铜绿假单胞菌cntRLMN操纵子介导锌离子摄取的功能鉴定

【目的】本研究以铜绿假单胞菌PAO1 (Pseudomonas aeruginosa PAO1,菌种编号ATCC15692)为对象,研究cntRLMN在锌离子摄取中的功能。【方法】在ΔznuBC的基础上,以同源重组的方法构建了cntRLMN的各种突变菌株,通过质粒接合转移的方法构建其互补菌株及lacZ转录融合报告菌株,运用β-半乳糖苷酶酶活检测研究了Zur蛋白对cntRLMN的转录调控,凝胶阻滞实验(EMSA)检验Zur蛋白与cnt启动子及cnt启动子的突变片段的体外结合,并进一步通过生长曲线分析对cntRLMN中cntR、cntL、cntN等基因的锌离子摄取功能进行了分析和鉴定。最终,通过构建大蜡螟幼虫的侵染模型来研究cntRLMN对铜绿假单胞菌毒力发挥的影响。【结果】lacZ转录融合的酶活分析显示cntRLMN受Zur蛋白的负调控,其表达以Zur蛋白依赖的方式受锌离子饥饿的诱导;EMSA实验的结果显示cntRLMN的启动子可以与His-Zur结合形成DNA-蛋白质复合体,结合位点为GCGTTATAGTATATCAT;生长曲线和大蜡螟幼虫侵染实验的分析结果显示ZnuBC和CntRLMN的功能存在互补性,仅znuBC和cntRLMN双缺失突变时菌株在限锌培养条件下的生长和对大蜡螟幼虫的毒性才受到显著抑制,说明CntRLMN代表另一种独立的锌离子摄取系统。【结论】cntRLMN是受Zur直接负调控的另一种独立的铜绿假单胞菌锌离子摄取系统,对铜绿假单胞菌毒力的发挥起重要作用。     

烟草青枯病劳尔氏菌与拮抗菌对根系分泌物的竞争作用

[目的]研究青枯病病原菌与拮抗菌的营养特性及其对烟草根系分泌物的响应差异,对提高拮抗菌定殖能力、有效生物防控烟草青枯病具有非常重要的意义。[方法]本研究通过筛选与鉴定贵州烟区青枯病病原菌株及拮抗菌株,通过Biolog表型芯片技术分别检测病原菌与拮抗菌的特征性碳、氮源,利用气质联用(GC-MS)检测烟草主栽品种K326根系分泌物的主要物质,在此基础上进行病原菌与拮抗菌对其利用能力、利用强度以及共培养的研究。[结果]经鉴定,分离、筛选到的病原菌株和拮抗菌株分别为青枯劳尔氏菌(Ralstonia solawacearum)和枯草芽孢杆菌(Bacillus subtilis);在含量为0.01μg/mL以上的根系分泌物中,12种物质的含量从高到低排序为:果胶>葡萄糖>木糖>阿拉伯糖>半乳糖>核糖>蔗糖>苯甲酸>果糖=D-甘露醇>棕榈酸>富马酸,果胶含量最高且明显高于其他物质;拮抗菌(LX4)对碳源利用能力高于病原菌(Rs)的碳源有阿拉伯糖、木糖和核糖,分别是病原菌利用能力的1.22、1.95和2.17倍;前12 h拮抗菌利用果糖强度高于病原菌,不同碳源共培养24 h后LX4对gfp-Rs(绿色荧光蛋白标记后的青枯病病原菌)抑制率为18.34%(阿拉伯糖)、53.23%(木糖)、63.53%(核糖)和52.09%(果糖)。[结论]拮抗菌对烟草根系分泌物的利用不及病原菌,但在特定碳源条件下拮抗菌能够利用根系分泌物中的某些碳源产生某种拮抗物质抑制病原菌,拮抗菌与病原菌之间同时存在利用性竞争和干扰性竞争关系,研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

相容溶质四氢嘧啶与羟基四氢嘧啶的代谢调控研究进展

四氢嘧啶(Ectoine)及其衍生物羟基四氢嘧啶(5-hydroxyectoine,5-HE)是嗜盐微生物胞内合成的一类能够抵抗外界高盐胁迫的相容溶质,具有细胞、细胞膜、蛋白质和核酸的保护作用,可抵抗高盐、高温、冷冻和干燥等极端环境因素的刺激,从而倍受关注。本文对不同类型微生物Ectoine/5-HE(Ects)生物合成代谢、分解代谢以及吸收/转运系统涉及的调控机制进行综述,以期为Ects合成产量的提升与高效积聚策略的优化,提供一定的理论参考依据。     

