拮抗菌BS—98分泌抗菌蛋白的条件及其发酵液特性

由本室分离得到一株强烈抑制芦笋茎枯病等植物病原真菌的拮抗菌ＢＳ—９８菌株（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）。用环柱法检测该菌株的抗菌活性表明,该菌株除抑制芦笋茎枯病菌ＰｈｏｍａａｓｐａｒａｇｉＳａｃｃ外,对小麦赤霉病菌（Ｆｕｓａｒｉｕｍｇｒａｍｉｎｅａｒｕｍ）,棉花枯萎病菌（Ｆｕｓａｒｉｕｍｏｘｙｓｐｏｒｕｍｆｓｐ．Ｖａｓｉｎｆｅｃｔｕｍ）、棉花黄萎病菌（Ｖｅｒｔｉｃｉｌｌｕｍａｌｂｏ—ａｔｒｕｍ）、黄瓜灰霉病菌（Ｂｏｔｒｙｔｉ     

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     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

枯草杆菌内切葡聚糖酶基因的克隆及表达

以质粒Ｐｕｃ１８为载体,从枯草杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）ＤＢ１０４基因组中克隆到一个内切葡聚糖酶基因。限制酶切分析表明重组质粒中的插入片段为３.５ｋｂ,其中各含有一个ＥｃｏＲＩ,ＨｉｎｄⅢ和ＰｖｕⅡ位点。该插入片段含有一完整的内切葡聚糖酶基因,其自身启动子能被大肠杆菌转录系统所识别。当加入ｌａｃＺα基因启动子的诱导物ＩＰＴＧ后,内切葡聚糖酶表达量提高２.８倍。加入０.５％葡萄糖能抑制该基因的表达。该基因在大肠杆菌ＤＨ５αＦ′中表达的内切葡聚糖酶分布为：胞外６７.３％,胞间周质３.９％,胞内２８.８％。Ｓｏｕｔｈｅｒｎ杂交证实了该插入片段来自供体菌Ｂ．ＳｕｂｔｉｌｉｓＤＢ１０４。     

蒙自桤木根瘤内生菌的分离培养

从蒙自桤木（Ａｌｎｕｓｎｏｐａｌｅｎｓｉｓ）根瘤中分离得到４株Ｆｒａｎｋｉａ,对其进行了形态学研究,并对Ａｎ１２菌株进行了生长曲线测定,结果表明从蒙自桤木根瘤中分离Ｆｒａｎｋｉａ,除所采用的培养基和方法外,宿主植物所处的生长状态也可能是一个影响分离成败的重要因素;“Ｓ”培养基是一种较为理想的分离培养基;在离体条件下,Ｆｒａｎｋｉａ生长缓慢,约１０ｄ左右达到其生长的稳定期。     

pNK289衍生质粒在牙孢杆菌中的分离稳定性

报道关于一系列pNK289衍生质粒分离稳定性研究结果。这些起源相同的质粒在Bacillus,subtilis AS1.1176中的分离稳定性存在差异,这种差异与质粒的大小和复制方式无关,而与质粒的考贝数有一定的关系。由于不稳定质粒pNK219在B.subtilis BD224宿主中能稳定遗传,所以推测宿主的遗传背景可能影响质粒的分离稳定性,这些研究不仅为进一步寻找与pNK289衍生质粒稳定性相关的基因奠定了基础,而且为在芽孢杆中构建稳定的重组质粒提供了理论依据。     

