Biosynthetic studies on N-acetylmannosaminuronic acid containing teichuronic acid in Bacillus megaterium.

The particulate enzymes obtained from four strains of Bacillus megaterium AHU 1240, AHU 1373, AHU 1375, and T catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid, UDP-N-acetylglucosamine, and UDP-glucose. Chemical studies involving Smith degradation, acid hydrolysis, and N-acetylation revealed that the polysaccharide product has a backbone made up of trisaccharide repeating units comprising glucose, N-acetylmannosaminuronic acid, and N-acetylglucosamine and that the main oligosaccharide moieties of the glycolipids were identical with N-acetylmannosaminuronosyl-N-acetylglucosamine and glucosyl-N-acetylmannosaminuronosyl-N-acetylglucosamine. Incubation of the disaccharide-linked lipid with each particulate enzyme in the presence of UDP-glucose produced the trisaccharide-linked lipid and a polysaccharide. It is therefore suggested that in this polysaccharide-synthesizing system the repeating unit is formed on a carrier lipid from appropriate nucleotide derivatives first and the polymerization of the units then occurs to synthesize the backbone while the growing chain remains in pyrophosphate linkage to the carrier lipid presumed to be undecaprenol.

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts.

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.

Effect of a hexosylceramide fraction of the hemodialysate Solcoseryl on experimental granulation tissue.

This work was carried out to study the effects of a hexosylceramide fraction (Hex-Cer) of the hemodialysate Solcoseryl on developing granulation tissue in rats. Subcutaneously implanted cylindrical hollow viscose-cellulose sponges were used as an inductive matrix for the growth of granulation tissue. In the control animals, the implants were treated daily by withdrawing 1 ml of wound fluid from the central dead space of the implant and by injecting back 1 ml of physiological saline. In the experimental animals, the aspirated wound fluid was replaced by a corresponding volume of a solution containing 0.08 microgram of Hex-Cer in physiological saline. Analyses of the wound fluid and granulation tissue were carried out 4, 10 and 21 days after the implantation. Statistically significant increases in the mean amounts of granulation tissue DNA and RNA were observed in the Hex-Cer group as compared with the controls, indicating an augmented cellularity. Concurrently, the mean amounts of collagen hydroxyproline in the Hex-Cer group were significantly higher than the respective control values. Similarly, on days 4 and 10, the amounts of uronic acids were higher in the Hex-Cer group than in the controls, reflecting an enhanced accumulation of glycosaminoglycans. The tissue hemoglobin reflecting the degree of vascularization rose gradually as the healing progressed, the mean amounts being generally higher in the Hex-Cer group than in the controls. Wound fluid pO2, pCO2 and pH as well as wound fluid hemoglobin and lactate concentrations showed no essential differences between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of three intermediates in the biosynthesis of teichuronic acid of Micrococcus luteus.

Teichuronic acid, the Micrococcus luteus cell wall polysaccharide which consists of D-glucose and N-acetyl-D-mannosaminuronic acid, is synthesized in vitro from uridine diphosphate N-acetyl-D-glucosamine, uridine diphosphate N-acetyl-D-mannosaminuronic acid, and uridine diphosphate D-glucose in a series of reactions catalyzed by a particulate enzyme preparation. Several lipid-linked intermediates are formed, of which the first three are called components A, B, and C. The formation of these intermediates is inhibited by tunicamycin. The lipid moiety of the intermediates is approximately 95% undecaprenol and 5% dodecaprenol as determined by mass spectrometry. The oligosaccharide moieties of components B and C are the disaccharide, N-acetyl-D-mannosaminuronyl-(1,3)-N-acetyl-D-glucosamine, and the trisaccharide, N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminuronyl++ +-(1, 3)-N-acetyl-D-glucosamine, respectively, as determined by the complete degradation of the former and partial degradation of the latter by the alkaline beta-elimination reaction. The saccharide and lipid moieties of the intermediates are linked through pyrophosphate. Thus, component A is P1-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, component B is P1-N-acetyl-D-mannosaminuronyl-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate, and component C is P1-N-acetyl-D-mannosaminuronyl-(1,4)-N-acetyl-D-mannosaminurony l-(1, 3)-N-acetyl-alpha-D-glucosaminyl P2-undecaprenyl diphosphate.

