Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases

In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.     

产SEB金黄色葡萄球菌α-溶血毒素基因缺失菌株的构建

构建产肠毒素B(Staphylococcal enterotoxin B ,SEB)的金黄色葡萄球菌α-溶血毒素（α-hemolysin, α-HL)缺失菌株。首先构建用于α-HL基因敲除的同源重组质粒pMHL-α,经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代,最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证明最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为野生型金黄色葡萄球菌的体内遗传操作及构建产超抗原药物金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。     

DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.     

Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.     

An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae.

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL(+) marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.     

Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (DeltapyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (DeltapyrF DeltatrpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (DeltapyrF DeltatrpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (DeltapyrF DeltatrpE DeltahisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.     

Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori

In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.     

Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A

We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases. These strains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed in vitro by using the cloned gene. The methods used to construct the mutations involved the use of a plasmid vector which allowed the selection of chromosomal integrates and their subsequent excision by homologous recombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insert with a selectable marker (a cat gene conferring chloramphenicol resistance). The strains produced no subtilisin, no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as 0.5% of that of the standard strain from which they were derived. The strains proved to be superior host strains for the production of staphylococcal protein A, accumulating higher levels of intact protein than do previously available B. subtilis strains.     

Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A.

We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases. These strains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed in vitro by using the cloned gene. The methods used to construct the mutations involved the use of a plasmid vector which allowed the selection of chromosomal integrates and their subsequent excision by homologous recombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insert with a selectable marker (a cat gene conferring chloramphenicol resistance). The strains produced no subtilisin, no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as 0.5% of that of the standard strain from which they were derived. The strains proved to be superior host strains for the production of staphylococcal protein A, accumulating higher levels of intact protein than do previously available B. subtilis strains.     

Improved oligonucleotide site-directed mutagenesis using M13 vectors.

An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.     

An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL⁺ marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.     

Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70⁺ strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.     

Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation

Haemophilus parasuis, belonging to the family Pasteurellaceae, is the causative agent of Glässer’s disease leading to serious economic losses. In this study, a successive markerless mutation system for H. parasuis using two sequential steps of natural transformation was developed. By the first homologous recombination, the target genes were replaced by a cassette carrying kanamycin resistance gene and sacB (which confers sensitivity to sucrose) gene using kanamycin selection, followed by the second reconstruction to remove the selection cassette, with application of sucrose to further screen unmarked mutants. To improve DNA transformation frequency, several parameters have been analyzed further in this work. With this method, two unmarked deletions in one strain have been generated successfully. It is demonstrated that this system can be employed to construct multi-gene scarless deletions, which is of great help for developing live attenuated vaccines for H. parasuis.     

Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.     

利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统

热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力,但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C. tropicalis ATCC 20336为出发菌株,通过化学诱变筛选获得了尿嘧啶缺陷型突变株C. tropicalis XZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase,乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisG- PDC1(PHUHP),并转化宿主菌株C. tropicalis XZX,筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子XZX02。在此基础上,将转化子XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上,筛选得到URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCm- URA3-PDCm,并转化C. tropicalis XZX03菌株,获得转化子C. tropicalis XZX04。经PCR和DNA测序确认转化子C. tropicalis XZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术,利用该技术成功敲除了细胞的PDC基因,为进一步利用代谢工程改造热带假丝酵母奠定了基础。     

A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans

The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage λ Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.     

变性双链法实现葡萄糖异构酶基因敲除的研究

对７号淀粉酶菌Ｍ１０３３的遗传背景进行分析,建立了其原生质体制备、转化的优化条件。构建了葡萄糖异构酶（ＧＩ）结构基因内插千方百计以链丝菌肽基因（ｔｓｒ）的置换型同源重组质粒,利用其变性双链片段实现与Ｍ１０３３菌株染色体上基因的同源重组,获得置换型葡萄异构酶缺陷型蓖Ｍ１０３３ＬＪ。为在染色体上引入突变位点实现染色体上分子定点改造造尊定了基础。     

A new mutation delivery system for genome-scale approaches in Bacillus subtilis

In Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour-intensive and not compatible with large-scale approaches. We have developed a new one-step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp, which encodes uracil phosphoribosyl-transferase, as a counter-selectable marker. We fused the upp gene to an antibiotic-resistance gene to create an 'upp-cassette'. A polymerase chain reaction (PCR)-generated fragment, consisting of the target gene with the desired mutation joined to the upp-cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp-cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter-selection of upp. This procedure was successfully used to deliver a point mutation, to generate in-frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two-component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.     

Simple and highly efficient BAC recombineering using galK selection

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (lambda) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive lambda prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.     

Split-marker 重组技术构建泛素C 末端水解酶基因缺失菌株

目的:对烟曲霉(Aspergillus fumigatus)泛素末端水解酶(creB)基因进行敲除。方法:通过氨基酸序列分析软件初步分析烟曲霉CreB蛋白结构.利用split-marker重组技术构建重组片段,并通过PEG-原生质体方法对烟曲霉野生菌株进行转化,采用PCR方法对转化子进行筛选,最后选取初步筛选的转化子进行测序鉴定。结果:结构分析显示烟曲霉CreB蛋白具有泛素特异蛋白酶(ubiquitin-processing protease)UBP亚家族六个结构域。本实验构建了转化片段并转化,在抗性平板中获得了25个Hyg抗性转化子,进一步采用PCR方法筛选到20个转化子,最终通过测序分析获得一株creB基因缺失菌株。结论:Split-marker重组技术是对烟曲霉creB基因进行敲除的快速有效的方法。获得的creB缺失菌株可用于基因功能研究。