Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid

The muramic acid assay is a powerful tool for detecting both intact bacteria and bacterial debris. Past use of aldononitrile acetate derivatization for determining muramic acid in complex samples by gas chromatography/mass spectrometry met detection needs in many instances; however, questions have been raised regarding the interpretation of the derivative structure and its electron ionization fragments. In this study, we applied different methods and proved that the aldononitrile acetate derivatized muramic acid yields a molecular weight of 398, associated with a lactam structure. We also presented evidence that the structure of aldononitrile acetate derivatized muramic acid is acetylated at four positions, 3 O-acetylations and 1N-acetylation. In practical manner, this communication provides a comprehensive reference to researchers using δ¹³C value or ion fragments of the muramic acid marker in biogeochemical studies.     

干湿交替条件下土壤氨基糖含量的动态变化

通过室内模拟培养试验,研究了恒湿和干湿交替条件下土壤中3种微生物来源氨基糖含量的动态变化,并且利用氨基葡萄糖和胞壁酸的比值分析了干湿交替条件下土壤真菌和细菌对土壤有机质转化的相对贡献.结果表明:恒湿条件下,细菌来源的胞壁酸在土壤中的分解速率大于真菌来源的氨基葡萄糖,氨基半乳糖在土壤中的分解速率较慢;干湿交替改变了土壤中3种氨基糖的分解特征,与恒湿处理相比,干湿交替培养前期以胞壁酸为代表的细菌残余物的分解速率高于以氨基葡萄糖为代表的真菌残余物,随着干湿交替频率的增大,以氨基葡萄糖为代表的真菌残余物分解速率高于以胞壁酸为代表的细菌残余物.可见,干湿交替条件改变了以氨基糖为代表的土壤氮素的微生物转化过程.     

Reliability of Muramic Acid as a Bacterial Biomarker is Influenced by Methodological Artifacts from Streptomycin

The muramic acid (MurA) assay is a powerful tool for the detection and quantification of bacteria with no need to enrich samples by culturing. However, the analysis of MurA in mixed biological and environmental matrices is potentially more complex than analysis in isolated bacterial cells. In this study, we employed one commonly used procedure for extraction of MurA from environmental samples and found that the presence of streptomycin interfered with the determination of MurA by creating chemical species that coeluted with the aldononitrile derivative of MurA prepared in this method. On a molar basis, streptomycin yields a signal that is approximately 0.67 times that of MurA. Mass spectrometry analysis confirmed that the interference from hydrolyzed streptomycin is not actually by MurA, but rather is likely to be N-methyl glucosamine. Because streptomycin is widely applied for selective growth of eukaryotes both in situ and in vitro, our findings may have implications for the significance of results from MurA assays. We conclude that MurA remains an effectual bacterial biomarker due to its unique bacterial origin, but care must be applied in interpreting results from the assay when performed in the presence of streptomycin.     

Analysis of muramic acid in holocene microbial environments by gas chromatography,electron impact,and fast atom bombardment mass spectrometry

Analytical procedures have been modified to determine the abundance of muramic acid in four different Holocene sediment samples. Muramic acid is specific to the peptidoglycan moiety of the cell walls of most eubacterial pro‐karyotic organisms. The following procedure seemed to be the most appropriate for the detection of muramic acid and amino acids, including diaminopimelic acid. Hydrolysis of the samples (in 6 N HCl, 4.5 h, at 100°C) was followed by separation and purification of amino sugars and amino acids using Amberlite XAD‐2 and then Bio‐Rad AG 50W‐X8 resins. The N,O‐heptafluorobutyryl‐n‐butyl ester derivatives were prepared by esterification in acidified (3 N HCl) n‐butanol for 3 h at 100°C, followed by acylation by refluxing with heptafluorobutyric anhydride in acetonitrile (2:1 v/v) for 12 min at 150°C. The derivatives were analyzed by gas chromatography (GC) and gas chromatography‐mass spectrometry. Fast atom bombardment (FAB) ionization was used for the muramic acid derivative to determine its molecular weight and structure, d‐and l‐amino acids were separated by GC and a capillary chiral column. By using this technique a stable N,O‐heptafluo‐robutyryl‐n‐butyl ester derivative of muramic acid was identified at picogram levels in Holocene sedimentary microbial communities. It has been reported previously that microorganisms in sediments rapidly degrade muramic acid from cell walls of dead prokaryotes. Kinetic experiments revealed that muramic acid was relatively stable in intact cell walls but decomposed rapidly in the free form. These investigations noted above showed that the concentration of muramic acid may be used as an indicator of the presence of the intact cell walls of cyanobacteria and most other bacteria in Holocene microbial communities, and of microbial contamination in samples older than the Holocene.     

引入固氮树种对辽东落叶松人工林土壤团聚体氨基糖的影响

土壤微生物残留物是稳定性碳库的重要组分,然而固氮树种引入对落叶松人工林土壤团聚体微生物残留物分布的影响还不清楚.为了阐明固氮树种对不同团聚体粒级内微生物残留物分布的影响,本研究以氨基糖作为微生物残留物的生物标识物,比较了辽东日本落叶松人工纯林和落叶松与固氮树种赤杨混交林土壤团聚体氨基糖的分布特征.结果表明: 赤杨引入不影响团聚体氨基糖的分布,但显著提高了团聚体氨基糖含量.与纯林相比,混交林土壤不同团聚体各粒级总氨基糖含量增加1.3～1.7倍.其中,混交林土壤团聚体内总氨基糖增加量的66.5%～66.9%来自氨基葡萄糖,30.0%～30.6%来自氨基半乳糖,2.5%～3.2%来自胞壁酸.赤杨引入显著提高了>2000 μm和<250 μm团聚体中的氨基葡萄糖/胞壁酸值,但不影响250～2000 μm粒级团聚体中真菌和细菌残留物的相对贡献.此外,赤杨引入增加了土壤不同团聚体内的氨基糖对土壤有机碳的贡献,但不影响团聚体粒级之间的微生物贡献,说明赤杨对微生物贡献的影响存在空间均一性.     

东北黑土氨基糖的矿化动态及其对外源物质添加的响应

采用间歇淋洗好气培养法研究了东北黑土中3种不同微生物来源氨基糖(氨基葡萄糖、胞壁酸和氨基半乳糖)的矿化动态以及对葡萄糖添加和葡萄糖与氮肥配施的响应.结果表明:土壤中不同种类的氨基糖具有不同的矿化特征.培养期间胞壁酸含量减少25.4%而氨基葡萄糖含量降低7.1%,表明细菌来源的胞壁酸在土壤中的矿化速率快于真菌来源的氨基葡萄糖,但氨基葡萄糖的矿化数量(68.4 mg·kg^-1)显著高于胞壁酸(15.4 mg·kg^-1).葡萄糖添加以及葡萄糖与氮肥配施均显著提高了土壤中氨基葡萄糖和胞壁酸的含量,但两种处理的影响有所不同.相比之下,氨基半乳糖在土壤中矿化较慢,并且受外源物质的影响较小,表现出较高的稳定性.     

三江平原湿地开垦对土壤氨基糖积累特征的影响

微生物残体在土壤有机质的形成和稳定过程中发挥着重要作用,但湿地开垦对土壤微生物残体积累特征的影响尚不清楚。本研究以三江平原小叶章湿地为对象,采集原始自然湿地和开垦改种豆科作物后不同耕作年限(5年、10年和25年)的土壤,以氨基糖为微生物残体的标识物,探讨湿地开垦对土壤微生物残体积累特征的影响。结果表明: 自然湿地开垦为农田后显著降低了土壤中氨基糖的含量,且随着开垦年限的增加,氨基糖的损失比例也增加。与自然湿地相比,开垦25年后土壤中的氨基葡萄糖、氨基半乳糖和胞壁酸含量分别下降38.0%、38.1%和35.9%,且在开垦最初5年中细菌来源的胞壁酸下降速率(25.8%)远高于真菌来源的氨基葡萄糖(14.9%),说明短期内湿地开垦对细菌的影响较真菌更加迅速。湿地开垦为农田5、15和25年后,土壤氨基糖总量分别下降21.1%、34.0%和38.0%;同时,氨基糖总量占土壤有机质的比例也受到湿地开垦的显著影响,由自然湿地中的4.8%降至开垦25年后的4.4%。这说明长期湿地开垦加速了土壤有机质中微生物来源有机组分的分解转化,进而改变土壤有机质的组成。这些变化将影响湿地生态系统中土壤有机质的长期稳定和功能演变。     

葡萄糖存在下选择性抑菌剂对土壤氮素转化的影响

采用2种氮源并分别加入选择性微生物抑制剂进行室内培养,通过测定样品中NH₄⁺-N和NO₃^--N及土壤中氨基葡萄糖和胞壁酸含量,研究土壤微生物氮素固持时间特征及其相对贡献.结果表明,加入青链霉素明显地降低了NH₄⁺-N的转化速率,且影响远大于真菌抑制剂放线菌酮;氨基葡萄糖和胞壁酸的相对比例急剧增加,而后趋于平衡;加入放线菌酮后NO₃-N转化速率持续下降,氨基葡萄糖的合成受到抑制,但加入细菌抑制剂青链霉素对其转化无显著影响.培养初期,细菌在葡萄糖存在下能快速固持NH₄⁺-N和NO₃^--N,并以NH₄⁺-N为首选氮源;培养后期,氮转化主要为真菌所推动,且真菌对NO₃^--N的利用能力显著大于细菌.     