Demonstration of uronic acid capsular material in the cerebrospinal fluid of a patient with meningitis caused by mucoid Pseudomonas aeruginosa.

A 39-year-old female with insulin-dependent diabetes mellitus developed Rhizopus infection of the maxillary sinus. Subsequent to successful treatment with amphotericin B and surgical debridement, she developed purulent meningitis due to a mucoid strain of Pseudomonas aeruginosa. Analysis of cerebrospinal fluid documented the presence of a uronic acid polymer at a concentration of 40 micrograms/ml. In spite of parenteral and intrathecal antibiotic therapy, the patient died. This case illustrates that mucoid strains of P. aeruginosa may result in fatal infection and that alginate capsule is produced in vivo in humans.

Influence of phosphate supply on teichoic acid and teichuronic acid content of Bacillus subtilis cell walls.

Bacillus subtilis 168 was grown in chemostat culture in fully defined media containing a constant concentration of magnesium and concentrations of phosphate that varied from those giving phosphate-limited growth to those in which phosphate was present in excess and magnesium was limiting. Phosphate-limited bacteria were deficient in wall teichoic acid and contained less than half as much cellular phosphate as did bacteria grown in excess of phosphate. Approximately 70% of the additional phosphate in the latter bacteria was present as wall teichoic acid, indicating that the ability of the bacteria to discontinue teichoic acid synthesis when grown under phosphate limitation permits a substantial increase in their growth yield. Since not all of the additional phosphate is present as wall teichoic acid other cellular phosphates may also be present in reduced amounts in the phosphate-limited bacteria. The content of phosphate groups in walls of magnesium-limited bacteria was similar to the content of uronic acid groups in walls of phosphate-limited bacteria, and walls of bacteria grown in media of intermediate composition contained intermediate proportions of the two anionic polymers. Phage SP50, used as a marker for the presence of teichoic acid, bound densely to nearly all of the bacteria in samples containing down to 22% of the maximum content of teichoic acid. Apparently, therefore, nearly all of these bacteria contain teichoic acid, and the population does not consist of a mixture of individuals having exclusively one kind of anionic polymer. Bacteria containing less than 22% of the maximum content of teichoic bound in a nonuniform manner, and possible explanations for this are discussed.

Elongation of teichuronic acid chains by a wall-membrane preparation from Micrococcus luteus.

A wall-plus-membrane preparation from Micrococcus luteus catalyzes the incorporation of [14C]glucose from UDP-[14C]glucose, into two fractions of teichuronic acid, which is the cell wall polysaccharide consisting of alternating residues of glucose and N-acetylmannosaminuronic acid (ManNAcUA). Membrane-associated teichuronic acid was extracted from the wall-membrane fraction of reaction mixtures by sodium dodecyl sulfate. The synthesis of membrane-associated teichuronic acid required UDP-glucose, UDP-ManNAcUA, and UDP-N-acetylglucosamine and was inhibited by tunicamycin. Glucose incorporated into wall-bound teichuronic acid remained in wall fragments after extraction with sodium dodecyl sulfate, and its incorporation required UDP-glucose and UDP-ManNAcUA (but not UDP-N-acetylglucosamine) and was insensitive to tunicamycin. Radioactive material incorporated into wall-bound teichuronic acid could be released by treatment with mild acid or by digestion with lysozyme, indicating that the wall-bound teichuronic acid was covalently linked to peptidoglycan. There were about 600 pmol of wall-bound teichuronic acid acceptor sites for glucose per mg of protein as measured in incorporation reaction mixtures lacking UDP-ManNAcUA. In the presence of both UDP-glucose and UDP-ManNAcUA, elongation of teichuronic acid acceptor sites occurred, with the addition of six to eight disaccharide units to each acceptor site.

