The identification of polypeptides synthesised during the acquisition of teichoic acid synthetic activity in Bacillus licheniformis

An attempt has been made to identify proteins synthesised during induction of teichoic acid synthesis in Bacillus licheniformis ATCC 9945. The proteins are recognised as those produced on the change from teichuronic acid to teichoic acid synthesis that occurs after the transfer of the bacteria from phosphate-limited to phosphate-rich conditions. B. licheniformis was grown in phosphate-limiting conditions in the presence of threonine to stimulate threonine uptake. The bacteria were then transferred to phosphate-rich conditions and were pulsed-labelled with [¹⁴C]threonine during the change to teichoic acid synthesis. All of the proteins were extracted from the cells with sodium dodecyl sulphate and were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by fluorography of the polyacrylamide gels. The radioactive polypeptides that were formed on change from teichuronic acid to teichoic acid synthesis were compared with the polypeptides present in a membrane sub-fraction that had high teichoic acid-synthesising activity. The labelling of nine polypeptides with [¹⁴C]threonine was dependent on new RNA synthesis. Of these nine polypeptides, five were also present in the membrane sub-fraction with the highest teichoic acid-synthesising activity.     

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Isolation of low-molecular-weight fragments from the soluble mucopeptide

1. Soluble mucopeptide was prepared by lysozyme treatment of acid-extracted walls of Bacillus licheniformis N.C.T.C. 6346 and separated into fractions differing in molecular size by chromatography on Sephadex G-25 and G-50. 2. About 16% of the weight of soluble mucopeptide has a weight-average molecular weight in excess of 20000. About one half has a weight-average molecular weight of less than 2000 and the balance of soluble mucopeptide is of intermediate size. 3. In the mucopeptide fractions isolated from Sephadex there is a correlation between the weight-average molecular weight, the number of non-reducing muramic acid residues and the proportion of diaminopimelic acid residues recovered after treatment with 1-fluoro-2,4-dinitrobenzene. 4. The extent of cross-linking between peptide side chains is relatively low, even in mucopeptide material of the large molecular size. 5. The small amount of residual phosphorus present in preparations of B. licheniformis soluble mucopeptide remains associated mainly with mucopeptide material of large molecular size. 6. The mucopeptide components of lowest molecular weight are not produced as artifacts during the preparation of soluble mucopeptide, but are apparently incorporated in the insoluble mucopeptide present in walls of exponentially growing cells. 7. Soluble mucopeptide isolated in a complex with acidic polymers after lysozyme treatment of walls of B. licheniformis N.C.T.C. 6346 and Bacillus subtilis W23 retains a high molecular weight when the covalent bonds between mucopeptide and the acidic polymers are broken. 8. Pure fragments were isolated from B. licheniformis soluble mucopeptide. A major component, C1, of the material of smallest size is made up of one residue each of N-acetylglucosamine, N-acetylmuramic acid, l-alanine, glutamic acid and diaminopimelic acid. The N-acetylglucosamine is in beta-glycosidic linkage with a reducing N-acetylmuramic acid residue. The peptide unit is probably amidated. A quantitatively minor component, C2, has amino acid and amino sugar composition identical with that of component C1, but probably lacks an amide group. Another fragment, B1, is made up of two molecules of component C1 or C2 that are joined together through a molecule of d-alanine.     

The isolation of structural components present in the cell wall of Bacillus licheniformis N.C.T.C. 6346

1. Four of the known components of wall preparations of vegative cells of Bacillus licheniformis N.C.T.C. 6346 have been isolated free of each other after successive treatments of the walls with trichloroacetic acid and lysozyme: (a) a mucopeptide consisting of glucosamine, muramic acid, alphain-diaminopimelic acid, glutamic acid and alanine in the molar proportions 1.0:0.8:1.0:1.2:1.7; (b) an insoluble protein; (c) teichoic acid containing phosphorus and glucose in equimolar amounts; (d) teichuronic acid containing equimolar amounts of N-acetylgalactosamine and glucuronic acid, as found by Janczura, Perkins & Rogers (1961). 2. Evidence has been obtained for the presence in the soluble fraction obtained by lysozyme treatment of whole walls of a stable covalent complex of the teichoic acid and the mucopeptide components. 3. The molar ratio of phosphorus to glucose in the teichoic acid present in intact walls or the soluble fractions obtained by extraction of the walls with lysozyme or trichloroacetic acid is 1.0:0.25, in contrast with values of about unity obtained for the purified teichoic acid. 4. Intact walls have been shown to contain polyribitol phosphate chains bearing different amounts of glucose substituents. 5. Trichloroacetic acid extracts of walls also contain polyribitol phosphate compounds of different chain lengths. Dialysis of trichloroacetic acid extracts removes the short chains of polyribitol phosphate that have been found to carry only very low amounts of glucose side chains. By contrast, the longer chains present in the non-diffusible fraction contain phosphorus and glucose in almost equimolar amounts.     

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.     

Sizes of isotopically labeled molecules released during lysis of tumor cells labeled with 51Cr and [14C]nicotinamide.

