Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp

Green fluorescent protein (GFP) is the most potential useful marker for the in situ monitoring of biofilm microbes. The objective of this study was to construct and compare the efficacy of transposon vectors containing native and foreign promoters in monitoring the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp. The promoter of nitrite reductase (Pnir) was cloned from LYS-86 and utilized to construct the transposon vector pUT/mini-Tn5-km2-Pnir-gfp. Another transposon vector, pUT/mini-Tn5-km2-Plac-gfp, containing the lactose promoter Plac was also constructed. These two transposon vectors and pUT-luxAB-gfp containing the promoter PpsbA were individually inserted into the chromosome of P. stutzeri LYS-86 by conjugation. Three GFP-tagged recombinant strains, LYS-Plac-gfp, LYS-Pnir-gfp, and LYS-PpsbA-gfp, were selected from the conjugants. Green fluorescence was observed only in LYS-Pnir-gfp, suggesting that the native promoter Pnir may be more suitable for GFP expression in P. stutzeri than the foreign promoters Plac and PpsbA. Indeed, LYS-Pnir-gfp maintained stable GFP fluorescence over 16 subcultures without significant changes in the denitrifying capacity.     

Chromosomal complementation using tn7 transposon vectors in enterobacteriaceae

Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn7 transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn7 systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn7 and transposase vectors to provide a more versatile system. Transposition of Tn7 at the chromosomal attTn7 site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn7 vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.     

遗传稳定型六六六、多菌灵降解基因工程菌构建

通过PCR的方法从六六六降解菌Sphingomonas sp.BHC-A扩增出完整的脱氯化氢酶基因linA.将其克隆到含有mini-Tn5的自杀性质粒pUT4K上,构建成质粒pUT/mini-Tn5-linA.通过三亲杂交,在辅助质粒RK600的帮助下,将pUT/mini-Tn5-linA转移到一株高效降解多菌灵菌株Rhodococcus sp.DJL-6中.利用mini-Tn5的转座作用将linA基因整合到DJL-6的染色体DNA上,得到工程菌株DJL-6A.该工程菌具有同时降解多菌灵和六六六的功能,且对于初始浓度为0.05 μg/mL和5 μg/mL的六六六的降解活性与亲本菌株BHC-A相当.在不加任何选择压力的条件下工程菌株进行连续传代,结果证明linA基因可以持续稳定的存在于宿主的染色体DNA上.     

Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

Pseudomonas putida strain BNF1 was isolated to degrade aromatic hydrocarbons efficiently and use phenol as a main carbon and energy source to support its growth. Catechol 2,3-dioxygenase was found to be the responsible key enzyme for the biodegradation of aromatic hydrocarbons. Catechol 2,3-dioxygenase gene was cloned from plasmid DNA of P. putida strain BNF1. The nucleotide base sequence of a 924 bp segment encoding the catechol 2,3-dioxygenase (C23O) was determined. This segment showed an open reading frame, which encoded a polypeptide of 307 amino acids. C23O gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Km^r) to get a novel transposon vector pUT/mini-Tn5-C23O. With the helper plasmid PRK2013, the transposon vector pUT/mini-Tn5-C23O was introduced into one alkanes degrading strain Acinetobacter sp. BS3 by triparental conjugation, and then the C23O gene was integrated into the chromosome of Acinetobacter sp. BS3. And the recombinant BS3-C23O, which could express catechol 2,3-dioxygenase protein, was obtained. The recombinant BS3-C23O was able to degrade various aromatic hydrocarbons and n-alkanes. Broad substrate specificity, high enzyme activity, and the favorable stability suggest that the BS3-C23O was a potential candidate used for the biodegradation of crude oil.     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

呋喃丹降解菌CDS-1的双标记菌株的构建

用Sau3AI消化呋喃丹降解菌Sphingomonassp.CDS-1的基因组DNA,将所得DNA片段与BamHⅠ酶切的启动子探针载体pRobe-GFP酶连后转化E.coliDH5α感受态细胞,在选择性平板上培养,从大约1×104个菌落中筛选到50个含启动子片段的阳性克隆。挑选其中一个发光强度最强的阳性克隆F7,将它的重组质粒pF7用EcoRⅠ和HindⅢ双酶切后得到包含Sphingomonassp.CDS-1启动子和gfp基因的DNA片段,将该片段克隆到广宿主载体pPZP201上,得到pPZP201-gfp质粒。将pPZP201-gfp通过三亲接合转移至Sphingomonassp.CDS-1中得到GFP标记菌株CDS-gfp,经荧光显微镜观察,gfp基因在CDS-gfp中表达量很高。对标记菌株进行连续传代10次(48h/次),发现pPZP201-gfp依然存在,而且发光明显。通过NotⅠ酶切位点把linA基因连接到pUT/mini-Tn5上构建新的转座子载体pUT/mini-Tn5-linA。以pRK600为辅助质粒将pUT/mini-Tn5-linA引入到CDS-1中,linA基因通过转座作用,插入到CDS-gfp的染色体中,得到双标记菌株CDS-GFP-LinA。该菌株是一株能同时降解γ-六六六和呋喃丹的基因工程菌,本研究的结果为研究Sphingomonassp.CDS-1的生态学行为奠定了基础。     

mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkholderia mallei ATCC 23344

The mini-Tn7 vectors are universally applicable in gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.     

Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads.

Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.     

Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells

The Gateway technology cloning system and transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools allows rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway technology-compatible transposon plasmid that combines the advantages of Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase, and provides highly efficient and precise transgene integrations into the host genome. A Gateway-compatible transposon plasmid was generated in which the potential target gene can be fused with a yellow fluorescent protein (YFP) tag at the N-terminal. The vector utilizes the CAGGS promoter to control fusion protein expression. The transposon expression vector encoding the YFP-interferon-β protein (IFNB1) fusion protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). ASCs and HEK293 cells stably expressed and secreted the human IFNB1 for up to 4 weeks after transfection. The generated Gateway-compatible transposon plasmid can be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications.     

mini-Tn7 insertion in bacteria with secondary, non-glmS-linked attTn7 sites: example Proteus mirabilis HI4320

We previously constructed a series of mini-Tn7 chromosome integration vectors that, when provided only with the site-specific transposition machinery, generally transpose to a naturally evolved, neutral attTn7 site that is located 25-bp downstream of the glmS gene. Here we provide a protocol for application of the mini-Tn7 system in Proteus mirabilis as an example of a bacterium with a secondary attTn7 site that is not linked to glmS but, in this case, located in the carAB operon. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. mirabilis by transformation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. When transposon-containing cells are selected on rich medium, insertions occur at both attTn7 sites with equal efficiency and frequency. Because carA mutants are arginine and pyrimidine auxotrophs, single-site insertions at the glmS attTn7 sites can be obtained by selection on minimal medium. From start to verification of the insertion events, the whole procedure takes 5 d. This chromosome integration system in P. mirabilis provides an important tool for animal and biofilm studies based on this bacterium. Vectors are available for gene complementation and expression, gene fusion analyses and tagging with a green fluorescent protein (GFP)-encoding reporter gene.     

利用转座子将parDE与pCPP430体内重组提高生防工程菌的遗传稳定性

带有梨火疫欧氏杆菌 (Erwiniaamylovora)完整的hrp基因簇的重组粘粒pCPP43 0转化到植物的附生菌成团泛菌 (Pantoeaagglomerans) 3 0 8R中后可以使成团泛菌获得在烟草等植物上引发过敏反应和诱导植物抗病性的能力。pCPP43 0携带着约 40kb的梨火疫欧氏杆菌的染色体DNA ,在成团泛菌 3 0 8R中不能稳定遗传。本文首先把广宿主范围质粒RK2的控制质粒稳定性的parDE片段插入到转座载体pUT mini Tn5Km的唯一克隆位点NotI上 ,然后利用Tn5的转座特性 ,将parDE体内重组到pCPP43 0上 ,经过在无抗生素选择压下反复传代和过敏反应活性测定 ,筛选到了遗传稳定性显著提高的重组生防工程菌。     

Bi-functional gfp- and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies

The gfp gene, encoding the green fluorescent protein, was combined with the gusA gene, coding for the beta-glucuronidase enzyme, in mini-Tn5 transposon derivatives for use in Gram-negative bacteria. These mini-Tn5 elements allow simultaneously monitoring of gene expression and localization of the marked bacteria. Introduction of the resultant mini-Tn5 transposons into Rhizobium etli, Azospirillum brasilense and Pseudomonas stutzeri allowed us to visualise the interaction of these bacteria with their host plant. The dual-marker mini-Tn5 transposons constitute a powerful new tool for studying gene expression and ecology of bacteria in the environment and during the interaction with plants.     

mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa

Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.     

Random transposon vectors pUTTns for the markerless integration of exogenous genes into gram-negative eubacteria chromosomes

A set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS50_R transposase tnp gene in cis, but they are external to the mobile element. The vectors' conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker (sacB), a 430-bp repeat of the sacB gene 3′ end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene (mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene (oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

美洲棉铃虫细胞中dsRNA介导的egfp基因沉默分析

为了分析在美洲棉铃虫细胞( HzAM1)内RNAi的效果,将egfp基因克隆到含有双向T7启动子/终止子的质粒载体中,在体外合成全长的增强型绿色荧光蛋白(egfp)基因dsRNA,将dsRNA和含有能在昆虫细胞内表达eGFP的质粒一起转染HzAM1细胞,分析dsRNA对eGFP表达的抑制作用.结果显示,由egfp基因转录的长dsRNA能有效抑制HzAM1细胞内eGFP的表达,而且该抑制作用表现为剂量依赖效应.但是抑制作用并不彻底,在高剂量的dsRNA处理下,仍有部分细胞内能观察到eGFP的表达.     

根癌农杆菌介导的增强型绿色荧光蛋白基因在淡紫拟青霉中的转化

为了实现增强型绿色荧光蛋白基因 (egfp) 在生防真菌淡紫拟青霉9410菌株中的转化,借助中间质粒pcDNA3.1(-) 构建nptⅡ-egfp融合基因的表达载体pUPNGT,然后采用根癌农杆菌介导的转化法将egfp基因转化到淡紫拟青霉9410菌株中。PCR检测和Southern blotting分析结果表明,egfp基因以单拷贝形式整合到淡紫拟青霉9410的基因组中。荧光显微镜观察结果显示,转化子在488 nm下能产生绿色荧光。这些结果说明egfp基因已成功转化至淡紫拟青霉9410菌株并获得表达。这些工作可为淡紫拟青霉在不同条件下的防效评价、环境安全评价等提供新的途径和方法。