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.     

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.     

sacB-5-Fluoroorotic Acid-pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB—5-fluoroorotic acid (5FOA)—pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura⁻) and hence resistant to 5FOA (5FOA^r). Although Ura⁺ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA^r to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA^r selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA^r colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose

Counterselectable markers are powerful tools in genetics because they allow selection for loss of a genetic marker rather than its presence. In mycobacteria, a widely used counterselectable marker is the gene encoding levan sucrase (sacB), which confers sensitivity to sucrose, but frequent spontaneous inactivation complicates its use. Here we show that the Escherichia coli galactokinase gene (galK) can be used as a counterselectable marker in both Mycobacterium smegmatis and Mycobacterium tuberculosis. Expression of E. coli galK, but not the putative M. tuberculosis galK, conferred sensitivity to 2-deoxy-galactose (2-DOG) in both M. smegmatis and M. tuberculosis. We tested the utility of E. coli galK as a counterselectable marker in mycobacterial recombination, both alone and in combination with sacB. We found that 0.5% 2-DOG effectively selected recombinants that had lost the galK marker with the ratio of galK loss/galK mutational inactivation of approximately 1:4. When we combined galK and sacB as dual counterselectable markers and selected for dual marker loss on 0.2% 2-DOG/5% sucrose, 98.6–100% of sucrose/2-DOG resistant clones had undergone recombination, indicating that the frequency of mutational inactivation of both markers was lower than the recombination frequency. These results establish a new counterselectable marker system for use in mycobacteria that can shorten the time to generate unmarked mutations in M. smegmatis and M. tuberculosis.     

A method for generating precise gene deletions and insertions in Escherichia coli

A simple and general method for disrupting chromosomal genes and introducing insertions is described. This procedure involves eliminating wild-type bacterial genes and introducing mutant alleles or other insertions at the original locus of the wild-type gene. To demonstrate the utility of this approach, the tig gene of Escherichia coli was replaced by homologous recombination with a cassette containing the chloramphenicol resistance gene and the sacB gene. The cassette was then removed and the tig mutant alleles were moved into the native tig location. Sequencing and Western blotting results demonstrated that insertions or deletions can be introduced precisely in E. coli using our approach. Our system does not require extra in vitro manipulations such as restriction digestion or ligation, and does not require use of specific plasmids or strains which are used to prevent false positive transformants caused by template plasmid transformation. This technique can be used widely in bacterial genome analysis.     

Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-δ in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.     

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.     

Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5′UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5′ sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.     

An Integrated System for Precise Genome Modification in Escherichia coli

We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl₂ for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. We demonstrate the power of this method to make a variety of genome modifications: the exact integration, without any extraneous sequence, of the lac operon (~6.5 kbp) to any desired location in the genome and without the integration of antibiotic markers; the scarless deletion of ribosomal rrn operons (~6 kbp) through either intrachromosomal or oligonucleotide recombination; and the in situ fusion of native genes to fluorescent reporter genes without additional perturbation.     

Prevalence of tetracycline resistance genes in Greek seawater habitats

The presence of selected tetracycline resistance (Tc^R) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, Tc^R bacteria, and exogenous isolated plasmids conferring Tc^R. The direct DNA-based analysis showed that tet(A) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fish-farm and coastal samples. Resistance genes tet(h), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the Tc^R genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring Tc^R genes in all the habitats tested. Plasmids were shown to carry tet(h), tet(C), tet(E), and tet(K) genes. It is concluded that Tc^R genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities.     

Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum

Bacillus subtilis sacB gene with its 463 bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum, but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum, and the native promoter of sacB is weak in C. glutamicum. The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter PlacM and a novel vector pDXW-3 were constructed. PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum. The pDXW-3 contains B. subtilissacB with the PlacM fused at the 5′-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for XhoI, NotI, EagI, SalI, SacI, BamHI, and NheI, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB-based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.     

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo^R expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo^R gene flanked by exon 3 of Hprt. The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration ~2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.     

Assisted large fragment insertion by Red/ET-recombination (ALFIRE)—an alternative and enhanced method for large fragment recombineering

Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4–5kb can be difficult to obtain and the risk of mutations or mismatches increases with the size of the template that is being amplified. This can be overcome by adding HAs by conventional cloning techniques, but with large fragments such as entire genes the procedure becomes time-consuming and tedious. Second, homologous recombination techniques often require addition of antibiotic selection genes, which may not be desired in the final construct. Here, we report a method to overcome the size and selection marker limitations by a two- or three-step procedure. The method can insert any fragment into small or large episomes, without the need of an antibiotic selection gene. We have humanized the mouse luteinizing hormone receptor gene (Lhcgr) by inserting a ~55kb fragment from a BAC clone containing the human Lhcgr gene into a 170kb BAC clone comprising the entire mouse orthologue. The methodology is based on the rationale to introduce a counter-selection cassette flanked by unique restriction sites and HAs for the insert, into the vector that is modified. Upon enzymatic digestion, in vitro or in Escherichia coli, double-strand breaks are generated leading to recombination between the vector and the insert. The procedure described here is thus an additional powerful tool for manipulating large and complex genomic fragments.     

Progress in lactic acid bacterial phage research

Background

The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

Results

In this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

Conclusions

By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.     

Construction of three new Gateway? expression plasmids for Trypanosoma cruzi

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway^® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway^® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).     

Isolation of plasmid deletion mutants and study of their instability

We describe a method which allows isolation of deletions within hybrid plasmids. It is based on the fact that the tetracycline resistance (Tc^R) gene of pBR322 can be inactivated by inserting foreign DNA into its HindIII site, and that the easily selectable Tc^R mutants of such plasmids are generally (>90%) due to deletions of certain hybrid plasmid sequences. We have found that Tc^R mutants are usually maintained within the cell recombined with the parental Tc^S plasmids. Such heterodimers dissociate in both Rec⁺ and in recA hosts. Parental rather than mutant plasmids are then retained by the host cell.     

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.