EXPRESSION OF THE DETOXIFYING GENE B1 IN ES‐CHERICHIA COLI AND SYNECHOCOCCUS1

Abstract We inserted the mosquito esterase B1 gene into the expression vector pRL‐439, which possesses the strong promoter PpsbA. The recombinant plasmid pRL‐Bl was used to transform E. coli HBlOl, and the positive clones were screened on LB medium plate containing 100 mg/mL ampicillin. The results of dot blotting and Southern hybridization demonstrated that these positive clones were transformed bacteria. Western blotting indicated that esterase B1 gene had been successfully expressed under the control of the PpsbA promoter in E. coli. A shuttle verruction‐B1 (pDGBl) was constructed by inserting B1‐cDNA from pRL‐Bl into polycloning site of plasmid pDc8. PDGBl was transferred into Synechoccus sp. PCC7942 through triparental conjugal transfer. Transformed single Synechococcus sp. PCC 7942 clone was obtained by neomycin screening, and large‐scale culture in liquid medium was carried out. Results of Southern blotting proved that pWB1 was transferred into Synechococcus sp. PCC 7942.     

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.     

Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.     

Transfer of plasmids fromEscherichia coli andPseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts

Several plasmids of incompatibility group P were transferred fromEscherichia coli andPseudomonas aeruginosa strains toMethylophilus methylotrophus and two other methylotrophs to test their recipient ability. The presence of plasmids in transconjugants was confirmed by electrophoretic analysis. Optimal conditions for detection of plasmid DNA in the strains tested based on alkaline lysis of cells at elevated temperature were established. Special behaviour of plasmids carrying the Mu phage in methylotrophic hosts is described.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Type IV secretion in Gram‐negative and Gram‐positive bacteria

Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram‐negative and Gram‐positive bacteria. They play important roles through the contact‐dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact‐independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F‐pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of ‘paradigmatic’ and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances.     

A Non-Restricting and Non-Methylating <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain for DNA Cloning and High-Throughput Conjugation to <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm⁻ Dam⁻ strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.     

High efficiency transduction of single strand plasmid DNA into enteric bacteria

Summary This report demonstrates high efficiency transduction of enteric bacteria using single strand plasmids packaged in M13 phage capsids. Transformation by plasmid DNA is usually a very inefficient process in many enteric bacteria other than Escherichia coli K12. Plasmids carrying an M13 origin of replication can be replicated and packaged when cells carrying such plasmids are infected with M13 or a derivative helper phage. By introducing an F plasmid into E. coli, Serratia marcescens, Citrobacter freundii, and Enterobacter aerogenes, these species can now be infected at high efficiency with M13 phage and with packaged single strand plasmids, yielding an efficient method to introduce cloned DNA fragments into these bacteria. The titer of colony forming units in a lysate was essentially equivalent in all the bacteria, demonstrating an equal efficiency of transduction of these other enteric bacteria compared to E. coli.     

枯草芽孢杆菌分子克隆系统受体的改造

实验证明枯草芽孢杆菌(Bacillus subtilis)BR151和MI112中的pUB110质粒,在液体中传代要比固体斜面传代稳定。通过NTG诱变获得了对pUB110稳定的受体BSG.来自大肠杆菌的穿梭质粒在BSG菌中转化频率较亲本高。小的外源DNA片段插入pBE2后在BSG菌中转管传代30次是稳定的。     

A convenient colony-based transformation method for high-throughput changing of plasmid hosts

We have developed a convenient Escherichia coli transformation method, termed colony-based transformation, in which a single fresh colony of plasmid-containing donor strain is used instead of extracted plasmid to transform E. coli recipient cell. Thus the need for plasmid extraction and competent cell preparation is avoided. Additionally, a high-throughput transformation process based on this method was designed in which samples are prepared in a 96-well PCR plate and hundreds of transformations can be performed simultaneously in a thermocycler. We therefore suggest that this method may serve as a substitute of current transformation methods based on plasmid extraction.     

Involvement of Rad52 in T‐DNA circle formation during Agrobacterium tumefaciens‐mediated transformation of Saccharomyces cerevisiae

Agrobacterium tumefaciens cells carrying a tumour inducing plasmid (Ti‐plasmid) can transfer a defined region of transfer DNA (T‐DNA) to plant cells as well as to yeast. This process of Agrobacterium‐mediated transformation (AMT) eventually results in the incorporation of the T‐DNA in the genomic DNA of the recipient cells. All available evidence indicates that T‐strand transfer closely resembles conjugal DNA transfer as found between Gram‐negative bacteria. However, where conjugal plasmid DNA transfer starts via relaxase‐mediated processing of a single origin of transfer (oriT), the T‐DNA is flanked by two imperfect direct border repeats which are both substrates for the Ti‐plasmid encoded relaxase VirD2. Yeast was used as a model system to investigate the requirements of the recipient cell for the formation of T‐DNA circles after AMT. It was found that, despite the absence of self‐homology on the T‐DNA, the homologous repair proteins Rad52 and Rad51 are involved in T‐DNA circle formation. A model is presented involving the formation of T‐DNA concatemers derived from T‐strands by a process of strand‐transfer catalysed by VirD2. These concatemers are then resolved into T‐DNA circles by homologous recombination in the recipient cell.     

A rapid method for the screening of plasmids in transformed yeast strains

A method for the rapid screening of plasmids in yeast cells has been developed. The method is an adaptation of the currently used alkaline lysis methods forEscherichia coli plasmids. Following the conditions described, several dozen ofSaccharomyces cerevisiae-transformed clones can be analyzed for their plasmid content in less than 2 h. The plasmids obtained by this procedure are suitable for restriction analysis or forE. coli andS. cerevisiae transformation.     

Characterization of putative circular plasmids in sponge‐associated bacterial communities using a selective multiply‐primed rolling circle amplification

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.     

Simple,fast and high‐efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme‐, phosphatase‐ and ligase‐free), fast (i.e. 1 day) and of high efficiency (i.e. ～10⁷ or ～10⁴ transformants per µg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two‐round PCR: (i) the mutagenized DNA fragments were generated by error‐prone PCR and linearized plasmids were made using high‐fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.     

Plasmid DNA production combining antibiotic‐free selection,inducible high yield fermentation,and novel autolytic purification

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage λR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non‐ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid–liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts. Biotechnol. Bioeng. 2009; 104: 505–515 © 2009 Wiley Periodicals, Inc.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Detection of nonsense and frameshift mutations in <Emphasis Type="Italic">BRCA1</Emphasis> gene using a new plasmid vector pPhoA-frame

The technique for detecting frameshift and nonsense mutations in the human BRCA1 gene has been suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment is placed in frame with alkaline phosphatase gene of Escherichia coli (phoA). A special plasmid pPhoA-frame was constructed for this analysis, and the plasmid contained the DNA fragment that encodes alkaline phosphatase of E. coli. The synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment that encodes alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. The occurrence of the frameshift or nonsense mutation in the tested DNA fragment can be detected after the transformation of E. coli by the recombinant plasmid that contains the tested fragment. E. coli colonies with newly constructed recombinant plasmids are plated out on the indicator agar. In the case of the frameshift or nonsense mutation, the colonies are not colored, and DNA fragments without these mutations result in the formation of blue colonies.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM flow cytometry - FITC fluorescein-iso-thiocyanate - LB Luria broth - MM minimal salt medium - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride