Creating random mutagenesis libraries using megaprimer PCR of whole plasmid

The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to avoid tedious screening, especially when producing a mutant gene library. To overcome these problems, we modified the QuikChange protocol so that each plasmid carries a single insert. Although the QuikChange was originally developed for site-directed mutagenesis using complementary mutagenic oligonucleotide primers in whole plasmid PCR, we found that the protocol also worked for megaprimers consisting of hundreds of nucleotides. Based on this discovery, we used insert fragments, which we wanted to clone, as the primers in the QuikChange reaction. The resultant libraries were virtually free from species with no inserts or multiple inserts. The present method, which we designated MEGAWHOP (megaprimer PCR of whole plasmid), is thus ideal for creating random mutagenesis megalibraries.     

A functional genomic analysis of type 3 Streptococcus pneumoniae virulence

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature-tagged mutants was created by insertion-duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty-six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.     

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.     

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.     

Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.     

Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis

For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions.     

A plant-transformation-competent BIBAC library from the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Landsberg ecotype for functional and comparative genomics

The genome of the model plant Arabidopsis thaliana has been sequenced to near completion. To facilitate experimental determination of the function of every gene in the species, we constructed a large-insert library from the Landsberg ecotype using a plant-transformation-competent binary BAC vector, BIBAC2. The library contains 11,520 clones with an estimated average insert size of 162 kb. Of a sample of 102 clones, 17.6% had no inserts; further, in the library as a whole, 287 clones contained chloroplast DNA, and 25 contained mitochondrial DNA. Thus it is estimated that 9,295 clones originated from the nuclear genome, representing a 11.5 x coverage. The library was further characterized by screening with probes corresponding to 180-bp repeats, 5S rDNA, 18S-25S rDNA and 23 single-copy RFLP markers. The results showed that 92 clones contained 180-bp centromeric repeats, 78 contained 5S rDNA and 95 contained 18S-25S rDNA, approximately 1%, 0.8% and 1%, respectively, of the nuclear clones in the library. Screening the library with the 23 RFLP markers showed that each one hybridized to an average of seven clones. This library is the first large-insert DNA library for the widely studied Landsberg erecta strain. It will greatly facilitate gene identification by complementation screening, and will enhance analysis of the structure, organization and evolution of the A. thaliana genome.     

Construction of a Bacterial Artificial Chromosome Library of Phytophthora infestans and Transformation of Clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

小麦抗叶锈病近等基因系TcLr41 SSH文库构建与分析

以小麦抗叶锈病近等基因系TcLr41和感病亲本Thatcher的叶片cDNA分别作为试验方和驱动方,利用抑制差减杂交技术,构建了一个包含2544个克隆的差减文库。随机提取阳性克隆质粒DNA后经PCR检测,插入片段大部分集中在200～1000bp之间,证明所构建的文库符合要求。在功能已知的基因中,推测过氧化氢酶（catalasc）基因、抗秆锈病基因（rust resistance gene）、铜蓝结合蛋白（blue copper—binding protein）基因、锌指蛋白（ring zinc finger protein）基因、胁迫反应蛋白（stress responsive protein）基因等可能是TcLr41中抗病相关差异表达基因。     

A new approach to random mutagenesis in vitro

Random mutagenesis is a powerful tool for studying the effects of a large number of permutations of a particular DNA sequence and its encoded products. Here we describe a new strategy of conducting in vitro random mutagenesis using ethyl methane sulfonate (EMS). The Bacillus aprN18 gene, coding for a serine protease with fibrinolytic activity, was used as a target gene. To study the mutations of the coding region, rather than the whole plasmid, the 1.4 kb gene fragment was cut out from an expression plasmid and treated with 10 mM EMS at 37 degrees C for 1 h. The treated fragment was then ligated back into the original expression vector and a library of random mutants was constructed in a protease-deficient Bacillus subtilis strain. A plate assay-based screening method was used to select for mutant clones with altered enzyme activity, and the change of activity was then confirmed by a semi-quantitative enzyme assay using liquid culture supernatant. The inserts of five clones with altered enzyme activity were randomly chosen for sequencing analysis. Among the point mutations detected, GC --> AT transition accounts for 42.1%, AT --> GC transition 34.2% and GC/CG transversion 23.7%, respectively. To our knowledge this is the first application of EMS for in vitro mutagenesis of a defined DNA sequence.     

一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离一株呼肠孤病毒（命名为EsRV905株）,采用Trizol试剂提取病毒核酸,经聚丙烯酰胺凝胶电泳,碎胶法回收基因组各节段。随机引物法合成第一节段的cDNA文库,胶回收试剂盒去除小片段,平端连接于载体,化学转化,利用蓝白斑筛选阳性克隆子,酶切鉴定重组质粒。从基因组第一节段的重组质粒中选择2个插入片段为1.5kb的质粒测序,结果得到包括RNA聚合酶主要特征性结构的一段序列。结果说明,这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段。     

Plasmid pU29, a vehicle for mutagenesis of the photosynthetic puf operon in Rhodopseudomonas capsulata

Plasmid pU21, which carries the reaction center and light-harvesting genes (puf operon) of Rhodopseudomonas capsulata, has been redesigned by site-specific mutagenesis. Five restriction sites have been removed and three unique restriction sites have been introduced into this 11,589-bp pBR322 derivative. The modifications divide the puf structural genes into four regions separated by five unique and nonmutagenic restriction sites. These four fragments have been subcloned into the M13-mp series of vectors to facilitate oligonucleotide-mediated site-specific mutagenesis experiments on the photosynthetic apparatus structural genes. The inserts can then be returned from the M13 replicative form to the redesigned pU21 derivative. The modified plasmid, pU29, greatly facilitates in vitro mutagenesis experiments since previously described techniques and screening procedures are more efficient with M13 derivatives carrying smaller inserts. Additionally, tandem homologous sequences (the reaction center L and M subunits) within the puf operon are now separated on different phage vectors, eliminating problems encountered in the targeting of mutagenic oligonucleotides to only one of the two homologous sites.     

Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation

Shuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gono-coccus; Gc). A size-restricted plasmid library of Gc strain FA1090 was mutated with the mini-transposon mTnEGNS. Randomness was tested by checking for transposon insertion bias between vector and insert DNA, Gc transformation efficiency of individual mutated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random shuttle mutagenesis were screened, using a colony-based polymerase chain reaction assay, for the ability to undergo pilin antigenic variation. Out of 8064 mutants screened, 22 unique transposon insertion mutants were found to be antigenic variation deficient (Avd). The Avd mutants were separated into five types according to recombination defect-associated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.     

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250?000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E.?coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E.?coli using regulated asRNA expression.     

QuikChange shuffling: a convenient and robust method for site-directed mutagenesis and random recombination of homologous genes

Here we describe a robust method, termed QuikChange shuffling, for efficient site-directed mutagenesis and random recombination of homologous genes. The homologous genes are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of complementary mutagenic primers, followed by assembly of the chimeric genes and linearized vector through QuikChange-like amplification to introduce recombinant plasmids with a site-directed mutation. The method, which can yield 100% chimeric genes after library construction, is more convenient and efficient than current DNA shuffling methods.     

Antimutagenic effects of para-aminobenzoic acid in experiments with bacterial cells. Noninterference in the mutation process controlled by plasmid pKM101

The dependence of expression of PABA antimutagenic action in bacterial cells on the character of genetic control of the mutagenic process was studied. PABA antimutagenic activity was largely connected with the negative control of SOS repair which is controlled by bacterial cell genes, but not by pKM101 plasmid genes. These results are in agreement with the idea that the systems of repair and mutagenesis specified by cell genome and plasmids are not identical.     

Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.     

一种温敏复制缺陷T载体的构建及在鸡白痢沙门氏菌基因敲除中的应用

基因敲除是基因功能研究的重要手段,载体是基因敲除的工具和核心。为获得有效的基因敲除载体以快速构建基因突变株及鉴定相应基因的必需性,在已有温敏复制缺陷pIDM1质粒的基础上,于EcoRⅠ和PstⅠ位点间插入串联的XcmⅠ酶切位点接头,构建了pIDM-T质粒;该质粒经XcmⅠ酶切可获得末端突出T的线性化pIDM-T载体。在验证了pIDM-T质粒复制的温敏特性基础上,应用构建的T载体克隆鸡白痢沙门氏菌CVCC527菌株的eno和ybdr两个基因,鉴定获得pIDM-T_eno和pIDM-T_ybdr两个重组质粒;将重组质粒转化527菌株,经IPC(Integration rate per cell)值计算,鉴定eno为必需基因,ybdr为非必需基因。挑取非必需ybdr基因527菌株重组菌(SalΔybdr),经PCR和测序,确认突变菌株重组位点的正确性。pIDM-T载体可快速克隆PCR产物,用于沙门氏菌的基因敲除及必需性鉴定,为沙门氏菌基因功能研究提供了一种有效快速的手段。