Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Combinatorial modulation of <Emphasis Type="Italic">galP</Emphasis> and <Emphasis Type="Italic">glk</Emphasis> gene expression for improved alternative glucose utilization

Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS⁻ strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.     

Common origin of plasmid encoded alpha-hemolysin genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Alpha (α)-hemolysin is a pore forming cytolysin and serves as a virulence factor in intestinal and extraintestinal pathogenic strains of E. coli. It was suggested that the genes encoding α-hemolysin (hlyCABD) which can be found on the chromosome and plasmid, were acquired through horizontal gene transfer. Plasmid-encoded α-hly is associated with certain enterotoxigenic (ETEC), shigatoxigenic (STEC) and enteropathogenic E. coli (EPEC) strains. In uropathogenic E. coli (UPEC), the α-hly genes are located on chromosomal pathogenicity islands. Previous work suggested that plasmid and chromosomally encoded α-hly may have evolved independently. This was explored in our study.     

Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions

Background  

Previous studies of gene amplification in Escherichia coli have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the lac operon was constructed, and its expansion into an amplified array was studied.     

Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event

Background  

Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage λ red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step.     

Exploring the evolutionary dynamics of plasmids: the <Emphasis Type="Italic">Acinetobacter</Emphasis> pan-plasmidome

Background  

Prokaryotic plasmids have a dual importance in the microbial world: first they have a great impact on the metabolic functions of the host cell, providing additional traits that can be accumulated in the cell without altering the gene content of the bacterial chromosome. Additionally and/or alternatively, from a genome perspective, plasmids can provide a basis for genomic rearrangements via homologous recombination and so they can facilitate the loss or acquisition of genes during these events, which eventually may lead to horizontal gene transfer (HGT). Given their importance for conferring adaptive traits to the host organisms, the interest in plasmid sequencing is growing and now many complete plasmid sequences are available online.     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.     

Use of the λ Red-recombineering method for genetic engineering of Pantoea ananatis

Background  

Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications.     

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

This paper describes a rapid method of constructing homologous recombinant baculovirus inE. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (Cm ^R ) to replaceorf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence oforf135, and the rest 20 bp was homologous to the each end sequence ofCm ^R . By using these primers, a linear fragment containing the completeCm ^R gene between 40 bp of homologous arms oforf135 was generated by PCR with the plasmid pKD3 which containsCm ^R as the template. By transforming the linear fragment into theE. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed inE. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.     

Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted for some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occuring during B-lymphocyte differentiation. The extent of the deleted region was determined using probes from the various IGLV subgroups and they each cover at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage V135 to the distal part of the IGLV gene region. Since the deletions are relatively small, the cell line will be valuable for mapping IGLV genes in the distal part of this region.     

Genomic transcriptional response to loss of chromosomal supercoiling in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.     

Gene replacement techniques for <Emphasis Type="Italic">Escherichia coli</Emphasis> genome modification

The subject of this review covers modern experimental procedures for chromosomal gene replacement in Escherichia coli and related bacteria, which enable the specific substitution of targeted genome sequences with copies of those carrying defined mutations. Two principal methods for gene replacement were established. The first “in–out” method is based on integration of plasmid into bacterial chromosome and subsequent resolving of the cointegrate. The “linear fragment” method (recombineering) is based on homologous recombination mediated by short homology arms at the ends of linear DNA molecule. Many new protocols and improvements in targeted gene replacement were introduced during the last 10 years. These methods are well suited for high-throughput functional gene studies and for many biotechnological applications.     

Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

Background  

Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the λ repressor-DNA complex for which structural and thermodynamic experimental data are available.     

Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

Background  

Oncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology.     

Joining of λ bacteriophage DNA moleculesin vitro using the gene product of the λint phage

The ability of cell extracts ofEscherichia coli 1100 prepared at different times after infection with bacteriophage λint 6 cI 857 or λ 857 to attach molecules of λ³²P-DNA to λ DNA adsorbed on a nitrocellulose membrane filter was compared. It was found that only with extracts from cells infected with bacteriophage λ cI 857 with an active int gene was it possible to attach molecules of λ³²P-DNA to λ DNA. This ability was reflected best in extracts prepared from cells 10 min after infection. A similar ability was observed also with extracts prepared from cells ofEscherichia coli 1100 (λ cI 857) after heat induction of the prophage. The maximum efficiency in this case was observed with cell extracts 15 min after the temperature rise.     

A molecular recombination map of <Emphasis Type="Italic">Antirrhinum majus</Emphasis>

Background  

Genetic recombination maps provide important frameworks for comparative genomics, identifying gene functions, assembling genome sequences and for breeding. The molecular recombination map currently available for the model eudicot Antirrhinum majus is the result of a cross with Antirrhinum molle, limiting its usefulness within A. majus.     

A method to generate recombinant <Emphasis Type="Italic">Salmonella typhi</Emphasis> Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy

Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated ‘targeting cassettes’ that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking ‘arms’ of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains.