Electrospray ionization multiple-stage tandem mass spectrometric analysis of diglycosyldiacylglycerol glycolipids from the bacteria Bacillus pumilus

Electrospray ionization (ESI) combined with multiple-stage tandem mass spectrometry (MSn) was used to directly analyze the glycolipid mixture from bacteria Bacillus pumilus without preliminary separation. Full scan ESI-MS revealed the composition of picomole quantities of glycerolglycolipid species containing C-14-C-19 fatty acids, some of which were monounsaturated, Two main components were identified from their molecular masses and fragmentation pathways. The fragmentation pathway of the known compound compared with the investigated compound verified the proposed structure as 1(3)-acyl-2-pentadecanoyl-3(1)-O-[beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-sn-glycerols. A comparison of the multiple tandem mass spectra of the different alkali-metal cation adducts indicates that the intensity of fragments and the dissociation pathways are dependent on the alkali-metal type, The basic structures of glycerolglycolipids were reflected clearly from the fragmentation patterns of the sodium cations, The intense fragments of the sugar residue from the precursor ions were obtained from the lithiated adduct ions. ESI-MSn spectra of [M + K](+) ions did not provide as much fragmentation as [M + Na](+) and [M + Li](+) adducts, but their spectra allow the position of glycerol acylation to be determined. On the basis of MS2 spectra of[M + K](+) ions, it was established that all components have a C-15:0 fatty acid at the sn-2 position of the glycerol backbone and C-14-C-19 acids at the sn-1 position of the glycerol backbone. Copyright (C) 1999 John Wiley & Sons, Ltd.

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

基于细胞行为的生物学图式形成的离散模型研究

生物学图式及其形成规律一直是生命科学特别是发育生物学的重要课题；同时也是组织工程中实现体外组织构建的核心科学问题之一。长期以来，对生物图式形成的模型研究的根本不足之处是以数理方法为基础的动力学模型研究和生物学背景的结合不够。因此，本文试图遵循生物图式本身的形成过程，寻求一条与生物学相适配的途径，即以哺乳动物组织发育／活组织工程化构建为目标，以细胞行为为基点，以力学一化学藕合作用为介导，以元胞自动机方法为基础，建立生物学图式形成的一个细胞一环境整体离散模型。应用这一整体离散模型，在不同的控制参数下，对盘基网柄菌的聚集图式和杆菌的生长图式进行了系统的分析，对血管发生（vasculogenesis）的自组装图式进行了初步的新的探索，得到了与实验研究定性上一致的结果。提出了“诱导开关”概念，对盘基网柄菌（Dictyostelium discoideu），杆菌（Bacillus）和血管内皮祖细胞（Endothelial Precursor cells，EPC）三种模式生物，分别以cAMP的信号波前，营养微粒，VEGF的浓度梯度等为诱导开关量。在对盘基网柄菌细胞接收到cAMP后分泌和定向迁移形成的聚集图式的模拟中，系统地考察了影响聚集图式的各种控制参数；一个重要的结果表明细胞初始响应间期对形成的聚集模式有十分显著的影响；引入聚集速度、回转半径、盒质量分布系数等概念对盘基网柄菌的聚集图式进行了一些定量描述的探索。在对杆菌因代谢、增殖、凋亡／衰亡而形成的生长图式的模拟中，系统地定量地分析了在初始营养浓度、营养／代谢物扩散快慢、代谢抑制三者藕合作用下的生长图式；引入定向流动边界，考察了杆菌向营养入口方向的优势生长。在对血管内皮祖细胞经vEGF分子浓度梯度场的诱导进行定向迁移，分化为血管内皮细胞，并自组装形成网状的原初毛细血管丛的模拟中，建立了一个微血管发生自组装图式的新的离散模型，为以后加入力与内皮细胞的相互作用以及血管再生等构建了一个前期模型框架；初步考察了细胞的浓度，细胞分泌vEGF分子的周期，vEGF分子的扩散时间尺度等对血管发生图式的影响因素。

