Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction

For the first time, the complete structure of the lipid A from the lipopolysaccharide of an Agrobacterium species is here reported. In particular, the structure of the lipid A from A. tumefaciens strain C58, a soil pathogen bacterium strictly related to Rhizobiaceae, was determined. The structural study, carried out by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy, revealed that lipid A fraction consisted of a mixture of species all sharing the bis-phosphorylated glucosamine disaccharide backbone that could be designated in two main structural motifs, according to the acylation pattern. The main species was a penta-acylated lipid A bearing two unsubstituted 14:0 (3-OH) fatty acids in ester linkage and two 16:0 (3-OH) in amide linkage; the one on GlcN II was O-acylated by a long chain fatty acid, 28:0 (27-OH). This in turn was esterified by a 3-hydroxy-butyroyl residue at its hydroxy group. The second species, in lesser amounts, was identified as a tetra-acylated lipid A and lacked the 14:0 (3-OH) residue on GlcN I. Other species deriving from these two lacked a phosphate group or 3-hydroxy-butyroyl residue or otherwise carried a 26:0 (25-OH) as long chain fatty acid. The lipid A structure of phytopathogen A. tumefaciens strain C58 presents deep structural analogies with lipid A of symbiotic Rhizobium, and the hypothesis is advanced that it can be a strategy of the bacterium to escape or attenuate the plant response.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide

Burkholderia cepacia, a Gram-negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions. This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients. An important virulence factor of B. cepacia is the lipopolysaccharide (LPS) fraction. Clinical isolates and environmental strains possess LPS of high inflammatory nature, which induces a high level production of cytokines. For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of B. cepacia is described. The structural studies carried out by selective chemical degradations, MS, and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns. The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide [Arap4N-l-beta-1-P-4-beta-D-GlcpN-(1-6)-alpha-D-GlcpN-1-P-1-beta-L-Arap4N]. Lipid A fatty acids substitution was also deduced, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide linkage, one of which was substituted by a secondary 14:0 residue at its C-3. Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.     

Structural studies of the O-specific polysaccharide from Plesiomonas shigelloides strain CNCTC 113/92.

The structure of the O-specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix-assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure: in which D-beta-D-Hepp is Dglycero-beta-Dmanno-heptopyranose and 6d-beta-D-Hep is 6-deoxy-beta-Dmanno-heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O-antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high-resolution magic angle spinning technique, 1H-NMR spectra were also obtained for the O-polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.     

Core oligosaccharides of Plesiomonas shigelloides O54:H2 (strain CNCTC 113/92): structural and serological analysis of the lipopolysaccharide core region, the O-antigen biological repeating unit, and the linkage between them.

The structure of the core oligosaccharide moiety of the lipopolysaccharide (LPS) of Plesiomonas shigelloides O54 (strain CNCTC 113/92) has been investigated by (1)H and (13)C NMR, fast atom bombardment mass spectrometry (MS)/MS, matrix-assisted laser-desorption/ionization time-of-flight MS, monosaccharide and methylation analysis, and immunological methods. It was concluded that the main core oligosaccharide of this strain is composed of a decasaccharide with the following structure: (see text) in which l-alpha-D-Hepp is l-glycero-alpha-D-manno-heptopyranose. The nonasaccharide variant of the core oligosaccharide ( approximately 10%), devoid of beta-D-Glcp substituting the alpha-D-GlcpN at C-6, was also identified. The core oligosaccharide substituted at C-4 of the outer core beta-D-Glcp residue with the single O-polysaccharide repeating unit was also isolated yielding a hexadecasaccharide structure. The determination of the monosaccharides involved in the linkage between the O-specific polysaccharide part and the core, as well as the presence of -->3)-D-beta-D-Hepp-(1--> instead of -->3,4)-D-beta-D-Hepp-(1--> in the repeating unit, revealed the structure of the biological repeating unit of the O-antigen. The core oligosaccharides are not substituted by phosphate residues and represent novel core type of bacterial LPS that is characteristic for the Plesiomonas shigelloides serotype O54. Serological screening of 69 different O-serotypes of P. shigelloides suggests that epitopes similar to the core oligosaccharide of serotype O54 (strain CNCTC 113/92) might also be present in the core region of the serotypes O24 (strain CNCTC 92/89), O37 (strain CNCTC 39/89) and O96 (strain CNCTC 5133) LPS.     