一株寒地高效解无机磷细菌的分离鉴定及拮抗作用

【目的】从北方寒地种植的不同农作物根际土壤中分离高效解磷的细菌,为微生物制剂和磷肥的开发提供适于本地区的优良菌种。【方法】通过初筛和复筛从26株解磷菌中筛选获得一株高效解磷细菌,对其进行生理生化和分子生物学鉴定,同时采用钼蓝比色法测定解磷能力。采用平板对峙法测定拮抗植物病原菌能力。【结果】通过筛选后获得的菌株B51-7经鉴定为伯克霍尔德菌属。菌株在发酵液中可溶性磷含量最高达到832.74 mg/L,同时具有很强的广谱抑菌作用,抑菌率最高为89.71%,可以显著促进水稻生长。【结论】菌株B51-7是一株具有生物防治作用的高效解磷细菌,可应用于生物菌肥和生防制剂中。     

顺反式肉桂醛对肉源隆德假单胞菌生物被膜和致腐性的抑制作用

【目的】为比较反式和顺式肉桂醛对肉源假单胞菌生物被膜和致腐性的影响。【方法】通过平板计数测定两种肉桂醛对隆德假单胞菌的最小抑菌浓度(MIC),结晶紫法、珠涡流法、激光共聚焦显微镜观察、福林法等检测亚抑菌浓度肉桂醛处理下隆德假单胞菌生物被膜形成、运动性和胞外酶活性变化。荧光定量RT-PCR检测肉桂醛对隆德假单胞菌粘附lapA、鞭毛fliC、蛋白酶aprX和脂肪酶lip基因表达量的影响。【结果】反式和顺式肉桂醛对隆德假单胞菌的MIC分别为200μg/mL和225μg/mL,1/8 MIC、1/4MIC、1/2MIC亚抑菌浓度肉桂醛显著降低隆德假单胞菌生物被膜结晶紫和粘附性,其中1/2MIC反式和顺式肉桂醛处理下被膜分别减少60.27%和52.05%,菌体粘附降低56.35%和61.10%。亚抑菌浓度肉桂醛显著减少被膜厚度,反式肉桂醛还能显著杀灭被膜菌。且肉桂醛能显著抑制菌体的泳动性,反式肉桂醛对生物被膜和泳动性的抑制效果更强。肉桂醛还能抑制隆德假单胞菌蛋白酶和脂肪酶活性,其中1/2MIC反式和顺式肉桂醛处理下菌体蛋白酶分别减少61.90%和76.19%,脂肪酶降低40.17%和47.01%。且发现肉桂醛显著降低lapA、fliC、aprX和lip表达量,其中1/2MIC反式和顺式肉桂醛分别降低4个基因表达量至对照组的0.05–0.16和0.02–0.12倍。【结论】两种亚抑菌浓度肉桂醛异构体显著抑制隆德假单胞菌生物被膜和致腐性,其中反式肉桂醛对生物被膜抑制较强,而顺式肉桂醛更有效地降低致腐酶活性,其与肉桂醛下调相应基因表达密切相关。     

A Computational Study of the Giant Local Electric Field Enhancement in Al-Au-Ag Trimetallic Three-Layered Nanoshells

The surface plasmon resonance (SPR)-induced local field effect in Al-Au-Ag trimetallic three-layered nanoshells has been studied theoretically. Because of having three kinds of metal, three plasmonic bands have been observed in the absorption spectra and the local electric field factor spectra. The local electric field enhancement and the corresponding resonance wavelength for different plasmon coupling modes and spatial positions of the Al-Au-Ag nanoshells with various geometry dimensions are investigated to find the maximum local electric field enhancement. The calculation results indicate that the giant local electric field enhancement could be stimulated by the plasmon coupling in the middle Au shell or the outer Ag shell and could be optimized by increasing the Ag shell thickness and decreasing the Au shell thickness. What is more, the local electric field enhancement also nonmonotonously depends on the dielectric constant of the environment; the local electric field intensity will be weakened when the surrounding dielectric constant is too small or too large.

     

FUNDC1 is a novel mitochondrial-associated-membrane (MAM) protein required for hypoxia-induced mitochondrial fission and mitophagy

Mitochondria need to be fragmented prior to engulfment by phagophores, the precursors to autophagosomes. However, how these 2 processes are finely regulated and integrated is poorly understood. We have shown that the outer mitochondrial membrane protein FUNDC1 is a novel mitochondrial-associated membrane (MAM) protein, enriched at the MAM by interacting with the ER resident protein CANX (calnexin) under hypoxia. As mitophagy proceeds, it dissociates from CANX and preferably recruits DNM1L/DRP1 to drive mitochondrial fission in response to hypoxic stress. In addition, knocking down of FUNDC1, DNM1L or CANX in hypoxic cells increases the number of elongated mitochondria and also reduces the colocalization of autophagosome and mitochondria, thus preventing mitophagy. These findings identify FUNDC1 as a molecular hub integrating mitochondrial fission and mitophagy at the MAM in response to hypoxia.     

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.     

Glucose‐based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N‐acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L‐tryptophan hydroxylase, a 5‐hydroxy‐L‐tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O‐methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co‐factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L^?1 in a 76h fermentation using simulated fed‐batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.     

Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.