果实采后病害生物防治及其机理研究

　　分离和筛选了5种能有效防治采后果实病害的拮抗菌。其中，季也蒙假丝酵母(Candida guiliermondii(Cast) Langeroret Guerra)从引种拮抗菌中筛选获得，枯草芽孢杆菌（Bacillus subtilis）B-912从土壤中分离筛选获得，膜醭毕赤酵母（Pichia membranefaciens hansen）从桃果实伤口上分离获得，隐球酵母(Cryptococcus albidus (Saito) Skinner)和丝孢酵母（Trichosporon sp.）从桃果实表面分离获得。本文主要研究了这些拮抗菌对桃、油桃、苹果、梨和柑桔等我国主要水果采后病害的防治效果，并对其可能的抑菌机理进行了初步研究。结果如下： 1. Sx108 CFU/mL的C．guiliermondii和P．membranefaciens悬浮液可完全抑制病菌孢子浓度为5x104个/mL时桃和油桃果实软腐病(Rhizopus stolonifer(Ehrenb.ex Fr.) Vuill.)在25℃，15℃和3℃下的发生。lx108 CFU/mL的C．albidus和Trichosporon sp.悬浮液可完全抑制孢子浓度分别为lx105个/mL和5x104个/mL时苹果灰霉病(Botrytis cinerea)和青霉病（Penicillum expansum）在23℃-25℃和1℃下的发生。C．albidus和Trichosporon sp.对梨灰、青霉病也有一定抑制效果。B-912对柑桔果实青霉病(Penicillium italicum)、绿霉病(Penicillium digitatum)和核果类果实褐腐病(Monilinia fructicola)也有极好的抑制效果。生物防治效果与拮抗菌的浓度成正比，与病菌孢子浓度成反比。 2．拮抗酵母菌在室温和冷藏条件下都能迅速在果实伤口定殖，接种酵母菌48 h后，数量可增加20倍以上。拮抗菌和病菌孢子的接种时间与生物防治效果有关，先接种拈抗菌的抑菌效果显著地好于同时或后于病菌接种的效果。 3．温度对拮抗酵母菌的抑菌活力没有明显影响，无论是在室温还是在冷藏条件下，拮抗酵母菌都有同样的抑菌效果。但拮抗细菌B-912的抑菌效果与温度有一定关系。较高的温度有利于细菌拮抗作用的发挥。 4．拮抗菌能与常规的果实采后处理措施如钙处理、化学杀菌剂、冷藏和气调贮藏相结合。酵母菌与2% CaC12配合能明显地增强其抑菌能力;拮抗菌与低浓度的杀菌剂如扑海因混合使用，可达到高浓度杀菌剂的抑病效果;C．albidus和Trichosporon sp.对果实采后气调贮藏环境有良好的适应性，它们在气调下对采后苹果、梨的灰霉病和青霉病的抑制效果比冷藏条件下的好。 5．细菌B-912的抑菌机理与其产生抗菌素有关，B-912的滤液在in vitro上能有效地抑制病菌孢子的萌发，在invivo上也能明显地抑制果实采后病害的发生。拮抗酵母菌的抑菌机理则较复杂，但主要与病菌竞争营养有关，同时，C．guilliermondii和P．membranefaciens对软腐菌的抑制还通过产生水解酶如β-1，3一葡聚糖酶和几丁酶与病原菌直接作用，并参与诱导寄主产生抗性等

