How Do Trichoderma Genus Fungi Win a Nutritional Competition Battle against Soft Fruit Pathogens? A Report on Niche Overlap Nutritional Potentiates.

We present a case study report into nutritional competition between Trichoderma spp. isolated from wild raspberries and fungal phytopathogenic isolates (Colletotrichum sp., Botrytis sp., Verticillium sp. and Phytophthora sp.), which infect soft fruit ecological plantations. The competition was evaluated on the basis of nutritional potentiates. Namely, these were consumption and growth, calculated on the basis of substrate utilization located on Biolog® Filamentous Fungi (FF) plates. The niche size, total niche overlap and Trichoderma spp. competitiveness indices along with the occurrence of a stressful metabolic situation towards substrates highlighted the unfolding step-by-step approach. Therefore, the Trichoderma spp. and pathogen niche characteristics were provided. As a result, the substrates in the presence of which Trichoderma spp. nutritionally outcompete pathogens were denoted. These were adonitol, D-arabitol, i-erythritol, glycerol, D-mannitol and D-sorbitol. These substrates may serve as additives in biopreparations of Trichoderma spp. dedicated to plantations contaminated by phytopathogens of the genera Colletotrichum sp., Botrytis sp., Verticillium sp. and Phytophthora sp.

The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition.

Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure-a ribitol in a phosphodiester linkage-for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains.

Active surfaces engineered by immobilizing protein-polymer nanoreactors for selectively detecting sugar alcohols.

We introduce active surfaces generated by immobilizing protein-polymer nanoreactors on a solid support for sensitive sugar alcohols detection. First, such selective nanoreactors were engineered in solution by simultaneous encapsulation of specific enzymes in copolymer polymersomes, and insertion of membrane proteins for selective conduct of sugar alcohols. Despite the artificial surroundings, and the thickness of the copolymer membrane, functionality of reconstituted Escherichia coli glycerol facilitator (GlpF) was preserved, and allowed selective diffusion of sugar alcohols to the inner cavity of the polymersome, where encapsulated ribitol dehydrogenase (RDH) enzymes served as biosensing entities. Ribitol, selected as a model sugar alcohol, was detected quantitatively by the RDH-nanoreactors with GlpF-mediated permeability in a concentration range of 1.5-9 mM. To obtain "active surfaces" for detecting sugar alcohols, the nanoreactors optimized in solution were then immobilized on a solid support: aldehyde groups exposed at the compartment external surface reacted via an aldehyde-amino reaction with glass surfaces chemically modified with amino groups. The nanoreactors preserved their architecture and activity after immobilization on the glass surface, and represent active biosensing surfaces for selective detection of sugar alcohols, with high sensitivity.

Structures of cell-wall phosphate-containing glycopolymers of Bifidobacterium longum BIM B-476-D.

Glycopolymers with oligosaccharyl phosphate repeats of two types and ribitol and glycerol teichoic acids were isolated from cell wall of Bifidobacterium longum BIM B-476-D by stepwise extraction with 10% CCl3CO2H. The following structures of the glycopolymers were established by sugar analysis, selective cleavage with aq 2% HOAc, dephosphorylation with 48% HF, 2D NMR spectroscopy, and high-resolution ESI MS: [structure: see text]. The ribitol teichoic acid also contains minor D-alanine, whose position was not determined.

Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a ß-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM ß-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.

Determination of strain-specific wall teichoic acid structures in Lactobacillus plantarum reveals diverse α-D-glucosyl substitutions and high structural uniformity of the repeating units.

The structural diversity of wall teichoic acid (WTA) was investigated using biochemical and NMR analyses among 19 strains of Lactobacillus plantarum, of which seven were previously established to contain a glycerol-type backbone, whereas the remaining 12 strains possess ribitol-containing WTA. Despite the fact that the WTAs consisted of identical components, namely phosphoric acid, alditol (glycerol or ribitol) and glucose, comparative analysis of the (1)H and (13)C NMR spectra indicated the presence of six different structures, based on the observed differences in the anomeric signals of glucose residues. To determine the six WTA structures, their repeating units were prepared by alkaline hydrolysis, followed by fractionation on HPLC, and analysis by NMR spectroscopy using synthetic molecules as a reference. The structures of the six isolates were established as 1-α-D-glucosyl-sn-glycerol 3-phosphate, 1-α-D-kojibiosyl-sn-glycerol 3-phosphate, 1-α-D-nigerosyl-sn-glycerol 3-phosphate, 4-α-D-kojibiosylribitol 1-phosphate and 1,5-linked di-(2,4-di-α-D-glucosylribitol) phosphate. The backbone structures appeared to be 3,6'-linked poly(1-α-D-glucosyl-sn-glycerol phosphate) for the glycerol-type WTA and 1,5-linked poly(ribitol phosphate) for the ribitol-containing WTA. Moreover, in the analysis of the alkaline hydrolysates on HPLC, only single structures of repeating units were released from each WTA, indicating the high structural uniformity of the WTA in each strain. Notably, analyses of lipoteichoic acid isolated from representative strains harbouring the six different WTAs revealed the universal presence of a 1,3-linked poly(glycerol phosphate) chain, substituted at C-2 of the glycerol residues with glucose residues. These findings provide fundamental information on WTA structural variability in Lb. plantarum, which seems likely to play a pivotal role in the physiology of this bacterial species.