尼罗罗非鱼致病性类志贺邻单胞菌(Plesiomonas shigelloides)的分离鉴定及其病理学观察

【目的】2013年5月广东番禺某养殖场罗非鱼出现大量死亡现象,临床症状表现为鱼体体色发黑、体表出血、鳞片脱落、鳍条溃烂等,解剖发现腹腔有大量腹水、胆囊肿大、肝呈黄色、脾脏呈暗红色。为确定病原,【方法】从具以上临床症状的病鱼组织中分离获得可疑菌株1株,编号PYS1。采用形态特征、生长特性、理化特征、16S rRNA基因序列分析等理化及分子生物学技术鉴定该菌株种类。通过人工回归感染及组织病理学研究确定该菌株的致病性,并开展药物敏感性试验筛选其敏感药物。【结果】结果表明PYS1菌株为类志贺邻单胞菌(Plesiomonas shigelloides),在16S rRNA基因序列构建的分子进化树中与其他鱼源类志贺邻单胞菌聚为一支。药敏试验结果表明该菌株已呈现多重耐药性,仅对少数检测抗生素(头孢曲松、头孢洛克和头孢唑啉等)敏感。人工回归感染结果显示PYS1菌株可使尼罗罗非鱼出现与自然发病鱼相似症状,其对尼罗罗非鱼半致死量为1.425×108CFU/尾,石蜡切片显示其对感染鱼的肠、肝、脾、肾和心脏等组织均可造成损伤。【结论】证明类志贺邻单胞菌为上述养殖场尼罗罗非鱼发病的病原,且为首次报道该菌对尼罗罗非鱼的致病性。     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 2. Lipid A, its structural variability, the linkage to the core oligosaccharide, and the biological activity of the lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identification of the putative lipopolysaccharide glycosyltransferases

Pasteurella multocida is an important multispecies veterinary pathogen. The cell surface lipopolysaccharide (LPS) is an important virulence factor and forms the basis of the serotyping scheme, although little structural information about it is known. The structure of the LPS from the Pasteurella multocida genome strain Pm70 was elucidated in this study. The LPS was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy, and mass spectrometry. The structure of the core oligosaccharide was determined on the basis of the combined data from these experiments. Identification of the core oligosaccharide structure enabled a search for glycosyltransferase homologs in the Pm70 genome and revealed a clustering of the genes putatively responsible for outer core oligosaccharide biosynthesis.     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

The complete structure of the core of the LPS from Plesiomonas shigelloides 302-73 and the identification of its O-antigen biological repeating unit

Plesiomonas shigelloides is a Gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal infections, which especially invades immunocompromised patients and neonates. The lipopolysaccharides are one of the major virulence determinants in Gram-negative bacteria and are structurally composed of three different domains: the lipid A, the core oligosaccharide and the O-antigen polysaccharide.In the last few years we elucidated the structures of the O-chain and the core oligosaccharide from the P. shigelloides strain 302-73. In this paper we now report the characterization of the linkage between the core and the O-chain. The LPS obtained after PCP extraction contained a small number of O-chain repeating units. The product obtained by hydrazinolysis was analysed by FTICR-ESIMS and suggested the presence of an additional Kdo in the core oligosaccharide. Furthermore, the LPS was hydrolysed under mild acid conditions and a fraction that contained one O-chain repeating unit linked to a Kdo residue was isolated and characterized by FTICR-ESIMS and NMR spectroscopy. Moreover, after an alkaline reductive hydrolysis, a disaccharide α-Kdo-(2→6)-GlcNol was isolated and characterized. The data obtained proved the presence of an α-Kdo in the outer core and allowed the identification of the O-antigen biological repeating unit as well as its linkage with the core oligosaccharide.     

Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway

The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcbeta1Cer --> Manbeta4Glcbeta1Cer [MacCer] --> GlcNAcbeta3Manbeta4Glcbeta1Cer [At(3)Cer] --> GalNAcbeta4- GlcNAcbeta3Manbeta4Glcbeta1Cer [At(4)Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galbeta3Manbeta4Glcbeta1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAcalpha4Galbeta3Manbeta4- Glcbeta1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAcalpha4 and Galbeta3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcbeta4 in the arthro-series penta- and hexaglycosylceramide structures (At(5)Cer and At(6)Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAcalpha4 and Galbeta3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galbeta3-T capable of using Manbeta4Glcbeta1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.     

Structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562

The structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562 is presented. LPS hydrolysis gave two oligosaccharides, OS-1 and OS-2, as well as lipid A. NMR spectroscopic data corresponded to the presence of one Kdo residue, one β-glucopyranose, three heptoses, one glyceric acid, one acetate, three PEtN, and one 5,7-diacylamido-3,5,7,9-tetradeoxynonulosonic acid residue (pseudaminic acid, Pse) in OS1. OS2 differed form OS 1 by the absence of glyceric acid, acetate, and Pse residues. Lipid A was analyzed for fatty acid composition and the following fatty acids were found: C14:0, C12:0-3OH, C16:0, C16:1, C14:0-3OH, C18:0, C18:1 in a ratio of 1:3:3:1:2.5:0.6:0.8.     

Structural characterization of the lipid A region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4'-monophosphorylated beta-2-amino-2-deoxy-d-glucopyranose-(1-->6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the alpha-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2',3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3'. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3' was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide.     

Identification of diacylglycerol-O-(N,N,N-trimethyl)-homoserine in the halotolerant alga, Dunaliella parva

An unusual zwitterionic polar lipid component isolated from the halotolerant alga, Dunaliella parva Lerche, has been identified as 1(3),2-diacylglyceryl-3(1)-O-4′-(N,N,N-trimethyl) homoserine by means of infrared spectrometry, ¹H- and ¹³C-NMR spectrometry and field desorption mass spectrometry of the intact lipid, as well as by its TLC mobilities and staining behaviour. Mass spectrometry of this lipid indicated the presence of the following major molecular species: 16:0–18:0 (24%); 18:3–18:3 (19%); 16:0–18:2 (16%); 16:0–18:1 (8%).     

The Structure of Uncommon Lipid A from the Marine Bacterium <Emphasis Type="Italic">Marinomonas communis</Emphasis> ATCC 27118<Superscript>T</Superscript>

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118^T with 1% AcOH. Using chemical analysis and 1D and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be β-1′,6-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-{(R)-3-hydroxydecanoyloxy)]decanoic acid and (R)-3-hydroxydecanoic acid at the C2, C2′ and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M .communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 404–413.Original Russian Text Copyright © 2005 by Vorob’eva, A. Dmitrenok, P. Dmitrenok, Isakov, Krasikova, Solov’eva.The article was translated by the authors.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Study of the Antifungal Ability of Bacillus subtilis Strain PY-1 in Vitro and Identification of its Antifungal Substance （Iturin A）

A Bacillus strain,denoted as PY-1,was isolated from the vascular bundle of cotton.Biochemical,physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis.The PY-1strain showed strong ability against many common plant fungal pathogens in vitro.The antibiotics producedby this strain were stable in neutral and basic conditions,and not sensitive to high temperature.From theculture broth of PY-1 strain,five antifungal compounds were isolated by acidic precipitation,methanolextraction,gel filtration and reverse-phase HPLC.Advanced identification was performed by mass spec-trometry and nuclear magnetic resonance spectroscopy.These five antifungal compounds were proved to bethe isomers of iturin A:A2,A3,A4,A6 and A7.In fast atom bombardment mass spectrometry/mass spec-trometry collision-induced dissociation spectra,fragmentation ions from two prior linear acylium ions wereobserved,and the prior ion,Tyr-Asn-Gln-Pro-Asn-Ser-βAA-Asn-CO~ ,was first reported.     

Structure and Properties of the Lipopolysaccharide of Pseudomonas fluorescens IMV 2366 (Biovar III)

The lipopolysaccharide (LPS) preparation isolated from the bacterial mass of Pseudomonas fluorescens IMV 2366 (biovar III) by Westphal's method and purified by repeated ultracentrifugation contained S- and R-forms of molecules. The structural components of the LPS molecule—lipid A, core oligosaccharide, and O-specific polysaccharide—were obtained in the individual state and characterized. The main components of the lipid A hydrophobic moiety were 3-hydoxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, and hexadecanoic fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic moiety. Rhamnose, glucose, galactose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as 2-amino-2,6-dideoxygalactose (FucN) and 3-amino-3,6-dideoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions. O-specific polysaccharide chains were composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl. Neither double immunodiffusion in agar not the immunoenzymatic assay revealed serological relations between the strain studied and the P. fluorescens strains studied earlier.