鸡源暹罗芽孢杆菌CML548的益生特性及全基因组分析

【背景】芽孢杆菌是用于动物微生态制剂的重要菌株之一。暹罗芽孢杆菌因具有较强的抑菌活性,近年来受到广泛关注。【目的】对实验室前期分离的鸡源暹罗芽孢杆菌CML548的益生特性进行评价,通过全基因组测序及生物信息学分析,探究其抑菌及潜在的益生机制。【方法】通过牛津杯法、稀释涂布平板法分别对菌株CML548的抑菌和产酶特性、酸和胆盐的耐受性进行研究。使用IlluminaHiSeq2500测序平台进行全基因组测序,并通过多种生物信息学工具和数据库对基因组进行注释和分析。【结果】体外实验表明该菌株具有优良的益生性状：能够有效抑制致病性大肠杆菌、鼠伤寒沙门氏菌、产气荚膜梭菌的生长;能够同时产生蛋白酶、淀粉酶和纤维素酶;具有较高的酸和胆盐耐受性。全基因组测序分析表明菌株CML548的基因组大小为4061741bp,GC含量为46.07%,预测到3 961个编码基因。分别有1 693、2 704、3 413、186、67、1、5个基因被GO、KEGG、COG、CAZy、DBAASP、CARD、VFDB数据库注释;通过antiSMASH预测到16个与次级代谢产物合成相关的基因簇。此外,还发现其含有抗菌活性...     

fliY基因失活减弱沙福芽孢杆菌ST7菌株的锰氧化能力

【背景】沙福芽孢杆菌ST7菌株具有较强的锰氧化能力,但其分子机制不清楚。【目的】着重研究鞭毛马达开关蛋白基因(fliY)对沙福芽孢杆菌锰氧化能力的影响。【方法】根据同源重组原理,以沙福芽孢杆菌ST7菌株为起始菌株,构建fliY基因敲除的突变株ΔfliY,测定菌落迁徙、细菌生物膜和锰氧化率等,研究fliY基因突变后菌株的运动能力、生物膜生成和锰氧化能力是否发生变化。【结果】经克隆测序,证实突变株ΔfliY中fliY基因的后半段被卡纳霉素抗性基因取代,fliY基因失活;与野生型菌株ST7相比,突变株ΔfliY在全营养的LB培养基中生长变化不大,但在含锰的PYCM培养基中,突变株的生长速度减慢、菌落较小、生物膜生成量显著下降,运动性和锰氧化能力分别下降65%和20%。【结论】fliY基因不仅影响菌株的生长和运动,而且参与细菌的趋化和锰氧化等生物学过程。     

玉米赤霉烯酮脱毒菌PA26-7的分离鉴定及其应用效果评价

【背景】玉米赤霉烯酮(zearalenone,ZEN)是广泛污染粮谷类作物的一种雌激素类真菌毒素,不仅给农业经济带来巨大损失,还能通过食物链对人和动物健康造成危害。【目的】从微生态制剂中筛选获得能够高效降解玉米赤霉烯酮的菌株,优化其脱毒条件,测定其在饲料中的实际脱毒效果及对饲料中植酸、维生素含量变化的影响。【方法】从微生态制剂中分离出玉米赤霉烯酮降解菌,通过细胞计数试剂盒-8 (cell counting kit-8, CCK-8)测定菌株降解玉米赤霉烯酮产物的细胞毒性和雌激素活性,通过高效液相色谱法测定分离株在培养基和饲料中的解毒效果,以及分离株在霉变的豆粕、麸皮和成品饲料中固态发酵前后维生素的含量变化,通过三氯化铁比色法测定饲料脱毒前后植酸的含量变化。【结果】从微生态制剂中筛选出一株通过分泌胞外酶高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌(Bacillus velezensis) PA26-7,该菌株在培养基起始pH 4.0-8.0、培养温度25-60℃条件下均可降解玉米赤霉烯酮,产物的细胞毒性和雌激素活性均较ZEN弱。PA26-7经固态发酵72h后,饲料原料(豆粕和麸皮)及霉变的成品鸡...     