枯草芽孢杆菌B034拮抗蛋白的分离纯化及特性分析

枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）Ｂ０３４分离自水稻叶面,对水稻白叶枯病菌具有较强的拮抗能力。除去菌体培养液以７０％饱和度硫酸铵沉淀所得的拮抗物粗提液对热稳定,对胰蛋白酶不敏感,对蛋白酶Ｋ、链霉蛋白酶Ｅ部分敏感,对氯仿部分敏感,其作用的活性ｐＨ范围低至４,高至１２以上,比较耐碱性。粗提液经ＰｈｅｎｙｌＳｅｐｈａｒｏｓｅＣＬ４Ｂ柱层析、ＤＥＡＥＳｅｐｈａｃｅｌ柱层析和ＨＰＬＣ的Ｓｕｐｅｒｄｅｘ７５ＨＲ１０／３０柱层析,得到二个拮抗活性峰：Ｐ１和Ｐ２。Ｐ２经ＳＤＳＰＡＧＥ和ＰＡＧＥＩＥＦ电泳显示为单一蛋白带,分子量５０３ｋＤ,等电点６２５。自动Ｅｄｍａｎ降解法从Ｐ２的Ｎ端测出残基序列为ＩｌｅＳｅｒＡｓｎＰｒｏＸＩｌｅＡｓｐＶａｌ     

脉冲电泳核型分析在酿酒酵母菌分类学研究中的应用

根据酵母属（ Ｓａｃｃｈａｒｏｍｙｃｅｓ Ｍｅｙｅｎ ｅｘ Ｒｅｅｓｓ） 分类学研究最新进展,核实并更新了保藏于中国普通微生物菌种保藏中心的该属菌株的种类归属。在形态和生理生化性状,包括对６ 种糖的发酵能力、对１８ 种碳源和３ 种氮源化合物的同化能力、在无维生素培养基中和３７ ℃下的生长情况、对放线菌素酮的抗性等常规分类学研究的基础上,对部分疑难菌株进行了脉冲电泳核型比较分析。酿酒酵母（ Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ） 、贝酵母（ Ｓ．Ｂａｙａｎｕｓ） 和巴氏酵母（ Ｓ．Ｐａｓｔｏｒｉａｎｕｓ） 三者与少孢酵母（ Ｓ．Ｅｘｉｇｕｕｓ） 在电泳核型上具有明显的差异,主要表现在前三者染色体ＤＮＡ 分子的大小范围均为２２５ ～２２００ ｋｂ ,而Ｓ．Ｅｘｉｇｕｕｓ 缺少小于３６５ ｋｂ 的染色体ＤＮＡ 分子。Ｓ．Ｃｅｒｅｖｉｓｉａｅ 的模式和权威菌株具有１２ ～１４ 条染色体ＤＮＡ 带;Ｓ．Ｂａｙａｎｕｓ 和Ｓ．Ｐａｓｔｏｒｉａｎｕｓ 的模式菌株均有１７ 条带,但在带型上存在一定差异。原归于Ｓ．Ｃｅｒｅｖｉｓｉａｅ 的株菌ＡＳ２－１００ 具有１６ 条带,与Ｓ．Ｃｅｒｅｖｉｓｉａｅ 区别明显而与Ｓ．…     

中华鳖白底板病和红底板病细菌的分离鉴定及致病性

从患白底板病和红底板病的病鳖中分离出７株细菌,应用一般细菌分离鉴定的方法和美国生物－梅里埃（Ｂｉｏ Ｍｅｒｉｅｕｘ ＵＳＡ）公司ＶＩＴＥＫ全自动微生物鉴定系统进行鉴定,结果显示有３株嗜水气单胞菌（Ａｅｒｏｍｏｎａｓ ｈｙｄｒｏｐｈｉｌａ）、１株温和气单胞菌（Ａｅｒｏｍｏｎａｓ ｓｏｂｒｉａ<.I>）、１株金黄色葡萄球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓａｕｒｅｕｓ）、１株肺炎克雷伯氏菌（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ）和 １株美人鱼弧菌（Ｖｉｂ     

维生素C混合发酵中产酸菌基因文库的构建

在对氧化葡萄糖酸杆菌ＳＣＢ３２９的纯培养方法进行了探索并获得了一定量的纯培养的ＳＣＢ３２９菌体的前提下,用常规方法抽提得到Ｇｌｕｃｏｎｏｂａｃｔｅｒ　ｏｘｙｄａｎｓ　ＳＣＢ３２９染色体ＤＮＡ。选用质粒ｐＫＳ作为载体,该载体具有氨苄抗性以及ｌａｃＺ基因．用限制酶对染色体进行部分消化,将一定范围内的消化片段回收后与载体连接,连接产物转化Ｅ.ｃｏｌｉＤＨ５ａ感受态细胞,利用蓝白斑特性选出重组子构建ＳＣＢ３２９基因组文库。用低熔点琼脂糖将ＳＣＢ３２９菌体包埋起来,对包埋在凝胶块中的细菌     

Instability of recombinant pUB110 plasmids in Bacillus subtilis: Plasmid-encoded stability function and effects of DNA inserts

Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.     

