Chemotaxonomic studies ofAsplenium sect.Hymenasplenium (Aspleniaceae)

We previously isolated and characterized a new free amino acid withd-configuration at the α-carbon,trans-3, 4-dehydro-d-2-aminopimelic acid and its related amino acids,d-2-aminopimelic acid and 4-hydroxy-l-2-aminopimelic acid fromAsplenium unilaterale. In this paper, we report that the biosynthetic relationshps among these three amino acids were studied using¹⁴C-and³H-labeled compounds as tracers. Glutamate and aspartate were shown to be good precursors and it was suggested that 4-hydroxy-l-2-aminopimelic acid is biosynthesized first and the twod-amino acids are derived from it. Furthermore, the distribution patterns of these non-protein amino acids inAsplenium sect.Hymenasplenium were examined in detail and they were evaluated by their biosynthetic pathway. Morphological characters especially on their rhizomes were also examined and their character phylogeny was determined by outgroup comparison. Taking all the characters available into account, the phylogenetic relationship among 7 species ofAsplenium sect.Hymenasplenium in Japan and Taiwan is discussed by the transformed cladistic method.     

Cell wall polymers of Actinomadura carminata INA 4281

The cell walls of Actinomadura carminata INA 4281 were found to contain peptidoglycan, teichoic acid, and nonpeptidoglycan amino acids. The peptidoglycan was of the A1 type and contained a small amount of ll-DAP in addition to m-DAP. The teichoic acid was an 1,3-poly(glycerol phosphate) chain composed of about eight glycerophosphate units, two of which had a 2-acetamido-2-deoxy--d-galactopyranosyl substituent and one, a 3-O-methyl--d-galactopyranosyl-(1 3)-2-acetamido-2-deoxy--d-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was identified by chemical analysis and ¹³C-NMR spectroscopy. The teichoic acid contained 3-O-methyl-d-galactose (madurose) — the first ever finding of this compound within a teichoic acid. The nonpeptidoglycan amino acids made up some 30% of the cell wall's dry weight, about a quarter of the amino acids being removable with sodium dodecyl sulfate. Further treatment of the cell walls with LiCl and guanidine hydrochloride caused only a small loss of the amino acids and slight changes in their molar ratio.Abbreviations Gro glycerol - GroP monophosphate glycerol - GroP₂ diphosphate glycerol - Gro2P -monophosphate glycerol - P_TA phosphorus of teichoic acids - P_NA phosphorus of nucleic acids - TA teichoic acid     

The lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum

The cell wall lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum was obtained by the phenol-chloroform-petroleum ether and the hot phenol-water methods, respectively. It contained mannose, glucose, galacturonic acid, glucosamine, glycine, and small amounts of rhamnose, galactose and glucuronic acid. In addition to d-glycero-d-mannoheptose, the corespecific constituents 2-keto-3-deoxyoctonate and l-glycero-d-mannoheptose were found. Polyacrylamide gel-electrophoresis in the presence of sodium deoxycholate gave no indication for the presence of O-specific repeating units. Degradation of the lipopolysaccharide required 10% acetic acid (100° C, 2 h). The lipid A moiety contained the total of glucosamine of the lipopolysaccharide as well as small amounts of 2,3-diamino-2,3-dideoxy-glucose. It was phosphate-free. The fatty acid spectrum comprised 3-OH-14:0, 3-OH-16:0, and iso-3-OH-18:0 besides little 12:0, 14:0 and 16:0. Hydroxylaminolysis and sodium methylate treatment revealed all of the three hydroxy fatty acids to be amidebound.Abbreviations DOC sodium deoxycholate - PAGE polyacrylamide gel-electrophoresis     