Carbon-13 nuclear magnetic resonance spectroscopic study of teichuronic acid from Micrococcus luteus cell walls. Comparison of the polysaccharide isolated from cells with that synthesized in vitro.

Teichuronic acid isolated from the cell walls of Micrococcus luteus has been examined by natural-abundance (13)C NMR spectroscopy. Proton-decoupled and proton-coupled spectra were obtained for native teichuronic acid and also after the teichuronic acid had been oxidized with periodate and reduced with borohydride. The spectra are consistent with the structure [ManNAcUAp-( beta -1,6)-G1cp-( alpha -1,4)]n. Teichuronic acid synthesized in vitro from suitable substrates by the particulate enzyme fraction obtained from M. luteus yielded a (13)C NMR spectrum which is indistinguishable from that of the native teichuronic acid, indicating a structural identity of the teichuronic acid synthesized in vitro with that isolated from cell walls.

The increase of glycosaminoglycan synthesis and accumulation on the cell surface of cultured skin fibroblasts in Werner's syndrome.

Glycosaminoglycans (GAGS) synthesis and accumulation on the cell surface of skin fibroblasts in two patients with Werner's syndrome were measured by the incorporation of 3H-glucosamine and 35S-sulfate. The strains of fibroblasts from Werner's syndrome (WF) had a much higher production of GAGS and accumulation on the cell surface than did the controls. Uronic acid was measured by means of the carbazole sulfuric acid method and orcinol method and it was revealed that WF fibroblasts had a large amount of total uronic acid in the cell layer, the largest portion being the iduronic acid fraction. Electrophoretic analysis of the GAGS of WF showed an increase of production. Increased release into the medium and accumulation of pericellular glycosaminoglycans was observed in hyaluronic acid (HA), heparan sulfate + dermatan sulfate (HS + DS), and chondroitin sulfate (CHS) fractions. No specific accumulation of GAGS in the intracellular pools was observed.

A note on the fermentation of pectin by pure strains and combined cultures of rumen bacteria.

Bacteroides ruminicola, pure or combined with Selenomonas ruminantium, and Lachnospira multiparus, pure or combined with Succinivibrio dextrinosolvens, were grown on a medium with pectin as energy source. There was a difference in fermentation products between the pure and combined cultures and efficiency of substrate utilization was better with the combined cultures.

The biosynthetic pathway of L-ascorbic acid in Euglena gracilis Z.

D-Glucose and D-galactose were the starting materials of L-ascorbic acid biosynthesis in Euglena gracilis as evidenced by feeding experiments of unlabeled and labeled sugars, but D-glucose was the more effective precursor. The addition of various acid derivatives of D-glucose and D-galactose, with the exception of D-glucono-delta-lactone, considerably augmented L-ascorbic acid formation. D-Galacturonic acid and L-galactono-gamma-lactone showed greater effects than did D-glucurono-gamma-lactone and L-gulono-gamma-lactone. The results of isotopic dilution experiments also showed the preference for the galacto-configuration. Fed U-14C-D-glucose was transformed into labeled D-galacturonic acid to a greater extent than into labeled D-glucuronic acid, and added D-galacturonic acid only caused extensive accumulation of labeled D-galacturonic acid. These results together show that the pathway involving D-galacturonic acid and L-galactono-gamma-lactone is the major one, the one involving D-glucuronic acid L-gulono-gamma-lactone being the minor one. A likely pathway for L-ascorbic acid biosynthesis in Euglena is proposed in the Scheme, which thus involves uronic acid intermediates and configurational inversion.

Control of teichoic and teichuronic acid biosynthesis in Bacillus subtilis 168trp. Evidence for repression of enzyme synthesis and inhibition of enzyme activity.