Previous studies of mechanisms of immune lysis have utilized the release of isotopically labeled molecules from cells labeled with Na₂⁵¹Cr0₄ and [¹⁴C]nicotinamide, among others. The interpretation of specific isotope release from such cells was hampered by the lack of knowledge about the molecular sizes of the released radioactive molecules. In the present study, P815 tumor cells were labeled with the above two isotopic compounds. The cells were then lysed by freezing and thawing, antibody and complement, or immune T-lymphocyte-mediated killing. Soluble supernatants from the lysates were chromatographed in the cold on Sephadex G-10, G-25, and G-200 using several elution media. No differences were detected due to different kinds of lysis or different eluting media. Eighty-five percent of ¹⁴C-labeled molecules released from the cells were indistinguishable from nicotinamide and clearly distinct from nicotinamide adenine dinucleotide. The results suggest that [¹⁴C]nicotinamide taken up by the tumor cells during overnight culture is released unaltered during lysis. Ninety percent of ⁵¹Cr-labeled lysate molecules had an apparent molecular weight less than 4000. Most of this material was distinct from inorganic chromate, but its chemical form is not known.     

Molecular sieve chromatography of biosynthetic cell (14C)glucomannan chains

Radioactive d-glucomannan chains, prepared using Phaseolus aureus enzymes, were acetylated and subjected to molecular sieve chromatography. A comparison with dextran acetates, of known molecular weight range, provided approximate molecular weight data. The [¹⁴C]glucomannan chains were not uniformly dispersed, but were separated into two major fractions. These fractions may be collections of polysaccharide chains incompletely resolved. The two fractions had a mobility similar to that of dextrans with molecular weights of 200,000–300,000 and 60,000–90,000. The molecular weight of the largest [¹⁴C]-glucomannan fragment is, consequently, around 200,000 at the minimum. Preliminary results suggest that the lower molecular weight components may be precursors of the higher molecular weight components. Mild acid and alkaline treatment cause the production of materials of much lower molecular size.     

Sugar utilization by citrus juice cells as determined by [14C]-sucrose and [14C]-fructose feeding analyses

Sugar utilization by mature citrus juice cells was investigated in light of previous reports suggesting the inability of these cells to phosphorylate hexoses. Grapefruit juice sac cells were incubated in solutions of [¹⁴C]-sucrose or [¹⁴C]-fructose for 16 h during which ¹⁴CO₂ evolution was measured by trapping into soluene. After the incubation period, tissue was extracted in 5 % trichloroacetic acid or 80 % ethanol and extracts separated and identified by thin layer chromatography. Fructose was taken up and metabolized more rapidly than sucrose. In both cases, significant amounts of ¹⁴CO₂, [¹⁴C]-pyruvic and [¹⁴C]-citric acid were recovered after incubation. In separate experiments, hexokinase activity in tissue extracts was found to co-sediment with mitochondrial fractions but was not detected in the soluble fractions as previously reported. The data indicated that, contrary to earlier observations, mature citrus fruit juice cells contain the enzymatic machinery to metabolize soluble sugars. This is consistent with the glycolytic utilization of sugars in cells undergoing anaerobic respiration.     

Control of teichoicand teichuronic acid biosynthesis in Bacillus subitlis 168trp−: Evidence for repression of enzyme synthesis and inhibition of enzyme activity

Phosphate starvatiion induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp^? which had previously been grown with excess phophate. This induction was prevented when protein synthesis 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 phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chlorampehnicol or amino acid starvation and cells continued to make teichuronic acid. The 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 phophate-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 intermediates do not act as repressors.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

N-Glycosylation is required for Na+-dependent vitamin C transporter functionality

The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased ¹⁴C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.     

In vivo incorporation of [U-14C]glycerol and [2-3H]glycerol into phospholipids of brain subcellular fractions in normal and malnourished animals

A double label design was used to study the in vivo incorporation of [U-¹⁴C] and [2-³H]glycerol into total and individual phospholipids of various brain subcellular fractions isolated from 20-day old normal and undernourished rats. In control animals, synthesis of glycerophospholipids of microsomes, mitochondria and nerve endings seems to occur through the glycerol-3-phosphate (G-3-P) pathway while a large part of the synthesis of myelin glycerophospholipids appears to proceed through the dihydroxyacetone phosphate (DHAP) pathway. In starved animals, on the other hand the incorporation of phospholipid precursors through the DHAP pathway was found to be lower than in controls while synthesis of phospholipids in the other subcellular fractions was unaffected.The possible relationship between the synthesis of glycerophospholipids and especially plasmalogens of the myelin membrane and microperoxisomes of oligodendroglial cells, where the enzymes of the DHAP pathway are located, is discussed.     

Synthesis of glycoproteins in a single identified neuron of Aplysia californica

Incorporation of L-[³H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [³H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [³H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [³H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [³H]fucose, gel profiles of particulate fractions labeled with [¹⁴C]valine were much more complex.     