Chemical composition of Eubacterium alactolyticum cell wall peptidoglycan

The mechanism of lysis of Eubacterium alactolyticum cell walls by Streptomyces albus G enzyme was studied. The analysis of the peptide terminal groups and peptide subunits isolated from the cell wall digest, released during solubilization of the cell walls, revealed that lytic action of S. albus G enzyme was mainly due to D-alanyl-A₂pm endopeptidase, N-acetylmuramyl-L-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase. E. alactolyticum cell wall peptidoglycan is composed mainly of glucosamine, muramic acid, D-glutamic acid, L- and D-alanine, meso-diaminopimelic acid and glycine. The peptide subunit consists of L-alanyl-D-glutamyl-meso-A₂pm-D-alanine. D-Alanine is connected directly with the amino group of the meso-A₂pm residue of another peptide subunit. All of the L-amino groups of meso-diaminopimelic acid are involved in cross-linking.The possible structure of the peptide moiety of E. alactolyticum cell wall peptidoglycan is presented.     

Rhizocticin A,an antifungal phosphono-oligopeptide of Bacillus subtilis ATCC 6633: biological properties

Rhizocticin A, the main component of the antifungal, hydrophilic phosphono-oligopeptides of Bacillus subtilis ATCC 6633, was used for sensitivity testing and experiments into the molecular mechanism of the antibiotic action. Budding and filamentous fungi as well as the cultivated nematode Caenorhabditis elegans were found to be sensitive, whereas bacteria and the protozoon Paramecium caudatum were insensitive. Rhizoctonia solani was inhibited in agar dilution tests but not in diffusion tests. The antifungal effect of rhizocticin A was neutralized by a variety of amino acids and oligopeptides. Oligopeptide influence was mainly understood as transport antagonism, and it was concluded that the antibiotic enters the recipeint cell via the peptide transport system. l- and d-cystine were also identified as potent, general antagonists of the oligopeptide transport. The rhizocticin-antagonism of four other amino acids was taken as a clue to the site of action. Provided that rhizocticin A is split by peptidases of the target cell into inactive l-arginine and toxic l-2-amino-5-phosphono-3-cis-pentenoic acid (l-APPA), the latter may interfere with the threonine or threonine-related metabolism.Abbreviations APPA (2-amino-5-phosphono-3-cis-pentenoic acid) - B. (Bacillus) - P. (Paecilomyces) - S. (Saccharomyces) Dedicated to Professor Dr. Hans Zähner for the 60th return of his birthdayThis work was supported by the Deutsche Forschungsgemeinschaft (DFG-Lo 320¹, SFB 323²)     

Modification of cell membrane lipids inMicrococcus lysodeikticus induced by pantoyl lactone

Summary Growth ofMicroccoccus lysodeikticus in the presence of pantoyl lactone brings about both qualitative and quantitative changes in cell membrane lipids. Significant amounts of the two major phospholipids (phosphatidylglycerol and diphosphatidylglycerol) are converted to lyso forms; the largest conversion occurs in the phosphatidylglycerol. In addition, amounts of several phospholipid fatty acids are changed. Physical alteration of the cell membrane can be demonstrated using differential scanning calorimetry. Although growth and transport are significantly inhibited when pantoyl lactone is present, cells possessing altered cell membrane phospholipids and phospholipid fatty acids, brought about by growth in the presence of pantoyl lactone, transportd-alanine,l-glutamic andl-aspartic acid normally when washed free of the pantoyl lactone.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid     

D-amino acid N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free D-amino acids

d-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on d-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyltransferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of d-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except l-lysine and d-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of d-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.     

Chemical analysis of Azospirillum lipopolysaccharides

Lipopolysaccharides (LPS) were extracted by hot phenol-water from five strains each of Azospirillum lipoferum and Azospirillum brasilense. Rhamnose, glucose, glucosamine and 3-deoxy-d-mannooctulosonic acid were comon sugar constituents of all LPS preparations. 2-O-Mefucose, 3-O-Me-fucose, 3-O-Me-rhamnose and 2-O-Megalactose were found in LPSs of some A. brasilense strains. Fatty acid spectra from all LPSs studied were almost identical with predominance of 3-hydroxymyristic and 3-hydroxypalmitic acids. 3-Hydroxypalmitic acid was the only amide-linked fatty acid. Lipopolysaccharides isolated from A. brasilense showed higher heterogeneity in sugar composition than those from A. lipoferum.Abbreviations glc gas liquid chromatography - ms mass spectrometry - LPS lipopolysaccharide - dOclA 3-deoxy-d-mannooctulosonic acid - 3-OH-16:0 3-hydroxypalmitic acid - nir^- nitrite reductase negative - nir⁺ nitrite reductase positive     