Phosphate starvation induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp-which had previously been grown with excess phosphate. This induction was prevented when protein systhesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid. The converse was true when phosphate was added to cells previously grown in a phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chloramphenicol or amino acid starvation and cells continued to make teichuronic acid. This suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phosphate-limited cells. The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs. Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing imtermediates do not act as repressors.

Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues.

L-[14C]Iduronic acid-containing sulfated galactosaminoglycans were formed by incubation of a fibroblast particulate fraction with UDP-D[14C]glucuronic acid, UDP-N-acetylgalactosamine, and sulfate donor (3'-phosphoadenylylsulfate). The formation of L-iduronic acid was strongly promoted by concomitant sulfation of the polymer. In the absence of sulfate donor 5 to 10% of the [14C]uronic acid residues were L-iduronic acid. However, when 3'-phosphoadenylylsulfate was included in the incubation mixture the amount of L-iduronic acid in the product increased 3 to 5-fold. Furthermore, approximately the same quantity of L-[14C]iduronic acid was recovered from the product formed in a pulse-chase experiment where incorporation of 14C-isotope preceded sulfation. It was therefore concluded that C-5 inversion of D-glucuronic acid to L-iduronic acid occurred on the polymer level as shown previously for the biosynthesis of heparin (Hook, M., Lindahl, U., Backstrom, G., Malmstrom, A., AND Fransson, L-A., J. Biol. Chem. (1974) 249, 3908). This conclusion was supported by the finding that no L[14C]iduronic acid could be detected in the UDP-hexuronic acid pool during this experiment. Nonsulfated and sulfated [14C]galactosaminoglycan products were degraded separately with chondroitinase-AC. The non-sulfated products afforded primarily disaccharide and a small amount of tetrasaccharide, while the sulfated products yielded, in addition, a considerable amount of larger oligosaccharides. Tetrasaccharides from nonsulfated products contained L-iduronic acid indicating that C-5 inversion at solitary sites can occur in the absence of sulfation of adjacent hexosamine moieties. The larger oligosaccharides obtained after chondroitinase-AC digestion of sulfated products yielded L-iduronic acid upon acid hydrolysis and were susceptible to chondroitinase-ABC digestion. The split products were almost exclusively 4-sulfated disaccharides. These results demonstrate that formation of blocks of L-iduronic acid-containing repeat periods is associated with 4-sulfation of adjacent hexosamine moieties.

Relationship between calcium and uroinic acids in the encystment of Azotobacter vinelandii.

Encystment of Azotobacter vinelandii (ATCC 12837) in modified Burk nitrogen-free medium (pH 7.0) containing 0.2 percent beta-hydroxybutyrate occurs optimally in 0.37 to 0.44 mM solutions of calcium ions. Suspension of cells in media deficient in calcium results in abortive encystment characterized by the release of viscous cyst coat material. Mature cysts rupture in ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, suggesting that calcium is a structural component of the cyst coat. Maximal stimulation of encystment by calcium ions occurs prior to the completion of the cyst exine or outer coat. The uronic acid composition of cyst components is dependent on calcium levels in the medium. Uronic acids account for 31.7 percent of the intine (inner coat) and 13 percent of the exine dry weight, and only mannuronic and guluronic acids are present in these fractions. These can be extracted as homo- and heteropolymeric sequence "blocks" characteristic of alginic acids. The polyuronic acid fraction of both the cyst coats contain approximately equal amounts of heteropolymeric (mannuronic acid/guluronic acid) blocks. The exine, however, is richer in polyguluronic acid and the intine is richer in polymannuronic acid. As a result, the mannuronic acid/guluronic acid ratio of the exine is lower than that of the intine. Slimes that form in abortive encystment are rich in polymannuronic acid and have a high mannuronic acid/guluronic acid ratio. A polymannuronic acid 5-epimerase is active in the mature cyst central body and the encystment culture fluid.