Chemical composition of Eubacterium nodatum cell wall peptidoglycan

The structure of Eubacterium nodatum cell wall peptidoglycan was investigated. The peptide subunit of E. nodatum peptidoglycan has the following structure: L-Ala-D-Glu (Gly)-L-Orn-D-Ala. The carboxyl group of alanine occupying position 4 is attached to the -amino group of ornithine of an other subunit by the cross-linking bridge L-Ala-L-Ala-L-Orn. All glycine molecules are connected with the -carboxyl group of glutamic acid with the ratio being 0.5–1. The hydrolysis of E. nodatum peptidoglycan by the S. albus G enzyme proceeds primarily due to the activity of alanyl-alanine endopeptidase, ornithyl-ornithine endopeptidase, ornithyl-alanine endopeptidase, N-acetyl-muramyl-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase.     

Brevibacterium divaricatum: Influence of nutritional conditions on the wall composition and release of uncross-linked peptidoglycan chains into medium

Purified cell walls, originating from penicillin-treated (3 g/ml, 1 h) and-untreated Brevibacterium divaricatum cells grown on complex (CM) and glucose minimal medium with (MM) or without (Ca-free MM) calcium carbonate, were isolated by two procedures. Electron micrographs and chemical analysis revealed no differences between identically isolated walls with respect to the presence or absence of either penicillin or calcium carbonate in the glucose growth medium. On the contrary, the appearance and peptidoglycan content of the walls was greatly dependent on the procedure used for their isolation and the walls isolated from the cells grown on complex medium contained more materials other than peptidoglycan. It was shown that the presence of calcium carbonate in the glucose minimal medium was essential for accumulation of large amounts of peptidoglycan chains into the medium. Penicillin-induced interruption of cell wall synthesis was prerequisite for manifestation of the calcium carbonate stimulating effect.Abbreviations CM complex medium - MM chemically defined minimal medium based on glucose and containing calcium carbonate - Ca-free MM MM modified only by the omission of calcium carbonate - ET-walls Enzyme treated walls - FPR-walls French press-ruptured walls     

大肠杆菌细胞壁肽聚糖的化学修饰及荧光标记

【目的】发展一种活细菌细胞壁荧光标记方法,为后期研究细菌肽聚糖的生物合成和代谢规律以及其与细菌感染致病的关系提供新的工具。【方法】对细菌肽聚糖的生物合成前体N-乙酰葡萄糖胺-1-磷酸(GlcNAc-1-P)进行化学修饰,设计并合成含有叠氮基的GlcNAc-1-P类似物(化合物5:Ac3GlcNAz-1-P)。将该类似物与细菌共同孵育,使其作为探针进入细菌肽聚糖天然合成途径。之后提取并酶解肽聚糖组分,用红外光谱(FTIR)和液质联用(LC/MS)检测探针是否通过代谢进入细菌肽聚糖结构中。同时用外源荧光素对代谢掺入细菌肽聚糖中的探针进行染色。在激光共聚焦显微镜下观察对活细菌的荧光标记效果。【结果】通过四步有机合成反应,以79%的总收率成功获得了化合物5。将大肠杆菌(Escherichia coli BL21)作为模式菌株与化合物5共孵育后,其肽聚糖组分的LC/MS和FTIR分析结果均显示探针可以被细菌利用并被代谢掺入到肽聚糖结构中。激光共聚焦显微镜观察结果显示,荧光素可以高效标记表面携带有生物正交探针的大肠杆菌。【结论】设计合成了一种新型探针,可用于活细菌成像,为深入研究细菌肽聚糖的生物学功能及其与细菌感染致病的关系提供了一种简便的方法。     

Chemical composition of cell walls as a taxonomical marker

Matrix sugar composition ofChlorella is species-specifically different. The rigid wall consists of either glucosamine or glucose and mannose. Ruthenium red stainability and anisotropy of cell wall are either plus or minus species-specifically. The cell wall is specifically degraded by the lytic enzyme of the cell itself.     