Condensed phosphate deposition,sulfur amino acid use,and unidirectional transsulfuration in Synechococcus leopoliensis

Cells of the cyanobacterium, Synechococcus leopoliensis, have previously been shown to exhibit diminished growth, increased condensed phosphate accumulation, enlarged polyphosphate bodies, and severe chlorosis when cultured under conditions of sulfur deficiency. These characteristics were used to identify which of several sulfur amino acids and a tripeptide served as a sole sulfur source for this unicellular microorganism. Completely serving sulfur compounds were l-cystine, dl-lanthionine, l-djenkolic acid, and glutathione. Sulfur amino acids serving poorly or not at all were l-cystathionine, dl-homocystine, l-methionine, l-cysteic acid, and taurine. This pattern of use suggests that the unidirectional transsulfuration pathway demonstrated in enteric bacteria and green plants, i.e. l-cysteine to l-homocysteine, operates as well in cyanobacteria of the Synechococcus type.     

Orinithine as a constituent of the peptidoglycan of Chloroflexus aurantiacus,diaminopimelic acid in that of Chlorobium vibrioforme f. thiosulfatophilum

L-Ornithine is the only diamino acid of the peptidoglycan of the gliding phototrophic Chloroflexus aurantiacus. The other constituents are L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (in part as muramic acid-6-phosphate), all in approximate equimolar ratios to L-ornithine, aside from small amounts of glycine and histidine. Furthermore unlike typical Gram-negative bacteria, protein is not bound to this peptidoglycan. Instead, the rigid layer (sodium dodecyl sulfate insoluble cell wall fraction) contained large amounts of a complex polysaccharide consisting of sugar O-methyl ethers, hexoses and pentoses. Its binding site is presumably muramic acid-6-phosphate of the peptidoglycan.In contrast, in Chlorobium vibrioforme f. thiosulfatophilium, meso-diaminopimelic acid was found as the only diamino acid of this peptidoglycan. As with other Gramnegative bacteria, L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (no muramic acid-6-phosphate) were observed in approximate equimolar ratios to meso-diaminopimelic acid, except a lower D-alanine content. The rigid layer of Chlorobium vibrioforme f. thiosulfatophilum contained protein, and there were no indications for a complex polysaccharide comparable to that of Chloroflexus aurantiacus.Abbreviations Ala alanine - A₂pm diaminopimelic acid - GC/MS combined gas-liquid chromatography/mass spectrometry - GlcNAc N-acetyl-glucosamine - Glu glutamic acid - Gly glycine - HF hydrofluoric acid - Lys lysine - MurNAc N-acetyl-muramic acid - Orn ornithine - SDS sodium dodecyl sulfate     

A new species of the lichen genusDiploschistes from Australia

Diploschistes hensseniae Lumbsch & Elix is described as new to science. It is characterized by its terricolous habitat, perithecioid closed ascomata, relatively small spores, cylindrical asci, and the presence of diploschistesic and orsellinic acids in addition to lecanoric acid. It occurs on soil in arid regions of Australia.     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

Chemical and biological studies on the lipopolysaccharide (O-antigen) of Anacystis nidulans

