-induced B cell differentiation into IgM-containing cells and their percentage correlated significantly with the extent of lymphocyte proliferation.

Adult

Characterization of cell wall polymers secreted into the growth medium of lysis-defective pneumococci during treatment with penicillin and other inhibitors of cell wall synthesis.

Autolysin-defective pneumococci secrete large quantities of choline-containing cell wall polymers into the growth medium during treatment with inhibitors of peptidoglycan synthesis. The secreted polymers were separated into three fractions by a combination of gel filtration on agarose and sodium dodecyl sulfate-gel electrophoresis. Fraction I had a high apparent molecular size and contained the Forssman antigen in complex with material exhibiting properties of cell wall teichoic acid. Choline-containing polymers of as yet uncharacterized structure were present in both fractions IIA and IIB, and fraction IIA also contained peptidoglycan components.

Antibodies, Bacterial

Secretion of cell wall polymers into the growth medium of lysis-defective pneumococci during treatment with penicillin and other inhibitors of cell wall synthesis.

Autolysin-defective pneumococci secrete into the growth medium choline-containing macromolecules during treatment with any one of a large number of inhibitors of cell wall biosynthesis, including beta-lactams, beta-halogeno-d-alanines, cephalosporins, and d-cycloserine. Secretion is closely related to the dose response of the bacteria to the various drugs: (i) secretion can already be detected at the minimum inhibitory concentration; (ii) the rate and extent of secretion is dependent upon the drug concentration; and (iii) secretion commences within minutes after the addition of the antibiotics to the cultures. Reversal of the growth-inhibitory effect of benzylpenicillin (by penicillinase addition) is accompanied by a halt in secretion just at the time when the bacteria resume normal growth. Secretion of the choline-containing macromolecules seems to be a specific consequence of the inhibition of peptidoglycan biosynthesis, since inhibition of growth by drugs affecting protein, ribonucleic acid, or deoxyribonucleic acid synthesis does not cause secretion. The choline-containing macromolecules include both the pneumococcal lipid-containing teichoic acid (Forssman antigen) and wall teichoic acids made after the addition of antibiotics. The appearance of these macromolecules in the growth medium is not due to the hydrolytic activity of an autolysin, since penicillin-induced secretion could be demonstrated in autolysin-defective mutants, in pneumococci grown on ethanolamine-containing medium (such cells are known to have defective autolytic systems), and in wildtype pneumococci grown under conditions nonpermissive for lysis.

Antimetabolites

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.

Cell Wall

Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria.

The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.

Bacillus

Plasma-dependent chemotaxis of macrophages toward BCG cell walls and the mycobacterial glycolipid P3.

BCG cell walls, associated with oil droplets in the form of emulsions in saline, generate macrophage chemotactic activity from fresh guinea pig plasma. Serum and heat-inactivated plasma were inactive, suggesting involvement of complement or fibrinogen-derived chemotactic factors. Suspensions of cell walls and oil droplets each generated chemotactic activity from plasma, and the activity of the cell wall vaccine was due to the additive effects of these two components. A mycobacterial glycolipid (P3), which is a constituent of BCG cell walls, also had plasma-dependent chemotactic activity. The results suggest that macrophage chemotaxis may be an important part of the immunopotentiating activity of these mycobacterial products.

Animals

High-Density Genome-Wide Association Mapping Identifies Candidate Loci Associated with Maize Stalk Cell Wall Composition.