Cell wall anionic polymers and peptidoglycan of Actinoplanes philippinensis VKM Ac-647

The cell wall of Actinoplanes philippinesis VKM Ac-647 harbours several carbohydrate-containing anionic polymers. (1) The main polymer of the wall is of a poly(glycosylglycerol phosphate) nature. Its monomeric units — O--d-mannopyranosyl-(14)--d-galactopyranosyl-(11)-glycerol monophosphates — are connected by phosphodiester bonds involving the hydroxyl groups at glycerol C3 and galactose C6. There also are chains without mannosyl substitutents. The teichoic acid structure has been established by chemical analysis and with ¹H and ¹³C NMR spectroscopy. This is the first finding of a teichoic acid with mannosyl residues in a bacterial cell wall. (2) The phosphorylated mannan contains mannose and 2-O-methylmannose. Its core chain has -1,2; -1,3; and -1,6 substitutions as revealed by ¹³C NMR spectroscopy.The peptide unit of the peptidoglycan contains no l-alanine, instead of which position 1 is occupied by glycine; and diaminopimelic acid is represented, besides its meso- (or DD) form, by small amounts of its LL isomer.Abbreviations Gro glycerol - Gro2P glycerol-2 phosphate - APT attached-proton-test - P_tot total content of phosphorus - P_lab phosphorus mineralized in 7 min at 100°C - P_NA phosphorus of nucleic acids - P_stab stable phosphorus - T trace amounts     

Lack of peptidoglycan in the cell walls of Methanosarcina barkeri

Neither muramic acid and glucosamine nor d-glutamic acid or other amino acids typical of peptidoglycan were found in cell walls of two strains of Methanosarcina barkeri. The main components are galactosamine, neutral sugars and uronic acids. Therefore, the structural component of the cell wall most likely consists of an acid heteropolysaccharide, resembling that of Halococcus morrhuae. It is, however, not sulfated.     

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.     

Effect of specific growth limitations on cell wall composition of Staphylococcus aureus H

Conditions are described for the continuous culture of a derivative of Staphylococcus aureus H in a fully defined minimal medium in which cysteine is the sole amino acid. The effects of growth under various nutrient limitations on the composition and properties of the cell wall have been studied. The proportion of ribitol teichoic acid present in the wall, and the extent to which it is substituted with N-acetylglucosamine, varies in bacteria grown under different conditions as does the composition and extent of cross-linking of the peptidoglycan. Neither the derivative nor the original strain H produced teichuronic acid when grown under phosphate limitation.Non-Standard Abbreviation SDS Sodium dodecyl sulphate     

Use of an industrial grade medium and medium enhancing effects on high cell density CO fermentation by Eubacterium limosum KIST612

Studies were made on the composition of the growth medium to increase the cell concentration in a cell-recycled continuous culture (Eubacterium limosum KIST612) with carbon monoxide as a sole energy source using phosphate-buffered basal medium (PBBM) and modified PBBM. One of major limiting factors in PBBM might be nitrogen during the high cell density culture. This limitation could be overcome by increasing of inorganic nitrogen or yeast extract concentration in the medium. Anaerobic digester fluid, which could replace the organic nitrogen in PBBM, was used to develop an industrial grade medium for conversion of CO to multi-carbon compound.     

Methanol conversion in Eubacterium limosum

The conversion of methanol by cell-free extracts of the acetogenic bacterium Eubacterium limosum was studied. Incubation of mixed cell-free extracts of both E. limosum and Methanobacterium formicicum resulted in methane formation from methanol in the presence of ATP and 2-mercaptoethanesulfonic acid. The separate extracts were not able to perform this reaction. Addition of ferredoxin obtained from Methanosarcina barkeri to the mixed extracts resulted in increased methane formation. The enzyme, responsible for methanol binding in cell-free extract of E. limosum, was inactivated by FAD under N₂ and exhibited maximal activity under an atmosphere of H₂. This enzyme contains a firmly bound cobalamin which was methylated by methanol in the presence of ATP. It was demethylated in the presence of methylcobalamin: coenzyme M methyltransferase obtained from M. barkeri under concomitant formation of methylated coenzyme M. These properties are similar to those of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase from M. barkeri. It was proposed that methylotrophic acetogens and methylotrophic methanogens use similar enzymes in the first step of methanol conversion.Abbreviations HS-CoM 2-mercaptoethanesulfonic acid - CH₃S-CoM 2-(methylthio)ethanesulfonic acid - BrES 2-bromoethanesulfonic acid - TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid - MT₁ methanol: 5-hydroxybenzimidazolylcobamide methyltransferase - MT₂ methylcobalamin - HS-CoM methyltransferase - DMBI 5,6-dimethylbenzimidazole and HBI, 5-hydroxybenzimidazole, are -ligands of corrinoids - (S-CoM)₂ 2,2-dithiodiethanesulfonic acid     

