Homologous Integration Research Articles

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

A versatile Cas12k-based genetic engineering toolkit (C12KGET) for metabolic engineering in genetic manipulation-deprived strains.

The genetic modification of microorganisms is conducive to the selection of high-yield producers of high-value-added chemicals, but a lack of genetic tools hinders the industrialization of most wild species. Therefore, it is crucial to develop host-independent gene editing tools that can be used for genetic manipulation-deprived strains. The Tn7-like transposon from Scytonema hofmanni has been shown to mediate homologous recombination-independent genomic integration after heterologous expression in Escherichia coli, but the integration efficiency of heterologous sequences larger than 5 kb remains suboptimal. Here, we constructed a versatile Cas12k-based genetic engineering toolkit (C12KGET) that can achieve genomic integration of fragments up to 10 kb in size with up to 100% efficiency in challenging strains. Using C12KGET, we achieved the first example of highly efficient genome editing in Sinorhizobium meliloti, which successfully solved the problem that industrial strains are difficult to genetically modify, and increased vitamin B12 production by 25%. In addition, Cas12k can be directly used for transcriptional regulation of genes with up to 92% efficiency due to its naturally inactivated nuclease domain. The C12KGET established in this study is a versatile and efficient marker-free tool for gene integration as well as transcriptional regulation that can be used for challenging strains with underdeveloped genetic toolkits.

NIH4215: A mutation-prone thiamine auxotrophic clinical Aspergillus fumigatus isolate.

Aspergillus fumigatus is the main cause of life-threatening invasive aspergillosis. Despite the availability of various antifungals, therapy remains challenging and requires further studies. Accordingly, the clinical A. fumigatus isolate NIH4215 deriving from a fatal case of human pulmonary aspergillosis has frequently been used in drug efficacy studies. Unexpectedly, our initial attempts to generate a bioluminescent reporter of strain NIH4215 for in vivo drug efficacy studies failed, as NIH4215 was unable to grow on defined minimal medium. Subsequent analyses discovered a previously undescribed thiamine auxotrophy of strain NIH4215 and transformation with thiamine biosynthesis genes from A. fumigatus strain Af293 identified the nmt1 gene as cause of the thiamine auxotrophy. Sequencing of the defective nmt1 gene revealed the loss of a cysteine codon within an essential iron-binding motif. Subsequently, the wild-type nmt1 gene was successfully used to generate a bioluminescent reporter strain in NIH4215 by simultaneously deleting the akuB locus. The resulting bioluminescent ΔakuB strains showed a high frequency of homologous integration as confirmed by generation of pyrG and niaD deletion mutants. When tested in a Galleria mellonella infection model, neither thiamine auxotrophy nor the deletion of the akuB locus had a significant effect on virulence. However, besides thiamine auxotrophy, sectors with altered morphology and albino mutants frequently arose on colony edges of strain NIH4215 and its derivatives, and stable albino mutants were successfully isolated. A proposed increased mutation rate of NIH4215 was confirmed by screening for spontaneous occurrence of fluoorotic acid resistant mutants. Independent mutations in the pyrG and pyrE gene were identified in the fluoroorotic acid resistant NIH4215 isolates and the frequency of mutation was by at least one order of magnitude higher than that observed for the clinical A. fumigatus isolate CBS144.89. In summary, despite its virulence in animal models, strain NIH4215 is a thiamine auxotroph and prone to accumulate mutations. Our results suggest that thiamine biosynthesis is dispensable for host infection and mutation-prone strains such as NIH4215 could potentially facilitate the evolution of azole resistant strains as increasingly observed in the environment.

A Homologous Recombination System to Generate Epitope-Tagged Target Genes in Chaetomium thermophilum: A Genetic Approach to Investigate Native Thermostable Proteins.

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50–52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.

Increased Agrobacterium-mediated transformation of Saccharomyces cerevisiae after deletion of the yeast ADA2 gene.

Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions, the yeast Saccharomyces cerevisiae and other yeasts and fungi can also be transformed, and Agrobacterium‐mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae, T‐DNA is integrated preferentially by homologous recombination and integration by non‐homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T‐DNA was mediated by homologous or non‐homologous recombination. This correlates with an increase in double‐strand DNA breaks, the putative entry sites for T‐DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52‐GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform.