芽孢杆菌FJAT-55034的鉴定、生长特性及对梨轮纹病菌的抑菌活性

【背景】由葡萄座腔菌(Botryosphaeria dothidea)引起的梨轮纹病是梨栽培生产中的主要病害之一,造成巨大的经济损失。【目的】对梨轮纹病菌具有较高拮抗活性的菌株FJAT-55034进行鉴定、生长特性及抑菌活性评价,为梨轮纹病的生物防治提供菌种资源。【方法】采用形态学观察、生理生化测定和16S rRNA基因序列分析进行拮抗菌株FJAT-55034的鉴定;通过生长曲线、培养温度和pH测定,研究该菌株的生长特性。采用抑菌圈法测定该菌株的抑菌谱;采用共培养、显微镜观察和果实回接,测定菌株FJAT-55034对梨轮纹病菌生长的抑制作用。【结果】拮抗菌株FJAT-55034鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis)。该菌株在20-50℃(最适温度30℃)和pH 5.0-9.0 (最适pH值7.0)均能够生长,NaCl添加量为0-5%时,该菌株均能够较好地生长。菌株FJAT-55034对6种果树不同病原真菌均具有不同程度的抑制作用,抑菌圈直径范围为19.8-29.1 mm;与梨轮纹病菌共培养后,菌株FJAT-55034对菌丝生长的抑制率为77.2%。菌株FJAT-5...     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

利用原生质体融合技术选育防治植物病虫害的基因重组菌株

本试验选用抗菌蛋白产生菌枯草芽孢杆菌(B.subtilis) TG26和晶体蛋白产生菌苏云金芽孢杆菌(B.thuringiesis subsp,pacifeus) AS1.904的营养缺陷型衍生株,在聚乙二醇的诱导下进行原生质体种间融合,获得了表现双亲遗传性状的种间融合菌株。融合率为7.52×10~6,融合子经传代10次,稳定率为19.5%。融合菌株的菌落和细胞形态与亲本株明显不同。通过SDS-聚丙烯酰胺凝胶电泳检测,融合菌株表达了亲本的抗菌蛋白和毒素蛋白。抑菌杀虫试验表明,融合重组菌株具有抑制多种植物病原菌和毒杀鳞翅目幼虫的能力。     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

一种具有纤溶活性的枯草杆菌蛋白激酶的研究──Ⅰ高产酶菌株的筛选与鉴定

本文主要阐述了一种具有纤溶活性的枯草杆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）蛋白激酶产生菌株的筛选与鉴定的研究结果。作者从初筛的１２株Ｂａｃｉｌｌｕｓｓｕｂｌｉｌｉｓ菌中,通过对固体发酵和液体发酵所产生的枯草杆菌蛋白激酶,用琼脂糖－纤维蛋白平板法测其活性,经比较不同菌株的活性,筛选出两株高产酶菌株：Ｂ．ｓｕｂｔｉｌｉｓＨＷ—１２和Ｂ．ｓｕｂｔｉｌｉｓＨＷ—３。同时对菌体和菌落形态特点、生理生化反应进行了鉴定,认为Ｂ．ＳｕｂｔｉｌｉｓＨＷ－１２菌株可用来做为发酵生产该酶的菌种。     

Cloning and Sequencing of a 27.8-kb Nucleotide Sequence of the 79{degrees}-81{degrees}. Region of the Bacillus subtilis Genome Containing the sspE Locus

The nucleotide sequence of a 27830-bp DNA segment in the 79°–81°.region of the Bacillus subtilis genome has been determined.This region contains 29 complete ORFs including the sspE gene,which encodes a small acid-soluble spore protein gamma and locateson the one side terminal of our assigned region. A homologysearch for the products deduced from the 29 ORFs revealed thatnine of them exhibit significant similarity to known proteins,e.g. proteins involved in an iron uptake system, a multidrugresistance protein, a chloramphenicol resistance protein, epoxidehydrolase, adenine glycosylase, and a glucose-1-dehydrogenasehomolog.     

地衣芽孢杆菌β－甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和ＳｅｐｈａｄｅｘＧ－１００离子交换柱层析以及制备ＰＡＧＥ筹步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点ＰＩ为５．０。酶反应的最适ｐＨ为９．０,最后温度为６０℃,稳定ｐＨ为６．０—９．０,稳定温度为４０℃。金属离子中Ｍｇ￣（２＋）、Ｃａ￣（２＋）、Ｆｅ￣（２＋）、Ｎｉ￣（２＋）对该酶有一定的激活作用;而Ｓｎ￣（２＋）、Ｚｎ￣（２＋）、Ａｌ￣（３＋）、Ａｇ￣＋和Ｈｇ￣（２＋）对该酶有强烈的抑制作用。ＮＫ－２７菌株的β－甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Ｋｍ值分别为７．１４和５．５６ｍｇ·ｍｌ￣（－１）;Ｖ＿（ｍａｘ）分别为２００．５３和１５７．４５μｍｏｌ·ｍｇ￣（－１）·ｍｉｎ￣（－１）。