Faecal virome of red foxes from peri-urban areas

Red foxes (Vulpes vulpes) are the most abundant carnivore species in the Northern Hemisphere. Since their populations are well established in peri-urban and urban areas, they represent a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. In this study, we evaluated the faecal virome of juvenile and adult foxes from peri-urban areas in central Croatia. The dominating mammalian viruses were fox picobirnavirus and parvovirus. The highest number of viral reads (N = 1412) was attributed to a new fox circovirus and complete viral genome was de novo assembled from the high-throughput sequencing data. Fox circovirus is highly similar to dog circoviruses identified in diseased dogs in USA and Italy, and to a recently discovered circovirus of foxes with neurologic disease from the United Kingdom. Our fox picobirnavirus was more closely related to the porcine and human picobirnaviruses than to known fox picobirnaviruses.     

Production of Avaroferrin and Putrebactin by Heterologous Expression of a Deep-Sea Metagenomic DNA

The siderophore avaroferrin (1), an inhibitor of Vibrio swarming that was recently identified in Shewanella algae B516, was produced by heterologous expression of the biosynthetic gene cluster cloned from a deep-sea sediment metagenomic DNA, together with two analogues, bisucaberin (2) and putrebactin (3). Avaroferrin (1) is a macrocyclic heterodimer of N-hydroxy-N-succinyl cadaverine (4) and N-hydroxy-N-succinyl-putrescine (5), whereas analogues 2 and 3 are homodimers of 4 and 5, respectively. Heterologous expression of two other related genes from culturable marine bacteria resulted in production of compounds 1–3, but in quite different proportions compared with production through expression of the metagenomic DNA.     

角质蛋白酶产生菌及其酶基因研究进展

角质蛋白酶是一种多功能酶、通用裂解酶。为角质蛋白酶产生菌及其活性蛋白酶的挖掘与利用提供理论参考,综述了角质蛋白酶产生菌的分类、角质蛋白酶结构和降解机理、角质蛋白酶产生菌酶活比较、通过宏基因组筛选角质蛋白酶基因、在农业植物病害防治上的应用等国内外研究进展,并进行了展望。     

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 10⁴ clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.     

一个新的内切葡聚糖酶基因umcel5K的克隆、表达及其表达产物的酶学特性

从水牛瘤胃内容物的添加滤纸为碳源的富集培养物中提取未培养微生物的总DNA,以柯斯质粒为载体构建了1个含约8000个克隆的宏基因组文库,对文库进行活性筛选获得1个既表达CMCase活性又表达4-MUCase酶活性的克隆。亚克隆及测序分析发现1个潜在的可编码333个氨基酸的ORF(Open Reading Frame),其蛋白质产物与1个来源于未培养细菌的糖苷水解酶家族5的纤维素酶Cel A的同源性最高,两者的一致性为53%,相似性为68%。将PCR扩增的该基因完整的ORF克隆入表达载体pET30a(+),在大肠杆菌中得到其过量表达产物。经过Ni-NTA纯化后,该表达产物(Umcel5K)具有CMCase活性和4-MUCase酶活性,其最适pH是4.5~5.0,最适温度是50°C。pH耐受性检测表明,该酶在pH4~4.5比较稳定。温度耐受性实验表明该酶不耐高温,在55°C以下比较稳定。经过镍柱纯化的酶液比活为26.15 U/mg。部分金属离子如Fe3+、Cr2+或Cu2+会抑制该酶的酶活,而另外一些金属离子如K+、Li+等对Umcel5K的活性影响不大。     

牦牛瘤胃微生物抗生素抗性基因对3种外源性刺激因子的响应

通过给牦牛投喂硫酸头孢喹肟(CEF)、盐酸二氟沙星(DIF)和黄曲霉毒素B1(AFB1),并进行瘤胃微生物宏基因组测序,旨在揭示这3种外源性刺激因子对抗生素抗性基因(ARGs)种类、抗性类型、抗性机制等的影响,对于深入研究微生物抗性组特征和抗性机制具有重要价值。选取15头牦牛,随机分5组。Cef组和Dif组分别根据说明书推荐剂量按体重计算、灌服CEF 1 mg·kg^-1和DIF 1 mL·kg^-1;E1组和E2组分别按采食量投喂AFB120、60μg·kg^-1;C组为对照组。处理7 d后,采集瘤胃液,提取DNA,Illumina HiSeq测序,对reads counts进行标准化得到TPM值,并进行方差分析。结果显示,对照组共获得132种ARGs,分属30种抗性类型,其中,四环素类tetQ和tetW基因丰度较高;Cef组tetW基因丰度增加(P<0.05),Dif组tetQ丰度增加(P<0.05);Cef组四环素类和头孢菌素类抗性基因丰度增加(P<0.05),Dif组四环素类和氨基香豆素类抗性基因丰度增加(P<0.05),E1组氨基香豆素和青霉烯类抗性基因丰度增加(P<0.05),E2组青霉烯类、头孢菌素类等9类抗性基因丰度均增多(P<0.05);Dif组Erm基因23S核糖体RNA甲基转移酶丰度增加(P<0.05),E2组中ATP结合盒超家族等3种抗性机制相关基因的丰度增加(P<0.05);3种因子均显著增加四环素类ARGs宿主的种类。结论:瘤胃是蕴含丰富ARGs的储藏库,其中,四环素类抗生素抗性基因tetQ和tetW是主要的ARGs。不仅CEF和DIF使部分ARGs的种类、抗性类型以及耐药机制相关酶等的丰度升高,增加瘤胃微生物的耐药性,而且AFB1也具有类似作用,且高剂量AFB1对抗性类型的影响范围较抗生素大。这3种因子还导致携带四环素类ARGs宿主微生物的种类数量增加,从而强化横向转移机制,加快ARGs传播,增强微生物对四环素类的耐药性。     