Viable strategies for enhancing acetone-butanol-ethanol production from non-detoxified switchgrass hydrolysates

A process engineering strategy was investigated towards developing a viable scheme for effective conversion of hydrothermolysis pretreated non-detoxified switchgrass hydrolysates (SH) to acetone butanol ethanol (ABE) using a metabolically engineered strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii_AKR. The engineered strain was modified by homologous integration into the chromosome and constitutive expression of Cbei_3974, which encodes an aldo–keto reductase. Intermittent feeding strategy was employed in which fermentation was initiated with 30% of the SH and the remaining 70% SH was added when the optical density (OD600nm) of C. beijerinckii attained 0.5. The ABE (14.9 g/L) produced from non-detoxified SH by the inhibitor-tolerant C. beijerinckii_AKR was comparable to the P2-glucose control medium (14.7 g/L). Using intermittent feeding, wildtype and C. beijerinckii_AKR produced similar amounts of ABE (about 17.5 g/L). This shows that intermittent feeding strategy and C. beijerinckii_AKR enhanced ABE fermentation and eliminated the need for SH detoxification prior to fermentation.

Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus.

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (ΔpyrF) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.

Recombination machinery engineering for precise genome editing in methylotrophic yeast Ogataea polymorpha

SummaryMethanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%–30% to 60%–70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the production of fatty alcohols in O. polymorpha. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

BackgroundPenicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus.ResultsPenicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome.ConclusionsIn this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

Fast and antibiotic free genome integration into Escherichia\xa0coli chromosome

Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges’ λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based “All-In-One” genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.

Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents.

DNA double-strand break (DSB) repair induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9-CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled invitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X-100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP-based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi-target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on invivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome-edited fungi for the food and medical industries, and has good prospects for commercialization.

Epistatic Effect of Regulators to the Adaptive Growth of Escherichia coli

Bacteria survive in the environment with three steps: a sensing environmental conditions, a responding to sensed signals, and an adaptation for proper survival in the environment. An adapting bacterial cell occurs cell division to increase the number of sister cells, termed adaptive growth. Two-component systems (TCSs), representing the main bacterial signal transduction systems, consist of a pair of one sensor kinase (SK) and one response regulator (RR), and RR genes are abundant in most bacterial genomes as part of the core genome. The OmpR gene family, a group of RR genes, is conserved in 95% of known bacterial genomes. The Escherichia coli genome has an estimated 34 RR genes in total, including 14 genes of OmpR family genes. To reveal the contribution of TCSs for fast growth as an adaptive growth strategy of E. coli, we isolated a set of gene knockout strains by using newly developed genome editing technology, the HoSeI (Homologous Sequence Integration) method, based on CRISPR-Cas9. The statistics of single cell observation show a knockout of an arbitrary pair of phoP, phoB, and ompR genes, stably expressed by positive feedback regulation, dramatically inhibit the optimum adaptive growth of E. coli. These insights suggest that the adaptive growth of bacteria is fulfilled by the optimum high intracellular level of regulators acquired during growth under environmental conditions.

Recombination Machinery Engineering for Precise Genome Editing in Methylotrophic Yeast <i>Ogataea Polymorpha</i>

Methanol biotransformation can expand biorefinary substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR/Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ), increase HR rates from 20-30% to 60-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated systems has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the first production of fatty alcohols in O. polymorpha. These findings will simplify the genetic engineering in non-conventional yeasts, and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.

Plasmid-based and -free methods using CRISPR/Cas9 system for replacement of targeted genes in Colletotrichum sansevieriae

The CRISPR-Cas9 system has a potential for wide application in organisms that particularly present low homologous integration rates. In this study, we developed three different methods using this system to replace a gene through homology-directed repair in the plant pathogenic fungus Colletotrichum sansevieriae, which has a low recombination frequency. The gene encoding scytalone dehydratase was used as the target so that mutants can be readily distinguished owning to a lack of melanin biosynthesis. First, we performed a plasmid-based method using plasmids containing a Cas9 expression cassette and/or a single-guide RNA (sgRNA) under the control of the endogenous U6 snRNA promoter, and 67 out of 69 (97.1%) transformants exhibited a melanin-deficient phenotype with high efficiency. Second, we performed a transformation using a Cas9 protein/sgRNA complex and obtained 23 out of 28 (82.1%) transformants. Lastly, we developed a hybrid system combining a Cas9 protein and donor DNA-sgRNA expression plasmid, which yielded 75 out of 84 (89.2%) transformants. This system was also applicable to four other genes at different loci of the fungus. This is the first study to establish a CRISPR/Cas9 gene replacement system in Colletotrichum spp. and it presents a potential application for a broad range of use in other species of the genus.

CRISPR/Cas9-Mediated Gene Replacement in the Fungal Keratitis Pathogen Fusarium solani var. petroliphilum.

