Genetic mobility of plasmids of Streptomyces erythraeus

Grossing of S. erythraeus 4 with S. erythraeus 1 resulted in transfer of genetic elements from strain 4 to strain 1 as evidence by the 20 and 18 kb fragments in the experiments on DNA-DNA hybridization. The presence of the genetic elements in strain 1 was the cause of plasmid pSE 21 mobility. In strain 6, a derivative of S. erythraeus 1 plasmid pSE 21 was accompanied by other extrachromosomal DNAs characterized by high instability. During storage of the strain at a temperature of 4 degrees C for more than 1 or 2 months the number of the plasmid pSE 21 copies decreased. When the strain was stored for longer periods (6 months or more) the plasmid DNA was not detectable even with the DNA-DNA hybridization procedure. The results of hybridization of a fraction of the extrachromosomal DNA of S. erythraeus 6, the Bam HIB fragment of plasmid pSE 21 with the total DNA of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus and hybridization of DNA of plasmid pSE 21 with the total DNA of S. erythraeus 6 and 1 showed that (1) strains 1, 5 and BTCC 2 had the same hybridization patterns, (2) the other extrachromosomal DNAs present in the fraction were homologous with the Bam HIA fragment of plasmid pSE 21, (3) chromosomes of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus also contained DNA homologous to the plasmid Bam HIA fragment. It was suggested that plasmid pSE 21 could be used as a basis for constructing the integrative vector for S. erythraeus.     

Development of integrative vectors for Actinomycetes--producers of antibiotics]

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.     

短小芽孢杆菌HZbp脂肪酶基因的克隆表达

以短小芽孢杆菌HZbp总DNA为模板以PCR的方式获得512 bp的脂肪酶基因,并在该基因的两端引入了EcoR1和Sal1的酶切位点,将该基因与大肠杆菌表达质粒pSE380连接,获得重组质粒pSE380-BPL。重组质粒转入大肠杆菌表达细胞株BL21,获得工程菌株BL21-BPL。序列分析显示所克隆的基因具有脂肪酶的保守G-X-S-X-G序列,SDS-PAGE电泳显示该脂脂肪酶的分子质量约为20 kDa。在LB培养基中,IPTG诱导浓度为1.0 mmol/L,33℃诱导培养10 h后,发酵液酶活达到8 U/mL。     

鼠李糖乳杆菌D-(+)-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达

利用PCR的方法从鼠李糖乳杆菌基因组DNA中扩增到D-(+)-乳酸脱氢酶基因(ldhD),并连接到载体pSE380上,构建表达质粒pSE-ldhD,将重组质粒pSE-ldhD转化大肠杆菌BL21(DE3),重组菌株经IPTG诱导表达,SDS-PAGE电泳分析表明ldhD在大肠杆菌中实现了表达,表达产物的分子量约为37kD。同时采用紫外分光光度法测定D-乳酸脱氢酶的酶活,测得重组菌株的D-乳酸脱氢酶活力为5.4U/mL,最适反应温度为35℃,最适pH为5.6。     

Identification and characteristics of plasmids of the strains of erythromycin-producing Streptomyces erythreus

Presence of plasmid DNA was investigated in laboratory strains 2 and 4 (NRRL 2338) of S. erythreus, as well as in strains 1 and 3 of S. erythreus subjected to improvement with respect to erythromycin production. Families of plasmids close by their molecular weights were identified in S. erythreus strains 3 and 4 (NRRL 2338). A plasmid DNA fraction of S. erythreus strain 3 was studied with electron microscopy. It enabled to identify 5 plasmids: pSE11, pSE12, pSE13, pSE14 and pSE15 with length of 5.3, 12.4, 16.3, 29.6 and 86.9 kb respectively. Using of various procedures for isolation of extrachromosomal DNA did not provide its detection in S. erythreus strains 1 and 2. At least a part of the plasmids detected in S. erythreus strains 3 and 4 (NRRL 2338) was conjugative. 32R-Labeled plasmid DNA of S. erythreus strain 3 was subjected to hydridization according to Sauthern with total DNA of the 4 strains treated with restrictases BamHI, PstI and BgIII. The studies showed that the genome of S. erythreus strain 2 was not homologous with the probe while S. erythreus strain 1 contained one of the plasmids or its part in chromosome-integrated state. In strains 3 and 4 (NRRL 2338) of S. erythreus certain plasmid DNAs were present in both autonomous and chromosome-inserted states. 32P-Labeled gene of erythromycin resistance (ermE) was subjected to hybridization according to Southern with total DNA of the 4 strains and with DNA plasmid fraction of S. erythreus strain 3. The signal was positive only in hydridization of the probe with total DNA of S. erythreus strains 1, 3, and 4 (NRRL 2338).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of clusters of erythromycin biosynthesis genes of Streptomyces erythraeus (Saccharopolyspora erythraea)