氨基糖单体碳氮同位素的分析及其应用

氨基糖(AS)作为有机质中在分子水平识别的重要组分,研究其来源与转化能更好地认知微生物对有机质的调控作用。作为一种新兴技术,氨基糖单体同位素分析(CSIA-AS)为研究氨基糖各组分在自然环境中的变化特征提供了更详细的信息。本文系统总结了CSIA-AS技术的测定方法及其在氨基糖循环转化研究中的应用,气相色谱-同位素比值质谱法(GC-IRMS)和离子色谱-同位素比值质谱法(IC-IRMS)作为2种主要的氨基糖同位素测定方法,各有利弊,但进行相应的校正后均可实现可靠的测定结果。氨基糖各组分在土壤有机质中具有相对较低的周转时间,细菌来源的胞壁酸相对葡萄糖胺、半乳糖胺和甘露糖胺具有更高的矿化速率。氨基糖在环境中的来源和代谢转化受底物的影响,这与微生物群落对不同碳、氮源的特异性响应有关。CSIA-AS技术的推广需要进一步的方法优化并将其与微生物甄别等其他手段相结合,以此来更好地阐释有机质的来源、转化和归宿及其调控机制。     

Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis

It has previously been established that several glycopeptides of peptidoglycan origin are formed as a result of processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264. Although the formation of these glycopeptides could account for the humoral immune responses characteristic of bacterial peptidoglycans, their formation does not account for the cellular-mediated immune responses observed for water-in-oil emulsions of peptidoglycan or for lipophilic derivatives of glycopeptide fragments thereof. Therefore, the processing of peptidoglycan by macrophages was reexamined to establish whether the lipophilic derivative of any peptidoglycan-derived glycopeptide was formed. The experiments were performed by incubating B. subtilis cell walls radiolabeled in muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid residues in the presence of the macrophage-like cell line RAW264. The crude lipid fraction derived from the macrophages was further fractionated and analyzed, revealing the presence of two lipophilic glycopeptides that contained glucosamine, muramic acid, and alanine of bacterial origin.     

The rapid,quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas-liquid chromatography

O-Methyloximes have been prepared from 2-amino-2-deoxy-d-glucose, -d-mannose, and -d-galactose. The acetates of these derivatives yield stable compounds which are readily separated quantitatively by gas-liquid chromatography on a number of polar phases. The above compounds along with the aldononitrile acetates of neutral sugars can be easily separated from one another on a single column in one chromatographic run. The procedures developed were tested on a number of glycoproteins of known composition as reported by other workers utilizing more classical methodologies, resulting in excellent agreement in terms of sugar composition. An improved method is also described for converting neutral sugars to oximes which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.     

Determination of muramic acid in organic dust by gas chromatography-mass spectrometry

A method is described for the quantitation of muramic acid, a marker of bacterial peptidoglycan, in organic dust. House dust samples were hydrolysed in hydrochloric acid and then extracted with hexane to remove hydrophobic compounds. The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography-mass spectrometry. The muramic acid derivative gave two peaks upon injection into the gas chromatograph-mass spectrometer. Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5–1500 ng of muramic acid. In a house dust sample, 40 ng of muramic acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed). The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust.     

Study of the formation of N-glycolylmuramic acid from Nocardia asteroides (author's transl)]

Nocardia asteroides was grown in Sauton medium containing sodium [carboxy-14C]acetate. The biosynthesis of the peptidoglycan was inhibited by adding penicillin or phosphonomycin to the growth medium. These antibiotics give an accumulation of radioactive nucleotidic precursors of the peptidoglycan. In the presence of penicillin, there was an accumulation of uridine diphosphate-N-glycolylmuramyl peptide (UDP-MurNGlyc peptide) and of a mixture of uridine diphosphate-N-acetyl and N-glycolylmuramic acid (UDP-MurNAc) and UDP-MurNGlyc). In the presence of phosphonomycin, the biosynthesis of muramic acid was blocked and there was an accumulation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-N-glycolyglucosamine (UDP-GlcNGlyc). Thus the formation of a N-glycolyl group can be performed upon the neucleotidic derivatives of glucosamine and muramic acid. However in the peptidoglycan synthesized in vivo in the absence of antibiotic, only muramic acid was glycolyated. So, glycolylation seems to take place essentially on UDP-MurNAc. When the binding of peptide chain to muramic acid is achieved, all the muramic acid is glycolylated, then the polymerisation of glycan and peptidoglycan units by the mean of particulate enzymes is carried out on the N-glycolylated derivative of muramic acid. A cell-free preparation from Nocardia asteroides was obtained which can hydroxylate the acetyl group of UDP-MurNAc. The activity was localised in the soluble fraction. This system acts as a hydroxylase and requires the presence of NADPH.     