Environmental control of protein export of Bacillus licheniformis NCIB 6346 reveals two types of extracellular proteins

Teichuronic acid was the major anionic polymer of Bacillus licheniformis NCIB 6346 durign phosphate-limited (P-limited) growth in the chemostat. This polymer was also present in significant quantities when B. licheniformis was grown under carbon-limited (C-limited) or magnesium-limited (Mg-limited) conditions where teichoic acid predominated in the cell wall. However, the cell wall composition was not of significance in protein export and the parameters for the excretion process were found to be environmental. In particular, two types of extracellular proteins were identified: the first type of enzyme, penicillinase, was only weakly catabolite repressed; was maximally synthesized and secreted during P-limited growth; was unaffected by growth in high Na⁺ media but its production was inhibited by gramicidin. The second type of enzyme, -amylase, was strongly catabolite repressed and its export was markedly inhibited during P-limited growth or in the presence of Na⁺ or gramicidin. It is noteworthy that the penicillinase carries a glyceride-cysteine modification at its N-terminus whilst the -amylase does not.     

NEURONAL AND GLIAL SYSTEMS FOR γ-AMINOBUTYRIC ACID TRANSPORT

—The uptake of [2,3-³H]γ-aminobutyric acid (GABA) by bulk prepared neuronal perikarya, nerve endings and glial cells has been studied in an in vitro-system. The uptake in the different fractions had a similar dependence for sodium, potassium and magnesium. Calcium stimulated the synaptosomal GABA uptake at concentrations which inhibited the glial uptake. Bicuculline strongly inhibited the uptake in all fractions. Picrotoxin and strychnine had little effect on the neuronal uptake whereas glial uptake was stimulated. l -2,4-di-aminobutyric acid and chlorpromazine inhibited GABA uptake in all fractions. The effect of cyclic AMP was not significant.     

Plasmodium berghei: uptake of clindamycin and its metabolites by mouse erythrocytes with clindamycin-sensitive and clindamycin-resistant parasites.

Tritiated Clindamycin was used to compare the uptake of Clindamycin in plasma and red cells of mice infected with clindamycin-sensitive or clindamycin-resistant Plasmodium berghei and in uninfected mice. Red cells infected with either sensitive or resistant parasites have a higher concentration of [³H]clindamycin and its active metabolites 1 hr after drug administration than uninfected red blood cells. There was no significant difference in uptake of Clindamycin by red blood cells parasitized by sensitive or resistant parasites. Levels of Clindamycin and its metabolites were consistently higher in red cells than in plasma, both in infected and uninfected mice, but the drug was readily removed by washing red cells with phosphate buffered saline in either case. It is concluded that resistance to Clindamycin is not due to an impaired uptake of the drug by the parasitized red cell as has been shown for chloroquine resistance in P. falciparum and P. berghei.     

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.     

The Effect of Sugars on the Binding of [Hg]-p-Chloromercuribenzenesulfonic Acid to Leaf Tissues

Replacement of mannitol with sucrose decreases the binding of [²⁰³Hg]-p-chloromercuribenzenesulphonic acid (PCMBS) to Vicia faba leaf discs without epidermis. This decrease is optimal for 20 minutes on incubation, is concentration-dependent, and is also found with maltose and raffinose. In parallel experiments, the addition of sucrose, maltose, and raffinose during PCMBS pretreatment was shown to increase subsequent uptake of [U-¹⁴C]sucrose. In contrast, d- or l-glucose, 3-O-methylglucose, galactose, fructose, palatinose, turanose, or melibiose had no effect either on PCMBS binding or on [¹⁴C]sucrose uptake. The sucrose-induced decrease of PCMBS binding is retained after a cold and ionic shock. Measurements of specific activities of membrane fractions prepared from tissues incubated in labeled PCMBS show that the decrease concerns the 120,000 gravity pellet, but that very mild procedures must be chosen to prevent redistribution of label in the supernatant. Altogether, the data provide new support to the hypothesis that the active site of the sucrose carrier contains a group sensitive to PCMBS.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Biogenesis of mitochondria. The effects of catabolite repression on the in vitro phospholipid synthetic capacities of subcellular fractions of respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae.

A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2-³H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.     

Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10

Bacillus licheniformis HS10 is a good biocontrol agent against Pseudoperonospora cubensis which caused cucumber downy disease. To identify and characterize the antifungal proteins produced by B.licheniformis HS10, the proteins from HS10 were isolated by using 30–60% ammonium sulfate precipitation, and purified with column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. And the SDS–PAGE and MALDI-TOF/TOF-MS analysis results demonstrated that the antifungal protein was a monomer with molecular weight of about 55 kDa, identified as carboxypeptidase. Our experiments also showed that the antifungal protein from B. licheniformis HS10 had significantly inhibition on eight different kinds of plant pathogenic fungi, and it was stable with good biological activity at as high as 100 °C for 30 min and in pH value ranged from 6 to 10. The biological activity was negatively affected by protease K and 10 mM metal cations except Ca²⁺.