Fungal keratitis (FK) is a site-threatening infection of the cornea associated with ocular trauma and contact lens wear. Members of the Fusarium solani species complex (FSSC) are predominant agents of FK worldwide, but genes that support their corneal virulence are poorly understood. As a means to bolster genetic analysis in FSSC pathogens, we sought to employ a CRISPR/Cas9 system in an FK isolate identified as Fusarium petroliphilum. Briefly, this approach involves the introduction of two components into fungal protoplasts: (1) A purified Cas9 protein complexed with guide RNAs that will direct the ribonuclease to cut on either side of the gene of interest, and (2) a “repair template” comprised of a hygromycin resistance cassette flanked by 40 bp of homology outside of the Cas9 cuts. In this way, Cas9-induced double strand breaks should potentiate double homologous replacement of the repair template at the desired locus. We targeted a putative ura3 ortholog since its deletion would result in an easily discernable uracil auxotrophy. Indeed, 10% of hygromycin-resistant transformants displayed the auxotrophic phenotype, all of which harbored the expected ura3 gene deletion. By contrast, none of the transformants from the repair template control (i.e., no Cas9) displayed the auxotrophic phenotype, indicating that Cas9 cutting was indeed required to promote homologous integration. Taken together, these data demonstrate that the in vitro Cas9 system is an easy and efficient approach for reverse genetics in FSSC organisms, including clinical isolates, which should enhance virulence research in these important but understudied ocular pathogens.

Chromosomal integration of aldo-keto-reductase and short-chain dehydrogenase/reductase genes in Clostridium beijerinckii NCIMB 8052 enhanced tolerance to lignocellulose-derived microbial inhibitory compounds

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.

Characterization of the function of murine guanylate-binding protein 9 in the context of bacterial and parasitic infections

Abstract Interferon-γ (IFNγ) signaling induces high expression of immunomodulatory molecules in most cell types. Among these molecules, the members of the murine guanylate-binding protein (mGBP) family comprise a group of eleven proteins (mGBP1-mGBP11) shown to have important functions in cell-autonomous immunity. Upon IFNγ stimulation, mGBPs localize in vesicle-like structures distributed within the cytoplasm. Previous studies have shown that after infection with several intracellularly replicating pathogens, mGBPs rapidly relocate towards the pathogen-containing vacuoles (PCV), such as the parasitophorous vacuole (PV) of Toxoplasma gondii or the inclusions of Chlamydia trachomatis. Accumulation of mGBP2 at the PV membrane leads to loss of membrane integrity and subsequent restriction of parasite replication. Although mGBP proteins share a high sequence identity, differences in localization frequencies could be detected. For instance, mGBP1 and mGBP2 show the highest frequencies of recruitment to both Toxoplasma and Chlamydia PCVs. However, mGBP9 recruitment was more frequent to chlamydial inclusions than to T. gondii PVs. Thus, the importance of individual mGBPs might vary depending on the type of the pathogen. To analyze these findings in more detail, we started with the generation of mgbp9−/− cell- and mouse lines. Therefore, the CRISPR/Cas9 gene editing system with homologous directed integration of a single-stranded oligodesoxynucleotide is used to generate mGBP9 deficient ES cells for mouse and cell line development. In-depth analyzes of mgbp9−/− cells and mice in T. gondii and C. trachomatis infection will lead to a better understanding of immunity against intracellular pathogens.

Nuclear transformation of the versatile microalga Euglena gracilis

Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.

Genome-Wide Screening for Novel Candidate Virulence Related Response Regulator Genes in Xanthomonas oryzae pv. oryzicola.

Two-component regulatory system (TCS), a major type of cellular signal transduction system, is widely used by bacteria to adapt to different conditions and to colonize certain ecological niches in response to environmental stimuli. TCSs are of distinct functional diversity, genetic diversity, and species specificity (pathovar specificity, even strain specificity) across bacterial groups. Although TCSs have been demonstrated to be crucial to the virulence of Xanthomonas, only a few researches have been reported about the studies of TCSs in Xanthomonas oryzae pathovar oryzicola (hereafter Xoc), the pathogen of rice bacterial streak disease. In the genome of Xoc strain GX01, it has been annotated 110 TCSs genes encoding 54 response regulators (RRs), 36 orthodox histidine kinase (HKs) and 20 hybrid histidine kinase (HyHKs). To evaluate the involvement of TCSs in the stress adaptation and virulence of Xoc, we mutated 50 annotated RR genes in Xoc GX01 by homologous vector integration mutagenesis and assessed their phenotypes in given conditions and tested their virulence on host rice. 17 RR genes were identified to be likely involved in virulence of Xoc, of which 10 RR genes are novel virulence genes in Xanthomonas, including three novel virulence genes for bacteria. Of the novel candidate virulence genes, some of which may be involved in the general stress adaptation, exopolysaccharide production, extracellular protease secretion and swarming motility of Xoc. Our results will facilitate further studies on revealing the biological functions of TCS genes in this phytopathogenic bacterium.