松材线虫伴生细菌宏基因组fosmid文库构建及特征分析

 松材线虫是松材线虫病的病原,且与其伴生细菌之间存在互作。为研究松材线虫和伴生细菌的互作关系,本研究用Nycodenz介质离心和SDS裂解法富集松材线虫的伴生细菌,以酚/氯仿抽提法提取DNA,构建了松材线虫伴生细菌fosmid宏基因组文库。文库克隆的插入片段大小分布在30~45kb,平均长度为40kb。该文库包含19200个克隆,共计包含7680000kb DNA。文库稳定性检测表明插入的DNA片段能够在fosmid质粒中稳定遗传,没有发现插入片段丢失或重排。随机挑选96个克隆进行末端测序,BLAST分析表明:该文库中松材线虫序列占5.2%,细菌序列占64.6%,无同源序列14.6%。对伴生细菌多样性分析表明:Stenotrophomonas为优势种群,Sphingomonas、Cupriavidus和Pseudomonas为次优势种群。该文库的建立为揭示松材线虫与其伴生细菌的互作关系及伴生细菌的生态功能奠定了基础。     

基于宏基因组学方法分析施肥模式对设施菜田土壤微生物群落的影响

  【目的】  采用宏基因组测序技术,研究施肥对细菌、真菌和古菌群落组成和结构的影响,为设施菜田可持续健康发展提供科学依据。  【方法】  设施蔬菜施肥长期定位试验始于2009年,试验地位于天津市西青区,为春季番茄和秋冬季芹菜轮作体系。在春茬番茄 (第20茬蔬菜) 盛果期,选择定位试验中的6个等氮磷钾投入处理,包括全部使用化肥氮 (4/4CN) 处理和5个有机替代处理 (3/4CN+1/4MN、2/4CN+2/4MN、1/4CN+3/4MN、2/4CN+1/4MN+1/4SN、2/4CN+2/4SN),其中M和S代表猪粪和秸秆。采集0—20 cm土壤样品,测定土壤微生物相关指标。  【结果】  有机替代处理的土壤微生物量碳(MBC) 和微生物量氮(MBN) 含量较4/4CN处理平均分别增加101.5%和134.6%,以2/4CN+1/4MN+1/4SN、2/4CN+ 2/4SN处理的土壤MBC和MBN含量相对较高,较4/4CN平均分别增加158.8%和210.9%。与4/4CN相比,有机替代处理的土壤细菌和真菌丰度平均分别增加8.6%和11.6%,古菌平均降低21.7%;高碳有机替代模式(1/4CN+3/4MN、2/4CN +1/4MN+1/4SN、2/4CN+2/4SN) 土壤细菌和真菌丰度较低碳有机替代模式(3/4CN+1/4MN、2/4CN+2/4MN) 平均分别增加12.3 %和12.0%,古菌丰度平均降低12.9%。与4/4CN处理相比,低量、中量有机替代模式（3/4CN+1/4MN、2/4CN+2/4MN）土壤细菌、真菌Shannon指数没有显著增加,而有机替代模式古菌Shannon指数平均增加了9.0%,高碳有机替代处理古菌Shannon指数平均增加了11.9%。与4/4CN处理相比,有机替代模式提高了土壤细菌中变形菌门相对丰度,降低了放线菌门、绿弯菌门相对丰度;提高了土壤真菌中子囊菌门、壶菌门和球囊菌门相对丰度,降低了担子菌门相对丰度;提高了古菌中的广古菌门和深古菌门相对丰度,降低了奇古菌门相对丰度。主成分分析表明,土壤细菌和真菌群落结构组成对施肥模式的响应弱于古菌。冗余分析表明,有机碳和硝态氮分别解释土壤细菌群落结构变异的22.3%和16.1%,解释真菌群落结构变异的10.4%和8.9%,解释古菌群落结构变异的36.0%和34.7%。  【结论】  同等养分投入量下,以有机肥、秸秆氮替代部分化肥氮可提高土壤细菌和真菌丰度,降低古菌丰度,改变土壤优势菌群的组成,增加微生物多样性。土壤硝态氮和有机碳含量是影响土壤细菌、真菌和古菌群落结构的共同因素,古菌对施肥模式的反应强于细菌和真菌。其中,以2/4化肥+1/4猪粪+1/4秸秆施肥模式可获得最多样的微生物群落结构。     

土壤宏病毒组的研究方法与进展

土壤是病毒遗传多样性的储存库,但由于土壤自身特性及技术手段的限制,基于传统培养方法对土壤病毒的研究及功能认知存在局限性。宏病毒组学技术能直接从土壤环境样品中获取病毒基因组,随后通过高通量测序、拼接组装、ORF预测,最终可对病毒基因进行功能注释,极大地丰富了对土壤病毒功能的认识。本文在阐释土壤病毒DNA提取、测序与病毒判别、功能基因注释等研究方法的基础上,重点探讨了单株噬菌体基因组,及近年来国内外土壤与极端陆地环境中宏病毒组研究进展。并对土壤宏病毒基因组研究的前沿和发展趋势进行了总结,强调了土壤病毒研究的整体化、技术流程规范化以及病毒资源库完善化的重要性。