Homologous Recombination Techniques Research Articles

Genetic Engineering of Bacillus subtilis Using Competence-Induced Homologous Recombination Techniques.

Bacillus subtilis is one of the best-studied bacteria and serves as a Gram-positive model system to address fundamental biological processes. Depending on conditions, a B. subtilis cell can initiate one out of various distinct differentiation processes to cope with changing environmental conditions. One of these differentiation processes is natural competence that allows cells to adsorb exogenous DNA and subsequently incorporate it into its chromosome by homologous recombination. Due to competence development, the genome of B. subtilis can be easily manipulated, and this has contributed to B. subtilis being a model system. In this chapter, we describe some of the most common genetic tools that can be used in combination with natural competence to tailor the genome of B. subtilis.

Effects of ppk1 deletion on the drug susceptibility of uropathogenic Escherichia coli producing ESBLs

To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital, meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney U test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (Z=0,P>0.05). The expression levels of ESBLs-enconding genes blaTEM and blaCTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated, while the expression of omp A and omp C were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes tol C, mdt A and mdtG were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of ppk1 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.

Rapid Construction of an Infectious Clone of Fowl Adenovirus Serotype 4 Isolate.

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis-hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors.

Characterization of Brucella abortus Mutant A19mut2, a Potential DIVA Vaccine Candidate with a Modification on Lipopolysaccharide.

Brucella abortus is the main causative agent for bovine brucellosis. B. abortus A19 is a widely used vaccine strain to protect cows from Brucella infection in China. However, A19 has a similar lipopolysaccharide (LPS) antigen to that of the field virulent Brucella strain, whose immunization interferes with the serodiagnosis of vaccinated and infected animals. [Aim] To develop a novel Brucella DIVA vaccine candidate. The B. abortus mutant A19mut2 with the formyltransferase gene wbkC is replaced by an acetyltransferase gene wbdR from E. coli O157 using the bacterial homologous recombination technique, generating a modified O-polysaccharide that cannot induce antibodies in mice against wild-type Brucella LPS. The biological phenotypes of the A19mut2 were assessed using a growth curve analysis, agglutination tests, Western blotting, and stress resistance assays. Histopathological changes and bacterial colonization in the spleens of vaccinated mice were investigated to assess the residual virulence and protection of the A19mut2. Humoral and cellular immunity was evaluated by measuring the levels of IgG, IgG subtypes, and the release of cytokines IFN-γ and IL10 in the splenocytes of the vaccinated mice. ELISA coated with wild-type LPS can distinguish mouse antibodies induced by A19 and A19mut2 immunization. The A19mut2 showed a decreased residual virulence in mice, compared to the A19 strain, but induced significant humoral and cellular immune responses, as the A19 immunization did. The protection efficacy of A19mut2 immunization against B. abortus S2308 NalR infection was similar to that of A19 immunization. The A19mut2 has potential as a novel DIVA vaccine candidate in the future.

Generation of a Null Mutation of the &lt;i&gt;toothrin&lt;/i&gt; Gene by Targeted Homologous Recombination in &lt;i&gt;Drosophila melanogaster&lt;/i&gt;

Drosophila toothrin gene is related to the conserved d4 family of genes that encode specific transcription factors. In early works, we investigated the effect of tth gene overexpression and characterized the specific pattern of its expression in the nervous system, but mutations in the tth gene have not yet been found. Null mutants are essential for analyzing gene function and screening of partner genes. Here we report the generation of the first tth gene knockout in Drosophila using a homologous recombination technique.

The Small Non-coding RNA rli106 Contributes to the Environmental Adaptation and Pathogenicity of Listeria Monocytogenes.