Kinetic analysis of the effects of plasmid multimerization on segregational instability of CoIE1 type plasmids in Escherichia coli B/r

The effect of plasmid multimerization on segregational instability was investigated using a structured, segregated model of genetically modified Escherichia coli cells. By including the multimerization of plasmids, the model can predict the proportion of each multimer in the total plasmid population. Simulation results suggest that the plasmid copy number is controlled by the total plasmid content (i.e., total number of plasmid origins) in the host cell and that multimerization reduces the total number of independent, monomeric segregation units. However, multimerization is found to have a minor effect on decreasing plasmid segregational stability for multicopy plasmids with average copy number per cell greater than about 25. Also model predictions were used to test whether or not a nonrandom plasmid distribution at cell fission could cause segregational instability. Even in the case of severely biased partitioning, plasmids whose copy number is above 45 per cell do not show significant segregational instability. The results suggest that when the ColE1-type plasmid does not encode and express any large or disruptive foreign proteins, the copy number of 45 per cell may be the threshold at which only growth rate-dependent instability is responsible for overall plasmid instability.     

Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis.

The Bacillus plasmid pLS11 partitions faithfully during cell division. Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis. The cloned par gene conferred upon the vector plasmid a high degree of segregational stability. The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined. The cloned par fragment regulated the partition of several different Bacillus replicons, and it only functioned in cis; it did not contain the replication function nor elevate the plasmid copy number in B. subtilis. The expression of par was orientation specific with respect to the replication origin on the same plasmid. We propose that the pLS11-derived par functions as a single-stranded site that interacts with other components involved in plasmid partition during cell division.     

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

Increased stability of recombinant plasmids by Tn1000 insertion in chemostat cultures of recombinantEscherichia coli GT123

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.     

Plasmid pTB913 derivatives are segregationally stable in Bacillus subtilis at elevated temperatures.

We studied the effects of temperature on the segregational stability of derivatives of the rolling-circle-type plasmid pTB913 in Bacillus subtilis. This 4.5-kb plasmid is a deletion derivative of pTB19, which was originally isolated from a thermophilic Bacillus. pTB913 derivatives carrying large inserts or lacking the minus origin for complementary strand synthesis were segregationally unstable at 37 degrees C. In contrast, at 47 degrees C all pTB913 derivatives tested were stably maintained in B. subtilis. The increased stability at 47 degrees C was attributed, at least partly, to increased copy numbers at this temperature. Although considerable amounts of single-stranded and high-molecular-weight plasmid DNA were formed at 47 degrees C, these products did not reduce plasmid stability at this temperature. The increased stability and increased copy number of pTB913 at elevated temperatures extend the use of this plasmid as a cloning vector in B. subtilis and other bacilli.     

Replication of staphylococcal multiresistance plasmids

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.     

Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.     

Characterization of two Bacillus thuringiensis plasmids whose replication is thermosensitive in B. subtilis

Abstract Two cryptic plasmids of 8.6 and 15 kb, originating from Bacillus thuringiensis , have been cloned in Escherichia coli . The determination of their physical map shows that the 8.6-kb plasmid harbors the transposon Tn 4430 and that the 15-kb plasmid carries Tn 4430 plus one copy of the IS 231 element. The replication regions were identified on the restriction maps and the segregational stability of derived plasmids containing these regions was analyzed in B. subtillis . The results indicate that the stability of these plasmids is negatively correlated to the temperature. After 30 generations, without selective pressure at 51°C, the two types of plasmids are lost.