The O-antigen (lipopolysaccharide) of Anacystis nidulans, strain KM, has been isolated from whole cells and from cell wall preparations by phenolwater extraction. The polysaccharide moiety consists of a D-mannose polymer accompanied by smaller amounts of 3- and 4-O-methyl-D-mannoses, D-galactose, D-glucose, L-fucose, D-glucosamine, mannosamine and 2-keto-3-deoxyoctonate. Aldoheptoses are lacking. The degraded polysaccharide is split from lipid A by acid hydrolysis (10% acetic acid, 100°C, 3 h) whereby 2-keto-3-deoxyoctonate is released in small amounts. Degraded polysaccharide forms only one major fraction by Sephadex G-50 gel-filtration. This fraction includes all the sugars mentioned above except L-fucose, which is released during the acetic acid degradation. Periodate studies and methylation analysis revealed that the poly-mannose chain consists of about 75% 13 linked and of 25% 14 linked D-mannose units.Lipid A of A. nidulans is phosphate-free. The main fatty acid, -hydroxypalmitic acid, is exclusively amide-bound, presumably to the amino group of D-glucosamine. Other fatty acids, found as minor constituents, are -hydroxymyristic, palmitic and stearic acids. Lipopolysaccharide of A. nidulans KM exhibits high anticomplementary activity in guineapig serum. It is about 800 times less toxic for adrenalectomized mice than endotoxin from Salmonella typhimurium.The isolated lipopolysaccharide reacts with rabbit antisera against living or heat-killed cells of A. nidulans in passive hemagglutination, when untreated or heated, but not when alkali-treated lipopolysaccharide is used for red blood cell sensibilization. It is concluded that lipopolysaccharide of A. nidulans KM is exposed on the surface of the cell.     

Regulation of pteridine biosynthesis and aromatic amino acid hydroxylation inDrosophila melanogaster

The relationship between high dietary levels of aromatic amino acid and regulation of pteridines inDrosophila eyes was examined by measuring changes in pool levels of six pterins in the wild type and mutants and amino acid pool levels in flies that carry mutations for pteridine biosynthesis. The effect upon relative viability and developmental times was also analyzed; relative viability was affected byl-phenylalanine,l-tryptophan, andl-tyrosine in decreasing order and thed-amino acids had little or no effect. The changes in concentration of biopterin, dihydrobiopterin, pterin, sepiapterin, drosopterins, and isoxanthopterin showed a characteristic pattern of increased and/or decreased amounts in response to each of the threel-amino acids. Pterin was regularly increased, and isoxanthopterin decreased.l-Tyrosine caused a 2.1-fold increase in dihydrobiopterin, the largest increase found in this study;l-tryptophan also caused dihydrobiopterin to increase butl-phenylalanine did not. Of 18 eye-color mutants examined, 2 were found to contain high levels of phenylalanine and/or tyrosine,Pu ² andHn ^r3. These two mutants, along withpr ^c4 cn/pr ^m2b cn, were shown to be very sensitive to dietaryl-phenylalanine, indicating that having low levels of certain pteridines makes them susceptible to toxic effects of these amino acids. Therefore, high levels of aromatic amino acids can perturb the balance among pteridine pools, and low levels of some pteridines in mutants are correlated with the inability to withstand the toxic effects of phenylalanine. From the patterns of change in the pteridines we suggest that tetrahydropterin may also be a cofactor for hydroxylation of phenylalanine, along with tetrahydrobiopterin.This work was sponsored in part by a grant from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

Streptomyces species with madurose (3-O-methyl-D-galactose) as a whole-cell sugar

Madurose, an actinomycete whole-cell sugar, was found in the strains of the genus Streptomyces: three strains of S. platensis, one strain each of S. platensis subsp. malvinus, and S. albus subsp. albus. The sugar was isolated from the hydrolysate of S. platensis IFO 14008 cells, and was identified as madurose (3-O-methyl-D-galactose) by chromatographic analyses, ¹H-NMR spectrometry, mass spectrometry as its alditol acetate, and demethylation with boron trichloride. Cell walls of the strain contained peptidoglycan and teichoic acids. ll-Diaminopimelic acid, glycine, glutamic acid, and alanine were present in the peptidoglycan fraction in molar ratios of 1.0:1.3:1.2:2.3. Madurose was detected in the teichoic acid fraction, which was composed of phosphorus, glycerol, galactose, and madurose in molar ratios of 9.3:8.5:2.9:1.0. Thus, madurose was found in the glycerol teichoic acid moiety of the cell walls of this strain.Abbreviations PC paper chromatography - TLC thin-layer chromatography - HPLC high performance liquid chromatography - GLC gas-liquid chromatography - TCA trichloroacetic acid     

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.