短棒状杆菌胞壁肽聚糖的实验研究

目的提取短棒状杆菌的有效成分,观察生物学效应,为研制新型治疗制剂奠定基础。方法将短棒状杆菌77—1株用冷热外压法获得细胞壁,再经苯酚及三氯甲烷萃取肤聚糖成分。结果提纯品经紫外吸收测定、淋巴细胞转化试验、抑瘤试验、脾激活试验、毒性试验证实,具有生物学活性及安全性。其主要成分经生化检测含有多肽及聚糖类物质。结论短棒状杆菌的有效成分是细胞壁上的肽聚糖类物质;实验采用的提纯工艺具有可操作性和实际应用价值,为新产品开发提供了可靠依据。     

Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10–15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala: Glu: GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M 1) alanine is replaced by threonine, however. Neutral sugars and-in some strains-additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or d-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three l-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min.The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.Non Standard Abbreviations SDS sodium dodecylsulfate - EDTA ethylenediaminetetra acetic acid - DNP dinitrophenyl Dedicated to Prof. Dr. Adolf Butenandt on the occasion of his 75th birthday     

Eubacterium aggreganssp. nov., a new homoacetogenic bacterium from olive mill wastewater treatment digestor

A strictly anaerobic, homoacetogenic, gram-positive, non spore-forming bacterium, designated strain SR12(T) (T = type strain), was isolated from an anaerobic methanogenic digestor fed with olive mill wastewater. Yeast extract was required for growth but could also be used as sole carbon and energy source. Strain SR12(T) utilized a few carbohydrates (glucose, fructose and sucrose), organic compounds (lactate, crotonate, formate and betaine), alcohols (methanol), the methoxyl group of some methoxylated aromatic compounds, and H2 + CO2. The end-products of carbohydrate fermentation were acetate, formate, butyrate, H2 and CO2. End-products from lactate and methoxylated aromatic compounds were acetate and butyrate. Strain SR12(T) was non-motile, formed aggregates, had a G+C content of 55 mol % and grew optimally at 35 degrees C and pH 7.2 on a medium containing glucose. Phylogenetically, strain SR12(T) was related to Eubacterium barkeri, E. callanderi, and E. limosum with E. barkeri as the closest relative (similarity of 98%) with which it bears little phenotypic similarity or DNA homology (60%). On the basis of its phenotypic, genotypic, and phylogenetic characteristics, we propose to designate strain SR12(T) as Eubacterium aggregans sp. nov. The type strain is SR12(T) (= DSM 12183).     

Eubacterium angustum sp. nov., a Gram-positive anaerobic,non-sporeforming,obligate purine fermenting organism

A strictly anaerobic, uric acid, xanthine, and guanine fermenting bacterium was isolated from sewage sludge requiring thiamine as a vitamin. Acetate, formate, ammonia and CO₂ were products. Cells were Gram-positive, straight rods, 3 to 6.5 m long and 1.1 to 1.5 m wide. They were non-motile, however, possessed flagella. Spore formation could not be obtained. The guanine-plus-cytosine content (G+C) of its deoxyribonucleic acid was 40.3 mol%. Based on these features, the organism belongs to the genus Eubacterium. Due to its restricted substrate spectrum and its inability to utilize arginine or to form cytochromes like E. lentum, this organism did not resemble any of the previously described species of Eubacterium. Therefore, it is proposed to form a new species Eubacterium angustum sp. nov.This paper is dedicated to Prof. Dr. Hans G. Schlegel on the occasion of his 60th birthday     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain

The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene. Mur1 is a 24.7-kDa protein endowed with a putative signal peptide. Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding. Mur1 appears to be expressed and cell-associated in S. thermophilus, as revealed by immunoblot analysis. These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.     

Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum

The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of glycine reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)⁻¹ with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)⁻¹ in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB. Dedicated to Prof. Dr. Gerhard Gottschalk.     

Short Communication: Lipopolysaccharide composition of Desulfovibrio cell wall

The lipopolysaccharide of the sulphate-reducing bacterium Desulfovibrio desulfuricans was analysed by GC, combined either with flame-ionization detection or with MS, and by standard chemical tests. The major sugar of the polysaccharide portion of the macromolecule was glucose (56%). Low amounts of mannose, galactose, rhamnose and amino sugar were also found. Six fatty acids were identified in the lipid A fraction: 9-octadecenoic, tetracosenoic, heptadecenoic, 10-octadecenoic, eicosenoic and 8-octadecynoic.     

Alterations in the cell wall of Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D

In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrinkled appearance 10–15 min after challenge by Bdellovibrion bacteriovorus 109D. This wrinkling effect is believed (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipoprotein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of Spirillum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun.