BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly

We have identified a potent antibacterial agent N-(4-sec-butylphenyl)-2-(thiophen-2-yl)-1H-benzod]imidazole-4-carboxa mide (BT-benzo-29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT-benzo-29 disassembled the cytokinetic Z-ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT-benzo-29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid-cell. Further, BT-benzo-29 bound to FtsZ with a dissociation constant of 24 +/- 3 m and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT-benzo-29 may bind to FtsZ at the C-terminal domain near the T7 loop. BT-benzo-29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT-benzo-29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT-benzo-29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT-benzo-29 did not affect the membrane integrity of mammalian red blood cells. BT-benzo-29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

Prediction and validation of gold nanoparticles (GNPs) on plant growth promoting rhizobacteria (PGPR): a step toward development of nano-biofertilizers

Several soil microbes are present in the rhizosphere zone, especially plant growth promoting rhizobacteria (PGPR), which are best known for their plant growth promoting activities. The present study reflects the effect of gold nanoparticles (GNPs) at various concentrations on the growth of PGPR. GNPs were synthesized chemically, by reduction of HAuCl 4, and further characterized by UV-Vis spectroscopy, X-ray diffraction technique (XRD), and transmission electron microscopy (TEM), etc. The impact of GNPs on PGPR was investigated by Clinical Laboratory Standards Institute (CLSI) recommended Broth-Microdilution technique against four selected PGPR viz., Pseudomonas fluorescens, Bacillus subtilis, Paenibacillus elgii, and Pseudomonas putida. Neither accelerating nor reducing impact was observed in P. putida due to GNPs. On the contrary, significant increase was observed in the case of P. fluorescens, P. elgii, and B. subtilis, and hence, GNPs can be exploited as nano-biofertilizers.

Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains

LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.

Manejo de tizón tardío (Phytophthora infestant (Mont.) de Bary) en el cultivo de papa cv calwhite en época de apante en la zona de Tisey, Esteli. 2007

El objetivo principal de este estudio fue la evaluación de diferentes estrategias de alternancia de fungicidas de contacto con fungicidas sistémicos. Además, se alternó el fungicida biológico Serenade 1.34 SC ( Bacillus subtilis QST 713) con los fungicidas de contacto Mancozeb y Bravonil para determinar el efecto de ésta en el manejo de tizón tardío en época de apante. El trabajo experimental se llevó a cabo en la localidad Tisey (Reserva Natural), del departamento de Estelí en el periodo comprendido entre noviembre del 2006 a febrero del 2007. La variedad de papa evaluada fue CalWhite, la cual es susceptible a tizón tardío. El diseño utilizado fue bloques completamente al azar con cinco tratamientos y cuatro repeticiones. Los tratamientos evaluados en este estudio fueron (Mancozeb+Serenade) - Equation Pro (Bravonil+Serenade) - Ridomil, (Mancozeb+Serenade)- Curzate, (Bravonil+Serenade) - Verita y un testigo absoluto. El manejo agronómico fue el que utiliza el productor. Las variables evaluadas fueron severidad y rendimiento. Con los datos de severidad se calculó el área bajo la curva de progreso de la enfermedad (ABCPE). No se encontraron diferencias significativas entre bloques para la variable severidad de tizón tardío, ni en la interacción tratamiento - bloque, pero si se observó diferencias altamente significativas entre tratamientos. La media de severidad en el tratamiento testigo fue más alta en comparación con las medias de los otros cuatro tratamientos, lo cual explica la diferencia entre tratamientos. Sin embargo, no se encontraron diferencias significativas en las medias de severidad de los tratamientos diferentes del testigo. Cuando se analizó la severidad en los tratamientos de acuerdo a la variable días después de la siembra (dds) se observaron diferencias altamente significativas entre tratamientos, en la variable dds y en la interacción tratamientos*d ds. Se encontraron diferencias altamente significativas entre el testigo y los demás tratamientos y entre días después de la siembra (dds) para los valores de la variable área bajo la curva de progreso de la enfermedad (ABCPE), así como también en la interacción tratamientos*dds. La mezcla del fungicida biológico Serenade 1.34 SC ( Bacillus subtilis QST713) con los fungicidas de contacto Mancozeb y Bravonil al parecer tuvo un efecto sinergístico al inhibir el desarrollo de P. infestans sobre la superficie de las hojas de papa. Los rendimientos fueron bastantes bajos en comparación con otros resultados encontrados en América Latina con la variedad de papa CalWhite. Es posible reducir los porcentajes de severidad de tizón tardío en el cultivo de papa cuando se combinan estrategias de rotación de fungicidas de contacto y sistémicos con la época de siembra.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores.

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks.

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 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.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks. © 2011 Elsevier B.V.

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.

微生物对抗菌肽耐受的诱导及耐受机理初步研究

抗菌肽是一类具有强大杀菌能力的肽类分子，同时还具有离子调节、免疫调节、蛋白酶抑制剂和自由基清除等其他生物活性。现已鉴定的抗菌肽超过1,200 种，几乎存在于所有生物种类中。在抗生素耐受严重的今天，抗菌肽极有潜力成为新型的有效抗菌药物，许多抗菌肽已进入临床前研究或临床研究。在本论文中，我们选择了无指盘臭蛙（Odorrana grahami）来源的三种抗菌肽（Brevinin 2E-OG1、Nigrocin-OG4 和Palustrin-OG1），单独或组合使用，以藤黄微球菌（Micrococcus luteus）、枯草芽孢杆菌（Bacillus subtilis）和白假丝酵母菌（Candida albicans）为研究对象，进行微生物对抗菌肽耐受性的实验诱导；并通过检测胞外蛋白酶活性、蛋白质组学等方法对微生物耐受抗菌肽机制进行初步的研究。将微生物培养于含有低浓度抗菌肽（单独使用或组合使用）培养基中，每日转接一次，每十次酌情提高抗菌肽浓度。80 次转接后，除藤黄微球菌未对 Palustrin-OG1 产生耐受外，其余所有的实验菌株均表现出对所用三种抗菌肽的耐受。但是Palustrin-OG1 与Brevinin 2E-OG1 或Nigrocin-OG4 联合使用能在一定程度上降低耐受性。将诱导后细菌于不含抗菌肽条件下培养，转接5 次后，对耐受现象无影响，说明这种耐受是可以稳定遗传的。抗菌肽耐受机制之一是分泌蛋白酶水解胞外抗菌肽，我们通过两种方式检测胞外蛋白酶活性，一种是检测发酵液的酪蛋白水解活性，另一种是检测发酵液处理抗菌肽后对抗菌活性的影响。结果发现枯草芽孢杆菌和藤黄微球菌发酵液存在着蛋白酶活性，推测胞外蛋白酶可能与二者对抗菌肽的耐受有关；而白假丝酵母菌发酵液中未检测到蛋白酶活性。另外，我们还通过蛋白质组学的手段对枯草芽孢杆菌耐受机制进行了初步的研究，鉴定了5 个差异表达的蛋白，表达上调的蛋白有yraA（功能未知）、Tpx （巯基过氧化物酶，Thiol peroxidase）、pdhD（二氢硫辛酰胺脱氢酶，dihydrolipoamide dehydrogenase），表达下调的有cotN/TasA（芽孢膜相关蛋白，spore coat-associated protein）和gapA（三磷酸甘油醛脱氢酶，Glyceraldehyde 3-phosphate dehydrogenase 1 ，GAPDH）。yraA 和Tpx 都由Spx 调控，yraA 可以水解小肽增加自由氨基酸，而自由氨基酸增多时gapA、tasA 表达水平会下降，Spx 是由sigma-M 因子调控的，所以我们推测sigma-M 因子在B. subtiis 对抗菌肽耐受中起到了重要的作用。总之，本研究发现抗菌肽的联合作用会减缓微生物对其耐受的程度，为抗菌肽类药物研发提供了一种新思路；同时对抗菌肽耐受机制的初步研究也为今后的深入研究打下了基础。另外，我们还设计了一种新型的抗菌肽系统命名方法，并构建了昆明动物研究所抗菌肽数据库。

海洋细菌3512A对黄瓜枯萎病的防治及其生物学特性的研究

黄瓜枯萎病(Cucumber fusarium Wilt)又称萎蔫病、蔓割病，是一种世界性的植物维管束病害。1925年Weber 首次报道在美国佛罗里达州发生。该病由半知菌亚门，镰刀菌属尖孢镰刀菌(Fusarium oxysporum)侵染所致，是一类主要经土壤传播的维管束病害，在我国瓜类种植区也普遍发生，在北方地区尤为严重。近年来随着蔬菜栽培面积的增加，土壤菌量逐年积累，此病的发生日趋严重。据统计，每年黄瓜枯萎病的发病率可达20%，严重时高达80%-90%，甚至全部毁种，导致了黄瓜的严重减产。 本论文从56株选自与海洋动、植物共生/共栖的细菌为资源，以黄瓜枯萎病菌(Fusarium oxysporum)为靶菌，通过平板对峙试验筛选出8株具有较强抑黄瓜枯萎病菌作用的菌株，其中以海洋细菌3512A的抑菌效果最好。室内模拟实验表明该菌株能够在灭菌土和有菌土中高密度定殖，促进黄瓜幼苗的生长。盆栽试验表明其能够有效的防治黄瓜枯萎病，防效达64.29％～73.62％，还能促进黄瓜的生长，提高叶绿素含量，增加黄瓜的产量，可认为是一株植物根际促生菌（PGPR）采用传统的细菌学和分子生物学的鉴定方法对其进行了菌种鉴定，为枯草芽孢杆菌（Bacilluss subtilis）。 通过对抗菌谱的研究，发现海洋细菌3512A除了对黄瓜枯萎病菌(Fusarium oxysporum) 有很强的抑菌活性外，对金黄色葡萄球菌、大肠杆菌、白色念珠菌和多种植物病原真菌都具有较强的抑制作用，抗菌范围广，可进一步做对其它农作物病害的防治实验。 海洋细菌3512A在盆栽试验中对黄瓜枯萎病的成功防治以及促生作用，为其良好的使用提供了指导并为其进一步的研究提供了基础。

中国南海深海沉积物中的微生物资源

由于采样条件和培养条件的限制，深海微生物研究的较少，因此目前海洋微生物资源的研究主要集中在近海和浅海。而深海独特的生态环境，使微生物形成了独特的代谢体系，成为新颖化合物的重要来源，具有很好的开发应用前景。本文首次较为系统的研究了深度为1758-3600m南海海底沉积物中深海微生物资源的分离和活性菌株的筛选情况，旨在为探索我国南海深海微生物资源提供一定的科学依据。 采用多种分离培养基从6个不同深度（1758、2620、3200、3500、3587和3600m）的南海深海沉积物样品中，分离到225株深海微生物，包括40株放线菌和185株细菌。以分离得到的225株深海微生物为研究对象，从产蛋白酶、抗真菌、杀虫三个方面进行活性菌株的筛选，研究南海深海沉积物中的微生物资源状况： （1）.将分离到的225株深海微生物，同时在10℃、28℃、45℃三个温度下进行产低温蛋白酶、中温蛋白酶、高温蛋白酶的筛选。初筛结果表明：这225株深海微生物大都有大小不同的产蛋白酶能力。10℃有产蛋白酶的有109株，28 ℃产蛋白酶的有160株，45℃产蛋白酶的有117株。筛选到一株在45℃下有较强产蛋白酶能力的菌株B1394，其酶活可达873U/ml，有一定的应用前景。通过形态观察、生理生化测定、16SrDNA序列测定，将B1394定名为枯草芽孢杆菌（Bacillus subtilis）。其粗酶液性质研究发现：最适酶活温度60℃，最适pH 8.0， 40℃、50℃和60℃热稳定性较好， Mn2+ 、Mg2+ 、Ca2+对该蛋白酶有激活作用，Hg2+ 、Fe3+ 、Cu2+ 、Zn2+ 、 Fe2+对其有抑制作用，苯甲基磺酰氟（PMSF）几乎完全抑制其活性，推断为丝氨酸蛋白酶。 （2）.对其中100株深海细菌和40株深海放线菌，以立枯丝核菌（ Rhizoctonia solani）和白色念珠菌（Candida albicans）为靶菌进行了抗真菌活性的筛选。初筛结果表明：分别有37株和35株深海细菌对立枯丝核菌和白色念珠菌有抑菌活性，分别占被检测细菌数目的37%和35%；有18株深海放线菌对立枯丝核菌有抑制作用，占被检测放线菌总数的45%。筛选到一株有较强抗真菌活性的深海放线菌SHA6，其发酵液对包括尖孢镰刀菌（Fusarium oxysporum）在内的10种植物病原菌有明显的抑制作用。对其发酵液的理化性质进行初步研究发现SHA6发酵液具有良好的热稳定性和酸碱稳定性, 对SHA6进行分子生物学鉴定后将其初步定名为为橙色单胞菌（Aurantimonas altamirensis）。 （3）.以卤虫为初步筛选模型，对其中的20株深海放线菌进行了杀虫方面的筛选，结果发现有5个深海放线菌菌株表现有较强的杀虫活性。其中深海放线菌SHA4发酵液的乙酸乙脂提取物杀卤虫的活性最强，在浓度为100μg/ml时杀卤虫活性为83%,而在浓度400ppm时，48h可以杀死38%的甜菜夜娥。对SHA4进行分子生物学鉴定后将其初步定为拟诺卡氏菌属（Nocardiopsis sp）。 总之，本论文阐明了我国南海深海沉积物中微生物资源状况的初步研究结果，为进一步开发利用我国南海深海微生物资源奠定了基础。