Maize (Zea mays L.) stalk cell wall composition is a key determinant of forage digestibility, lodging resistance, and biomass utilization efficiency. Although previous genome-wide association studies (GWAS) have identified loci associated with lignin (LIG), cellulose (CEL), and hemicellulose (HC), advances in genomic resources provide an opportunity to revisit existing phenotypic datasets at substantially higher resolution. Here, we re-analyzed a maize association panel consisting of 341 diverse inbred lines using an expanded genotype dataset containing 10.77 million SNPs, two derived compositional indices (CEL/HC and [LIG/(CEL + HC)], and six complementary GWAS models. Across all traits and models, we identified 855 unique significant SNPs associated with 579 candidate genes. Among the traits examined, LIG/(CEL + HC) yielded the greatest number of associations, suggesting that indices representing the relative balance among cell wall components may better capture the genetic architecture of cell wall composition than individual component measurements alone. Integration of multiple GWAS models with functional enrichment, haplotype, and selective sweep analyses prioritized three biologically relevant candidate genes encoding a MYB58 transcription factor, the glycosyltransferase Xt9, and a putative xyloglucan 6-xylosyltransferase. Haplotype analysis revealed significant effects of Xt9 and the xyloglucan 6-xylosyltransferase on cell wall composition, while selective sweep analysis identified Xt9 as a target of repeated selection during maize domestication, ecological adaptation, and modern breeding. Although these candidate genes provide promising targets for future investigation, the associations identified here are based on a single association panel and require functional and independent population validation. Collectively, our results demonstrate how high-density genotyping combined with complementary GWAS models can refine candidate associations and generate testable hypotheses from existing phenotypic datasets.

cell wall composition

The crystalline glycoprotein cell wall of the green alga Chlorogonium elongatum: a structural analysis.

Members of the Chlamydomonaceae, mostly single-celled green algae, have been shown to contain a crystalline glycoprotein cell wall component. Most of the species examined fall into a class of algae whose walls have an identical crystalline unit cell. Chlorogonium elongatum has been chosen as a representative of this class in order to investigate in more detail its cell wall structure. The alga has a spindleshaped cell wall which retains its asymmetric shape on isolation. Sections from walls fized in the presence of tannic acid clearly reveal a regular subunit monolayer, about 20 nm thick, within the wall. Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis shows the presence of at least 2 major glycoprotein species in the wall. Negatively stained purified cell walls demonstrate the crystalline nature of the cell wall. Optical diffraction of bright-field images and direct electron diffraction both give clear diffraction patterns whose spacings extend out to 3 nm and fall on a reciprocal lattice whose vectors describe a 2-dimensional unit cell within the wall 21.5 nm X 7.0 nm and an included angle of 80 degrees. Lattice defects within the cell wall are revealed by both negative staining and surface replication. Through-focal series were used to choose images with the optimal degree of underfocus for image processing. Linear integration and optical filtering of such images gave essentially the same result. A similar image was also obtained by computing the autocorrelation function of the amplitudes in the electron-diffraction pattern and the optical-diffraction pattern of the in-focus image. On the basis of these data a 2-dimensional model of the crystalline cell wall layer is presented.

Cell Wall

[The cell wall of Coelastrum (Chlorophycees)].

The cell wall of Coelastrum is usually composed of three layers. The outermost layer was studied most extensively. It consists of erect tubules which often bear long bristles whose function may be to stabilize the algae in its enviroment. The cell wall can modify its morphology according to the enviroment.

Cell Wall

Investigation into the effects of cell wall antigens of gram-positive bacteria on lymphocyte stimulation and on cell-mediated cytotoxicity.

The effect of Staph, epidermidis and Bac. subtilis cell walls as well as of cell wall teichoic acid, N-acetyl-muramic acid, N-acetyl-D-glucosamine, D-alanine, and DL-alpha, epsilon-diaminopimelic acid on lymphocyte stimulation and on cell-mediated cytotoxicity has been studied. Bac. subtilis cell wall preparations, N-acetyl-D-glucosamine and teichoic acid showed a slight but significant effect on the mitogenic response of pig lymphocyte cultures. When studied in combination with the mitogens PHA, ConA, and PWM significant synergistic effects were observed with N-acetyl-muramic acid. The most significant stimulation of the in vitro 51Cr-release from labelled P815 tumor cells in the presence of non-sensitized as well as specifically sensitized lymphocytes was exerted by D-alanine.

Animals