The erythromycin resistance gene (ermE) and part of erythromycin biosynthesis genes located in the same cluster with the ermE gene were cloned from S. erythraeus 3 subjected to improvement with respect to erythromycin production. For isolating the erythromycin biosynthesis genes, the plasmid vector pUC18 and the phage vector lambda EMBL3 were used. The ermE gene DNA was used as a labeled probe for analysis of the recombinant plasmids and phages. The recombinant phages lambda ermE1 and ermE4 containing fragments of the chromosomal DNA collinear to the genome DNA of S. erythraeus 3 were analyzed. The size of the cloned fragment of the chromosomal DNA of S. erythraeus 3 was about 20 kb. Subcloning with the vector pUS18 resulted in isolation of plasmids pSU235-pSU244 containing BamHI fragments of chromosomal DNA from S. erythraeus 3. The restriction map of the chromosomal region of S. erythraeus 3 containing the ermE gene was constructed. The cloned genes of erythromycin biosynthesis are useful in the study of their structure and functions, construction of integrative vectors, improvement of cultures producing macrolide antibiotics and isolation of genes responsible for biosynthesis of other polyketide antibiotics.     

Cloning,expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.     

Transformation of Streptococcus sanguis Challis with a plasmid of Streptococcus pneumoniae

Abstract The present work is concerned with plasmid transformation of Streptococcus sanguis strain Challis with derivatives of pDP1/pSMB1, the only plasmid found to occur naturally in Streptococcus pneumoniae . Two recombinant plasmids derived from the cryptic pSMB1 were used: pDP27 (4.5 kb) conferring resistance to chloramphenicol (Cm), and pDP28 (7.8 kb), a shuttle plasmid, conferring resistance to Cm in Escherichia coli , and resistance to erythromycin (Em) in pneumococcus. It could be shown that pSMB1 can replicate in S. sanguis ; in fact, Challis strain V288 was transformed to Cm-resistance and to Em-resistance by pDP27 and pDP28 respectively.
Shuttle plasmid pDP28 can transform S. sanguis both when isolated from pneumococcus and from E. coli , albeit with a different efficiency. The low frequency of transformation observed when pDP28 was isolated from E. coli DH1 ( recA ) was shown to be due to lack of multimeric forms of the plasmid in the DNA preparations obtained from this strain. When pDP28 was isolated from E. coli C600 (RecA⁺), multimeric forms were present, and transformations of S. sanguis was more efficiency Using pDP28 as vector in cloning experiments, where S. sanguis was the host of the recombinant DNA molecules, treatment of the vector with alkaline phosphatase inhibited the recovery of recombinant clones.     

Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.     

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.     

Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.     

Study of the structure of amplifying sequence of Streptomyces antibioticus

A low productive laboratory strain of S. antibioticus and a strain with an increased productivity of oleandomycin derived from it were studied comparatively with using restriction analysis and blotting hybridization. Amplification, site specific integration and segregation of the DNA sequence 32.0 kb in size were detected in the strains. The chromosomes of the laboratory strain contained one copy of the amplifying sequence AUD. After uniting of the end sequences AUD appeared to be capable of segregating from the chromosomes and its one copy per five genomes was present in the form of an extrachromosomal genetic element eSA1. The genome of the strain with increased productivity of oleandomycin contained in its chromosomes sequence ADS-Sa1 amplified to 150 copies and the eSA1 extrachromosomal genetic element in the form of mono-, di- and trimeric structures in the quantity of approximately one copy per genome. The BamHIB fragment of the eSA1 DNA 4 kb in size was identified. The fragment was able to participate in segregation or integration of eSA1 from or into the chromosomes since its subfragments were flanking AUD and ADS-SA1 in the chromosomes. The BamHIB fragment was hybridizing with a number of fragments of the chromosomal DNA of S. antibioticus, S. erythraeus. S. lividans and other strains of streptomycetes. It probably contained an IS-like element or a dispersed genetic element of another class. The DNA sequence of the eSA1 genetic element contained regions homologous to the sequence of the Erm E gene in S. erythraeus NRRL 2338.     

Site-specific integration in Saccharopolyspora erythraea and multisite integration in Streptomyces lividans of actinomycete plasmid pSE101.

An 11.3-kilobase-pair plasmid, designated pSE101, exists in Saccharopolyspora erythraea NRRL 2338 as an integrated sequence (pSE101int) at a unique chromosomal location and in the free form in less than an average of 1 copy per 10 chromosomes. The plasmid sequence is missing from S. erythraea NRRL 2359. Restriction maps of the free and integrated forms of pSE101 showed point-to-point correspondence. Plasmid pECT2 was constructed by ligation of pSE101, pBR322, and the gene for thiostrepton resistance (tsr). When introduced by polyethylene glycol-mediated transformation into protoplasts of S. erythraea NRRL 2359, all thiostrepton-resistant regenerants examined were found to carry a single copy of pECT2 in the integrated state at a single chromosomal site. The chromosomal site of pECT2 integration in strain NRRL 2359 (attB) corresponded to the chromosomal location of pSE101int in strain NRRL 2338. The plasmid crossover site (attP) was mapped to the plasmid site that corresponded to the site of interruption of the plasmid sequence in the host carrying pSE101int, indicating that site-specific integrative recombination had occurred. An additional 2.8-kilobase-pair chromosomal sequence homologous to a segment of pSE101 was also observed in strains NRRL 2338 and NRRL 2359. After introduction of pECT2 into Streptomyces lividans, approximately half of the transformants examined were found to carry the plasmid as a stable, autonomously replicating element. The other half carried a single copy of pECT2 as an integrated sequence, but the location of pECT2int in Streptomyces lividans varied from one transformant to another. In each case, integrative crossover used the attP site. A model is proposed to account for the determination of the particular state of pSE101 in Streptomyces lividans.     

The frequency of expression of pyelonephritis-associated pili is under regulatory control

The Escherichia coli urinary tract isolate C1212 contains two pyelonephritis-associated pili (pap) DNA sequences designated here as pap-17 and pap-21. Each of these pap sequences encodes antigenically-distinct pilin monomers, pilin-17 and pilin-21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin-21. Only a small fraction (5%) of strain C1212 cells expressed pilin-17. Most of the latter population simultaneously expressed pilin-21, but a low percentage of cells expressed pili composed of pilin-17 alone. In contrast, almost every E. coli K-12 cell containing multicopy pap-17 expressed pilin-17 at the cell surface. These results indicated that the regulation of pilin-17 expression observed for strain C1212 was lost when pap-17 was in the multicopy state. Transfer of pap-17 to a single copy vector resulted in a pilin-17 expression frequency lower than strain C1212 (1%). Using E. coli K-12 containing single copy pap-17, we found that the frequency of pilin-17 expression increased about 15-fold when pap-21 was present in multiple copies in trans. Subcloning of pap-21 showed that a 2.2 kilobase-pair DNA sequence adjacent to, but not including, the pilin-21 structural gene was sufficient for activation of pilin-17 expression.     

弗氏柠檬酸菌甘油脱水酶基因在大肠杆菌中的克隆和表达

以弗氏柠檬酸菌（Citrobacter freundii）基因组DNA为模板,通过PCR得到甘油脱水酶（glycerol dehydratase）基因dhaB、dhaC、dhaE,克隆到表达载体pSE380上,得到重组质粒pSn-dhaBCE。将此重组质粒转化到E．coli JM109中,重组菌株SDS-PAGE结果显示有明显的61kD、22kD、16kD三条特异性蛋白条带出现。重组菌株经诱导表达,酶活力为11．59U／mL。     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S. suis 2) 05ZYH33全基因序列进行生物信息学分析, 并与相关家族蛋白进行同源性比较, 设计合成引物, PCR法扩增出约1.3 kb的烯醇化酶编码基因 (enolase, eno), 将其克隆入pMD-18T载体中, 进一步亚克隆入表达载体pET32a。将重组表达质粒pET32a::eno转化E. coli BL21 (DE3), 经IPTG诱导表达后, SDS-PAGE初步检测到分子量约为75kD的蛋白带。通过His-Tag亲和层析纯化, 获得融合蛋白His-ENO。Western-blot表明该表达产物具有免疫原性。基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S. suis 2 05ZYH33细菌的表面。这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用。     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S.suis 2)05ZYH33全基因序列进行生物信息学分析,并与相关家族蛋白进行同源性比较,设计合成引物,PCR法扩增出约1.3 kb的烯醇化酶编码基因(enolase,eno),将其克隆入pMD-18T载体中,进一步亚克隆入表达载体pET32a.将重组表达质粒pET32a::eno转化E.coli BL21(DE3),经IPTG诱导表达后,SDS-PAGE初步检测到分子量约为75kD的蛋白带.通过His-Tag亲和层析纯化,获得融合蛋白His-ENO.Western-blot表明该表达产物具有免疫原性.基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S.suis 2 05ZYH33细菌的表面.这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用.     

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

D-乳酸高产菌菊糖芽胞乳杆菌Y2-8磷酸果糖激酶基因在大肠杆菌中的克隆和表达

以D-乳酸高产菌菊糖芽胞乳杆菌Y2-8基因组DNA为模板,通过PCR扩增得到960 bp的磷酸果糖激酶基因(pfk)。氨基酸序列比对分析表明,该磷酸果糖激酶(PFK)与其他乳酸菌PFK具有保守的底物结合位点,但是其变构效应物结合位点存在差异。将pfk基因克隆到表达载体pSE380上,获得重组菌E-pSE-pfk。进一步通过诱导条件的优化,重组菌的PFK比酶活达到4.89 U/mg,是优化前的4.79倍。采用低温诱导策略有助于实现菊糖芽胞乳杆菌pfk基因在大肠杆菌中可溶性表达。