Automated analysis of 2,3-diamino-2,3-dideoxy-D-glucuronic acid by cation exchange chromatography with fluorometric postcolumn derivatization

2,3-Diamino-2,3-dideoxy-D-glucuronic acid (diaminoglucuronic acid) occurs as its di-N-acetyl derivative as a unique constituent of some bacterial cell walls. A sensitive chromatographic method for its determination is described. Diaminoglucuronic acid was well separated from glucosamine and galactosamine in about 80 min on a Dionex DC-6A cation exchange column (0.9 x 18 cm, 50 degrees C) with a sodium citrate buffer (pH 5.28) containing boric acid (0.2 M). The amino sugars in the eluate were monitored fluorometrically by postcolumn derivatization with orthophthalaldehyde detection reagent. This method allowed the automated determination of 50-100 pmol of glucosamine, galactosamine, and diaminoglucuronic acid and was applied successfully to the analysis of diaminoglucuronic acid in Propionibacterium acnes cells.     

氮磷添加对长白山温带森林土壤微生物群落组成和氨基糖的影响

在长白山阔叶红松林中设置氮添加(N,50 kg N·hm^-2·a^-1)、磷添加(P,25 kg P·hm^-2·a^-1)和氮磷添加(NP,50 kg N·hm^-2·a^-1+25 kg P·hm^-2·a^-1)试验,分析氮磷添加对有机层和矿质层土壤微生物群落组成和氨基糖的影响。结果表明: 在有机层土壤中,N、P添加使总微生物生物量显著降低19.5%和24.6%,P添加还使细菌和真菌生物量分别显著降低23.8%和19.3%;在矿质层土壤中,N、P和NP添加使总微生物生物量显著增加94.8%、230.9%和115.0%,细菌和真菌生物量在施肥处理下显著增加。N添加下有机层土壤真菌与细菌生物量比值(F/B)显著增大,而NP添加使矿质层土壤F/B显著减小。革兰氏阳性菌与革兰氏阴性菌比对N、P和NP添加有显著正响应。土壤氨基糖对不同处理的响应不同。在有机层土壤中,N、P和NP添加使氨基葡萄糖含量分别减少41.3%、48.8%和36.4%,而N和NP添加分别使胞壁酸含量显著增加43.0%和71.1%;在矿质层土壤中,氨基葡萄糖和胞壁酸含量在N添加下无显著变化,而在P和NP添加下显著增加。在施肥处理下,有机层土壤中氨基葡萄糖与胞壁酸比值显著减小,表明N、P添加增加了细菌对土壤有机碳积累的相对贡献。N、P添加后土壤氨基糖含量的变化与微生物群落组成的变化密切相关,且二者均受到土壤化学性质变化的影响。     

Variations in Soil Microbial Communities and Residues Along an Altitude Gradient on the Northern Slope of Changbai Mountain,China

Altitudinally-defined climate conditions provide specific vegetation types and soil environments that could influence soil microbial communities, which in turn may affect microbial residues. However, the knowledge is limited in terms of the degree to which microbial communities and residues present and differ along altitude. In this study, we examined the soil microbial communities and residues along the northern slope of Changbai Mountain, China using phospholipid fatty acid (PLFA) and amino sugar analysis, respectively. Soil samples were taken from five different vegetation belts defined by climates. Principal component analysis (PCA) revealed substantial differences in soil microbial community composition among study sites, appeared to be driven primarily by soil pH and C/N ratio on the first principal component (PC1) which accounted for 50.7% of the total sample variance. The alpine tundra was separated from forest sites on the second principal component (PC2) by a signifiscantly higher amount of fungal PLFA (18:2ω6,9). Soil pH and C/N ratio were also correlated with the ratios of Gram-positive to Gram-negative bacteria (Gm⁺/Gm⁻), glucosamine to galactosamine (GluN/GalN), and glucosamine to muramic acid (GluN/MurA). Both total PLFAs and amino sugars were positively correlated with soil organic carbon, inorganic nitrogen, available phosphorus and potassium. We concluded that soil pH and C/N ratio were the most important drivers for microbial community structure and amino sugar pattern, while substrate availability was of great importance in determining the concentrations of microbial communities and residues. These findings could be used to facilitate interpretation of soil microbial community and amino sugar data derived from measurements in latitude or managed forests.     