拮抗菌和化学物质的抑病机理及果实抗病相关基因的克隆

真菌病害是造成采后新鲜水果损失的一个主要原因。生物拮抗菌能有效地防治果实采后腐烂，降低杀菌剂的用量，从而增加了食品安全性和降低了潜在的环境危害。然而，与化学杀菌剂相比，单独使用生物拮抗菌对果实采后病害的控制效果有时不如化学杀菌剂明显。因此，为了提高拮抗菌的生防效力，有效控制果实的采后病害，本文主要研究了拮抗菌与化学物质使用的防病机理，并从冬枣果实中克隆β-1，3-葡聚糖酶基因并对其特性进行了初步分析。研究结果表明： 1. 酵母菌Cryptococcus laurentii和枯草芽孢杆菌Bacillus subtilis能够有效的防治冬枣果实采后青霉病和黑霉病的发生，而且C. laurentii对病害的防治效果比B. subtilis好。拮抗菌的抑病效果与使用浓度成正比。在接种C. laurentii的伤口上再接种病原菌可以显著刺激酵母菌的生长。然而，在接种B. subtilis的伤口上接种病原菌则不增加拮抗细菌的群体数量。 2. 不同酵母拮抗菌对四种杀菌剂（Deccozil，Sportak，Iprodine和Stroby）的敏感程度不同。其中，R. glutinis对Deccozil，Iprodione和Stroby最敏感。将低剂量的杀菌剂与酵母菌配合能显著增强酵母菌对采后病菌的抑制作用。C. laurentii与100 µl/L的Stroby配合能完全抑制青霉和黑霉病菌的孢子萌发。2%（w/v）的碳酸氢钠（SBC）与C. laurentii或T. pullulans配合使用显著抑制采后病菌(Penicillium expansum或Alternaria alternata)的孢子萌发和芽管伸长。SBC显著增强拮抗菌对梨果实采后青霉病和黑霉病的防治能力。C. laurentii对采后病害的防治效果好于T. pullulans的防治效果。 3. C. laurentii和B. subtilis对冬枣果实抗病性的诱导与接种距离和接种时间密切相关。距接种拮抗菌近的部位，抗性诱导就越强。酵母菌诱导果实的这种抗病性与诱导果实几丁质酶，β-1，3-葡聚糖酶， PAL，POD和PPO活性有关。 4. 采前喷施2 mM的水杨酸（SA）和0.2 mM的茉莉酸甲酯（MeJA）显著降低甜樱桃果实采后褐腐病的病斑直径， 并能诱导甜樱桃果实β-1，3-葡聚糖酶， PAL， POD和PPO活性以及乙烯含量的增加。采前处理对果实抗病性的诱导效果要好于采后处理。采前和采后SA或MeJA处理，贮藏于25C的甜樱桃果实β-1，3-葡聚糖酶和PAL活性显著高于贮藏于0C的甜樱桃果实的酶活性。2 mM的SA显著抑制了Monilinia fructicola的孢子萌发和菌丝扩展;而0.2 mM的MeJA则对M. fructicola几乎没有抑制作用。在贮藏早期，MeJA对果实β-1，3-葡聚糖酶和PAL活性的诱导作用要强于SA的诱导作用。 5. 1 × 108CFU/ml的C. laurentii，以及5 × 107CFU/ml的C. laurentii与0.2 mM的MeJA 配合使用均可诱导桃果实的抗性，并显著降低果实青霉病和褐腐病的病斑直径。0.2 mM的MeJA能促进C. laurentii生长，抑制P. expansum的菌丝扩展， 但对M. fructicola基本没有抑制作用。在25和0C，MeJA和C. laurentii单独或配合使用都诱导了桃果实几丁质酶，β-1，3-葡聚糖酶，PAL和POD活性的升高。这些抗病相关酶活性的升高可能与病斑扩展的程度是直接相关的。 6. 通过设计简并引物，采用降落PCR，扩增出β-1，3-葡聚糖酶基因的同源片段，分别克隆到两个彼此间同源性很低的β-1，3-葡聚糖酶的cDNA全长（Glu-1和Glu-2）。RT-PCR结果表明，Glu-1基因的表达受酵母拮抗菌C. laurentii处理所诱导，这一结果与酵母拮抗菌诱导果实β-1，3-葡聚糖酶活性的增加相呼应;而Glu-2基因的表达则不受C. laurentii处理所诱导。

枯草芽孢杆菌生物学特性及其与稻瘟病菌互作的蛋白质组学研究

枯草芽孢杆菌（Bacillus subtilis）是革兰氏阳性细菌研究的模式菌株，为重要的生防菌剂。本论文以枯草芽孢杆菌ATCC6051作对照，首次对新型菌株KB-1111和KB-1122进行了生物学特性、生防潜力以及与水稻稻瘟病致病菌互作的蛋白质组学研究。 形态学观察表明，菌株KB-1111和KB-1122在细胞形态、芽孢的大小、运动性、菌落褶皱和色素的生成等方面与菌株ATCC6051相似，具有枯草芽孢杆菌的典型特征。生理生化测定以及对多种碳源的利用结果显示，三个菌株大部分指标检测结果相同，只在几个方面等存在差异。 体外平板对峙抑菌试验说明，枯草芽孢杆菌ATCC6051、KB-1111和KB-1122对8种作物、果蔬代表性病害致病真菌具有明显的拮抗效果。其中，菌株KB-1122的广谱抗真菌活性优于KB-1111，而KB-1111又强于对照菌株ATCC6051，尤其是对稻瘟病（M. grisea P131）和蔬菜菌核病（S. sclerotiorum）显现出强烈的抑制作用，具备生防拮抗菌的优秀性能。 比较蛋白质组学分析结果表明，液体悬浮培养枯草芽孢杆菌KB-1111、KB-1122二维蛋白质组表达谱至少有11个胞内蛋白和10个胞外蛋白出现丰度差异。其中，菌株KB-1122中胁迫或逆境反应相关ATP酶、顺乌头酸水合酶和alpha-淀粉酶前体在细胞内蛋白质组，以及分泌型蛋白―内切葡聚糖酶在胞外蛋白质组中的高丰度表达可能与菌株KB-1122的优势拮抗能力相关。 将对数生长期的枯草芽孢杆菌KB-1122与菌丝丰富期的稻瘟病菌P131悬浮混合共培养发现，在24小时的共培养过程中，稻瘟病菌P131菌丝体及芽管经历了致变、破裂、细胞质溢出直至菌丝体崩溃等一系列变化，枯草芽孢杆菌KB-1122表现出强烈的拮抗效应。差异显示蛋白质组学研究表明，共培养菌体蛋白质组至少有39个蛋白点丰度发生显著变化，其中33个蛋白点得到成功鉴定，包括12个上调蛋白和21个下调蛋白。根据鉴定结果分析，这些上调的蛋白质全部来源于枯草芽孢杆菌，而下调的蛋白全部属于稻瘟病菌。共培养过程中的培养液蛋白质组至少有20个蛋白点丰度发生显著变化，其中18个蛋白点得到成功鉴定。根据以上分析结果初步认为，3-磷酸甘油醛脱氢酶、丝氨酸蛋白激酶和内切葡聚糖酶在枯草芽孢杆菌KB-1122与稻瘟病菌P131相互作用的过程中可能是B. subtilis KB-1122发挥抗真菌活性的关键性蛋白。

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. (C) 2008 Elsevier Masson SAS. All rights reserved.

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K-i) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs. (C) 2007 Elsevier Inc. All rights reserved.

武汉东湖几丁质分解菌生态研究

对武汉东湖水柱和沉积物中几丁质分解菌的种类组成、数量分布及动态的研究结果表明:几丁质分解菌中.放线菌居于优势,在水柱中约为总数的50—72.3％;在沉积物中约为63—88.5％。经鉴定的102株放线菌中,链霉菌属(Steptomyces)占总数的70.6％;链孢囊菌属(Streptosporangium)、小单孢菌属(Micromonospora)、小多孢菌属(Micropolyspora)分别为8.8％、9.8％和10.8％。细菌中主要是芽孢杆菌属(Bacillus),占鉴定菌株数的42％,其次为沙雷氏

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp strain PCC 7120

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.

Cloning and characterization of the gene for UDPGlc dehydrogenase from the cyanobacterium, Microcystis aeruginosa FACHB 905

Using degenerate primers based on conserved regions of the UDP-glucose dehydrogenase (UDPGDH) gene, an initial 476-bp DNA fragment was amplified from the water-bloom forming cyanobacterium, Microcystis aeruginosa FACHB 905. TAIL-PCR and ligation-mediated PCR were used to amplify the flanking regions to isolate an about 2.5-kb genomic DNA fragment. Sequence analysis revealed an ORF encoding a putative 462 amino acid protein, designated Mud for Microcystis UDPGDH. The Mud amino acid sequence is closely related to UDPGDH sequences from cyanobacterium Synechocystis PCC6803 (73% identity, 81% similarity), and bacterium Bacillus subtilis (51% identity and 67% similarity). The cloned mud gene was expressed in Escherichia coli using the pGEX-4T-1 fusion expression vector system to generate a GST-Mud fusion protein that exhibited UDPGDH activity. The cytosolic fraction of M aeruginosa FACHB 905 was subjected to Western analysis with an anti-Mud antibody, which revealed a single band of approximately 49 kD, consistent with the deduced molecular mass of the enzyme. The Mud protein could thus be characterized as a UDP-glucose dehydrogenase, which was a key enzyme for polysaccharide synthesis and has, for the first time, been studied in algae.

Establishment and characterization of dual-species biofilms formed between a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities

In this study, the possibility of establishing a dual-species biofilm from a bacterium with a high biofilm-forming capability and a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading bacterium, Comamonas testosteroni A3, was investigated. Our results showed that the combinations of strain A3 with each of five strains with a high biofilm-forming capability (Pseudomonas sp. M8, Pseudomonas putida M9, Bacillus cereus M19, Pseudomonas plecoglossicida M21 and Aeromonas hydrophila M22) presented different levels of enhancement regarding biofilm-forming capability. Among these culture combinations, the 24-h dual-species biofilms established by C. testosteroni A3 with P. putida M9 and A. hydrophila M22 showed the strongest resistance to 3,5-DNBA shock loading, as demonstrated by six successive replacements with DMM2 synthetic wastewater. The degradation rates of 3,5-DNBA by these two culture combinations reached 63.3-91.6% and 70.7-89.4%, respectively, within 6 h of every replacement. Using the gfp-tagged strain M22 and confocal laser scanning microscopy, the immobilization of A3 cells in the dual-species biofilm was confirmed. We thus demonstrated that, during wastewater treatment processes, it is possible to immobilize degrader bacteria with bacteria with a high biofilm-forming capability and to enable them to develop into the mixed microbial flora. This may be a simple and economical method that represents a novel strategy for effective bioaugmentation.