Physiological and biochemical responses of green microalgae from different habitats to osmotic and matric stress.

Growth of five aeroterrestrial green algal strains (Trebouxiophyceae) in response to changing water availabilities-caused by osmotic (ionic) and matric (desiccation) stresses-was investigated in comparison with a freshwater and a marine strain. All investigated algae displayed good growth under brackish conditions while four out of the five aeroterrestrial strains even grew well under full marine conditions (28-40 psu). The comparison between growth responses in liquid medium, on solid agarose, and on glass fiber filters at 100% air humidity indicated a broad growth tolerance of aeroterrestrial algae towards diminished water availability. While two aeroterrestrial strains even grew better on solid medium which mimics natural biofilm conditions, the aquatic strains showed significant growth inhibition under matric stress. Except Stichococcus sp., which contained the C6-polyol sorbitol, all other aeroterrestrial green algae investigated synthesized and accumulated the C5-polyol ribitol in response to osmotic stress. Using (13)C NMR spectroscopy and HPLC, it could be verified that ribitol functions as an osmotically regulated organic solute. This is the first proof of ribitol in free-living aeroterrestrial green algae. The biochemical capability to synthesize polyols under environmental stress conditions seems to support algal life outside aquatic habitats.

Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes.

Environmental stress deleteriously affects every aspect of an ectotherm's biological function. Frequent exposure of terrestrial insects to temperature variation has thus led to the evolution of protective biochemical and physiological mechanisms. However, the physiological mechanisms underlying the positive impact of fluctuating thermal regimes (FTRs) on the fitness and survival of cold-exposed insects have not been studied. We have thus investigated the metabolic changes in adults of the beetle Alphitobius diaperinus in order to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds, such as free amino acids (FAAs) and polyols. The metabolic profile was analyzed during constant fluctuating thermal regimes (the beetles had daily pulses at higher temperatures that enabled them to recover) and compared with constant cold exposure and untreated controls. The increase of several essential amino acids (Lys, Iso, Leu, Phe and Trp) in cold-exposed beetles supports the conclusion that it results from the breakdown of proteins. Some FAAs have been shown to have cryoprotective properties in insects, but the relationship between FAAs, cold tolerance and survival has not yet been well defined. Instead of considering FAAs only as a part of the osmo- and cryoprotective arsenal, they should also be regarded as main factors involved in the multiple regulatory pathways activated during cold acclimation. Under FTRs, polyol accumulation probably contributes to the increased duration of survival in A. diaperinus.

Characterization of lipoteichoic acids as Lactobacillus delbrueckii phage receptor components.

Lipoteichoic acids (LTAs) were purified from Lactobacillus delbrueckii subsp. lactis ATCC 15808 and its LL-H adsorption-resistant mutant, Ads-5, by hydrophobic interaction chromatography. L. delbrueckii phages (LL-H, the LL-H host range mutant, and JCL1032) were inactivated by these poly(glycerophosphate) type of LTAs in vitro in accordance to their adsorption to intact ATCC 15808 and Ads-5 cells.

In vivo and in vitro NMR spectroscopy reveal a putative novel inborn error involving polyol metabolism.

In vivo NMR spectroscopy was performed on the brain of a patient with a leukoencephalopathy, revealing unknown resonances between 3.5 and 4.0 ppm. In addition, urine and CSF of the patient were measured using high-resolution NMR spectroscopy. Also in these in vitro spectra, unknown resonances were observed in the 3.5-4.0 ppm region. Homonuclear (1)H two-dimensional J-resolved spectroscopy (JRES) and (1)H-(1)H correlation spectroscopy (COSY) were performed on the patient's urine for more accurate assignment of resonances. The NMR spectroscopic studies showed that the unknown resonances could be assigned to arabinitol and ribitol. This was confirmed using gas chromatography. The arabinitol was identified as D-arabinitol. The patient is likely to suffer from an as yet unknown inborn error of metabolism affecting D-arabinitol and ribitol metabolism. The primary molecular defect has not been found yet. Urine spectra of patients suffering from diabetes mellitus or galactosemia were recorded for comparison. Resonances outside the 3.2-4.0 ppm region, which are the most easy to recognize in body fluid spectra, allow easy recognition of various sugars and polyols. The paper shows that NMR spectroscopy in body fluids may help identifying unknown resonances observed in in vivo NMR spectra.

Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection.

The stability and integrity of glycoconjugate vaccines requires determination of the total saccharide and quantification of the unbound or free saccharide present. The traditional assay for Hib conjugates, based on colorimetric determination of ribose, has been much improved by the use of base hydrolysis and analysis of the Hib subunit generated using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The production of this subunit was confirmed by NMR analysis. However, quantification of free Hib saccharide using this method was not possible in the combination vaccines evaluated due to interferences emanating from DPT. Thus a method based on TFA hydrolysis followed by the chromatographic separation and quantification of ribitol on a CarboPac MA1 column was developed. The method is selective, and with the use of ED40 electrode, requires only nanomole amounts for the chromatographic step, thereby ensuring that free saccharide can be monitored accurately in the formulated Hib-CRM vaccine alone and when in combination with other vaccines.

Quantification of C-polysaccharide in Streptococcus pneumoniae polysaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection.

A sensitive method, using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been developed for measurement of the C-polysaccharide contamination in Streptococcus pneumoniae capsular polysaccharides, which are part of the polysaccharides based vaccines for the prevention of pneumococcal infections. This method, based on the quantification by HPAEC-PAD of the ribitol released by aqueous hydrofluoric acid (HF) followed by trifluoro acid hydrolysis (TFA) of the pneumococcal polysaccharides is simple and provides both a qualitative and quantitative method for control of the polysaccharides.

Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov.

Earlier phylogenetic studies based on the inferred DNA sequences of the polC, rpoB and gyrA genes suggested that strains of the species Bacillus subtilis formed two clusters, indicating the presence two closely related taxa; one contained the laboratory strain 168 and the other the laboratory strain W23. Significant sexual isolation was found between strain 168 and members of the group containing W23, but no sexual isolation was observed between strain 168 and other members of the 168 group. DNA reassociation between the two groups ranged from 58 to 69% and intragroup DNA relatedness ranged from 82 to 100%. Because group 168 strains were highly related to the B. subtilis type strain, they were considered to be bona fide members of the species. About 99.5% sequence identity was observed between the 16S rRNA genes of the 168 and W23 groups. Ribitol and anhydroribitol were principal cell wall constituents of the W23 but not of the 168 group. These observations revealed two closely related but genetically and phenotypically distinct groups within B. subtilis that correspond to two historically important strains. Subspecies distinction is proposed for the 168 and W23 groups, with the names Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov., respectively. The type strain of the former is NRRL NRS-744T and the latter NRRL B-23049T.

[Structure of teichoic acids from marine microorganisms Bacillus subtilis and Bacillus licheniformis]. / Struktura teikhoevykh kislot iz morskikh mikroorganizmov Bacillus subtilis i B. licheniformis.

Teichoic acids from the cell walls of marine bacilli Bacillus subtilis CMM (Collection of Marine Microorganisms) 234 (R-1) and B. licheniformis CMM 454 (1-1G-2) were isolated and characterized. These teichoic acids were found to have identical structures and are composed of the glucose, ribitol, and phosphoric acid residues. On the basis of 13C NMR and 31P NMR spectra of the teichoic acids and the products of their dephosphorylation, we established the following structure for the biopolymer: poly[-->5)-4-O-beta-D-glucopyranosylribitol-(1-phospho].

Teichoic acid content in different lineages of Staphylococcus aureus NCTC8325.

A series of mec transformants of Staphylococcus aureus strain NCTC8325 were analysed for alterations in wall teichoic acid and lipoteichoic acid. Although the methicillin resistance determinant alters the autolytic behaviour of S. aureus, it had no effects on the cellular content, chain length, and alanine substitution of the lipoteichoic acid, or on the wall teichoic acid content and composition. However, independently of the presence or absence of the methicillin resistance determinant, level of methicillin resistance, or autolytic behaviour, a correlation was found between a 25% reduced cell wall phosphate content and either loss of prophages phi11 and 13 or a 30-kb deletion in the chomosmal SmaI-F fragment adjacent to the prophage φ11 attachment site.

The aqueous solution structure of a lipoteichoic acid from Streptococcus pneumoniae strain R6 containing 2,4-diamino-2,4,6-trideoxy-galactose: evidence for conformational mobility of the galactopyranose ring.

The 2D-NOESY spectra for the per-N-acetylated and the native lipoteichoic acid (LTA) oligomer from Streptococcus pneumoniae strain R6 clearly indicate a difference in conformation of the 2,4,6-trideoxy-galactopyranose ring. Whereas the 2,4-N-acetylated Gal24N adopts the usual 4C1 chair conformation, the native 2-N-acetyl-4-amino Gal24N exhibits conformational mobility with comparable populations in the 4C1 chair and 5S1 skew conformations, as determined using MD simulation for the partial trisaccharide Me-beta-D-Glc6P-(1-->3)-alpha-D-Gal24N-[6-PC]-(1-->4)-alpha- D-galNAc and from the intra-ring NOE effects. 31P-NMR spectra point to a strong electrostatic or hydrogen-bonding interaction between the free 4-NH2 group on the Gal24N and the negatively charged diester phosphate group between adjacent pentasaccharide repeating-units [Ribitol-(5-->6)-beta-D-Glc6P]. Molecular modelling and MD simulation experiments confirmed that such an interaction was feasible with the Gal24N galactopyranose ring in the inverted B1.4 or skew 5S1 conformation.

Immunochemical characterization of the carbohydrate antigens of serotype k and Lancefield group G "Streptococcus milleri".

Carbohydrate antigens of the serotype k/Lancefield group G "Streptococcus milleri" were extracted by autoclaving whole cells of the type k reference strain Streptococcus anginosus K214-2K. The type k and group G antigen molecules are separated from each other and partially purified by a DEAE-Sephadex A25 column chromatography followed by a Sephadex G-100 gel filtration. In the double diffusion and the immunoelectrophoresis, the type k and group G antigen preparations obtained yielded single bands with their homologous antisera respectively. The type k antigen preparation contained principally glycerol, rhamnose, ribitol, galactose and glucose in a molar ratio of 0.54:1.20:0.43:0.33:1.00. The quantitative precipitin inhibition test indicated that beta(1-6)glucosyl-glucose (gentiobiose) sequence played a major role in immunodeterminant structure. Thus, the type k antigen of "S. milleri" appears to be a new carbohydrate type antigen that is chemically different from any Ottens type antigen. In contrast, the group G antigen preparation contained high proportion of rhamnose in addition to glucose, N-acetylglucosamine and N-acetylgalactosamine in a molar ratio of 6.45:1.00:0.24:1.15, and the rhamnose residue was involved in the immunodominant epitope, being in good agreement with the previously proposed chemical structure of the group antigen.

Rapid identification of Haemophilus influenzae serovar b by gas liquid chromatography using carbohydrate fingerprints.

Carbohydrates from whole-cell hydrolysates of 18 strains of the species Haemophilus influenzae (5 strains belonging to serovar b) were analysed by gas-liquid chromatography. The identity of the carbohydrate components was confirmed by comparison with the retention times of reference sugars and by gas chromatography mass spectrometry. Evidence was obtained that Haemophilus influenzae serovar b can easily be identified by the presence of one large peak representing ribitol. The method described can be routinely applied in bacteriological laboratories equipped with a gas chromatograph. It gives results within approximately 4 h, it is reproducible and easy to perform. Even single colonies isolated directly from agar plates can be used for analysis without further subculturing.

Quantitative analysis of cell walls of nutritionally variant streptococci grown under various growth conditions.

Strains of nutritionally variant streptococci are usually isolated from patients with subacute bacterial endocarditis. Only recently have these strains been subdivided into three serotypes; however, no group-specific antigen has been described. To understand the immunochemical basis for the serology of these microorganisms as well as set the groundwork for adherence studies, quantitative analysis of the cell walls of nutritionally variant streptococci was undertaken. The bacteria were grown in semisynthetic medium or pyridoxal-supplemented Todd-Hewitt broth and harvested during the exponential or stationary phase. Cell walls were isolated and analyzed for amino sugars, sugars, polyalcohols, amino acids, and phosphorus by gas chromatography, high-pressure liquid chromatography, or colorimetric assays. The peptidoglycans of the cell walls of the prototype strains from the three serotypes were representative of other streptococcal cell walls, including the presence of alanine as the possible cross-bridge. The composition of the peptidoglycan was similar for all three strains and included a decreased concentration of peptidoglycan in their cell walls during the stationary phase. Glucosamine, glucose, galactose, ribitol, and a small amount of rhamnose were found in each of the cell wall polysaccharides. Galactosamine was only found in serotype II and III cell walls and might be responsible for the previously described cross-reaction between these strains. The concentration of the other sugars and amino sugars varied in each of the cell wall preparations, depending on the growth conditions. Finally, all three strains expressed both ribitol and phosphorus in their cell walls, characteristic of the presence of a ribitol teichoic acid. Therefore the cell wall composition of the nutritionally variant streptococci varies depending on the growth conditions, and their composition appears similar to that of strains of Streptococcus mitis.

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.