Improved method of determining muramic acid from environmental samples

Muramic acid is an amino sugar that forms part of the peptidoglycan in prokaryotic cell walls. Since muramic acid is found only in prokaryotes it has been used as a measure of bacterial and cyanophyte biomass. Successful application of sensitive capillary gas‐liquid chromatographic (GLC) analysis required neutralization of the acid‐hydrolysate of a biomass sample followed by centrifugation to remove humic acids. After a further fractionation on a cation exchange column followed by derivatization and GLC analysis, recoveries of 98 ±9.5 (X± S.D.) % of authentic muramic acid from estuarine sediments with sensitivities of 10^‐13 mol were achieved. The structure of the GLC derivative was established by GLC infrared analysis and GLC mass spectrometry. The improvements in reproduci‐bility and sensitivity have allowed detection of ¹³C enrichments in muramic acid from the detrital microbiota incubated with relabeled precursors.     

Components of the cell wall of Clostridium welchii (type A)

1. The cell wall of Clostridium welchii (type A) contains alanine, 2,6-diaminopimelic acid, glutamic acid, glycine, glucosamine, muramic acid, galactosamine, mannosamine, ethanolamine, rhamnose, galactose and phosphorus. 2. Heating with formamide at 150 degrees resolved the wall into a formamide-soluble polysaccharide fraction and a formamide-insoluble mucopeptide fraction. 3. The formamide-soluble fraction contained two components: an electrophoretically neutral polysaccharide made up of galactose, rhamnose, galactosamine and phosphorus and an electrophoretically acidic polymer containing mannosamine, ethanolamine and phosphorus. 4. The formamide-insoluble residue has been digested by lysozyme to give soluble fragments of high molecular weight. 5. All fractions contain an unknown ethyl acetate-extractable substance that can be oxidized by sodium metaperiodate. 6. The amino acid compositions of the fragments produced by lysozyme are compatible with a mucopeptide structure which has cross bridges containing all of the constituent amino acids.     

Trace analysis of muramic acid in indoor air using an automated derivatization instrument and GC-MS(2) or GC-MS(3)

An automated derivatization instrument has been developed for the preparation of alditol acetates from bacterial hydrolysates for analysis by gas chromatography-mass spectrometry (GC-MS). The current report demonstrates the utility of the automated instrument for the more demanding task of trace analysis of muramic acid (Mur) in airborne dust using gas chromatography-tandem mass spectrometry (GC-MS(2)). Conditions for efficient derivatization of Mur, vital for trace analysis, are rigorous including lactam and imido group formation under anhydrous conditions. Furthermore, as the detection limit is lowered, possible contamination or carry-over of samples becomes an increasingly greater consideration and must not occur. The instrument meets these criteria and was successfully used for assaying the levels of Mur in laboratory air, which were found to be much lower than in the previous studies of heavily occupied schools and agricultural environments. The potential for GC-MS(3) in further lowering the detection limit was also demonstrated.     

不同施肥处理下水稻根际和非根际土壤中氨基糖积累特征

以水稻长期定位施肥试验土壤为研究对象,选取不施肥(CK)、化肥(NPK)、秸秆还田+化肥(NPKS)、30%有机肥+70%化肥(LOM)和60%有机肥+40%化肥(HOM)5种处理,分析水稻分蘖旺期根际土和非根际土中氨基糖积累特征.结果表明: 与CK和NPK处理相比,长期施用有机物料(NPKS、LOM、HOM)显著增加了水稻根际土和非根际土中有机碳、总氨基糖及其氨基单糖(胞壁酸、氨基葡萄糖和氨基半乳糖)含量.不同施肥处理下3种氨基单糖的积累规律不同,说明不同微生物对施肥处理的响应趋势和强度有所不同.受稻田翻耕等均匀化土壤的农事操作影响,各处理总氨基糖含量在根际土与非根际土间无显著差异.氨基糖碳对土壤有机碳积累的贡献范围为24.0～28.3 mg·g^-1,且以NPKS处理最高,HOM和CK处理最低.真菌氨基葡萄糖/胞壁酸比值范围为24.4～36.6,说明该试验点所有处理的根际土与非根际土中有机质的降解与转化过程以真菌为主导,且与NPK和CK相比,NPKS处理的真菌参与度提高,而施用HOM处理的细菌参与度提高.