Listeria monocytogenes (LM) is an important food-borne pathogen, and the risk of its ingestion is a serious public health issue. The better its environmental adaptation mechanisms and pathogenicity are understood, the better the risk it poses can be countered. The regulatory role of the small non-coding RNA (sRNA) rli106 in the environmental adaptation and pathogenicity of LM is still unclear and this study investigated that role through its biological function. An LM-Δrli106 gene deletion strain and an LM-Δrli106/rli106 gene complementation strain were constructed using the homologous recombination technique. Then, the adaptation of these strains to temperature, alkalinity, acidity, salinity, ethanol and oxidative stressors, their biofilm-forming ability and their pathogenicity in mice were investigated to show the regulatory roles of sRNA rli106 in LM. The target gene of rli106 was also predicted, and the interaction between it and rli106 was verified by a two-plasmid co-expressing system based on E.coli and Western blot analysis. The adaptation of LM-Δrli106 to environmental stressors of pH 9, 5% NaCl and 8% NaCl, 3.8% ethanol and 5 mM H2O2 was significantly reduced when compared to the parental (LM EGD-e) and complementation strains. Also, the biofilm formation, cell adhesion, invasion, intracellular proliferation and pathogenicity of LM-Δrli106 in mice were significantly reduced. The results of two-plasmid co-expression and Western blot showed that rli106 can interact with the mRNA of the predicted DegU target gene. The sRNA rli106 may positively regulate the expression of the DegU gene in LM. This study sheds light on its regulatory roles in environmental adaptation and pathogenicity, providing new insights into the molecular mechanism of sRNA mediation in LM .

The function of the gene loiP in transformation efficiency and outer membrane permeability change of Escherichia coli treated by Ca2+ ions.

The preparation of Escherichia coli competent cells by calcium chloride is a common method in molecular biology, but the mechanism is poorly understood. In a previous study, using transcriptomics and proteomics approaches, we found that the expression pattern of the gene loiP was upregulated by CaCl2. In order to investigate the function of the loiP gene in Ca2+- mediated formation of competent cells of E. coli DH5α, the loiP gene deletion strains were constructed by the lambda-derived Red homologous recombination technique. Then, the cell morphology, transformation efficiency, and cell membrane changes of the competent cells of the mutant strain were further explored. Compared with the wild-type E. coli DH5α, the mutant strains have no significant differences in the morphology, growth characteristics, and the permeability of the intracellular membrane. However, the transformation efficiencies of the mutant strains to plasmids of different sizes were significantly reduced, and the permeability of the outer membrane decreased by 2.94 times. These results indicate that the deletion of gene loiP may directly affect the transformation efficiency and outer membrane permeability of E. coli competent cells.

Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation.

Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation-a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.

Quantitative proteomics reveal the potential biological functions of the deubiquitinating enzyme Ubp14 in Saccharomyces cerevisiae

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

Genome analysis of Bacillus subtilis JCL16 and the synergistic relationship among its metabolites reveal its potential for biocontrol of Nocardia seriolae

Nocardia seriolae is one of the major causative agents of fish nocardiosis, responsible for large-scale fish die-offs and economic losses in the aquaculture industry worldwide. Therefore, research efforts towards developing efficacious alternatives to aquatic antibiotics, such as vaccines, immunomodulatory additives including aquatic probiotics, to control this disease are of high importance. Recently, we isolated a soil-derived strain of Bacillus subtilis JCL16, and the 21-day challenge tests results showed that the addition of B. subtilis JCL16 culture to the basal control diet significantly increased the survival rate of largemouth bass with nocardiosis from 21.67% to 68.3%. To reveal the putative functional factors, we determined the genome sequence of B. subtilis JCL16 using PacBio RS II and Illumina HiSeq 4000 platform, and predicted its secondary metabolites using antiSMASH, which showed that it contains 11 functional gene clusters, three of which, srf, bac and sbo, have previously been verified to have antagonistic bacterial functions. Subsequently, mutants Δsrf, Δbac, Δsbo were constructed using homologous recombination techniques, and HPLC results further elucidated that the three gene clusters are responsible for the synthesis of surfactin, bacilysin and subtilosin A, respectively. To our surprise, a significant reduction in the inhibition of N. seriolae occurred in all three mutants compared to the wild type, so we speculated that surfactin, bacilysin and subtilosin A might have adopted a synergistic strategy. Next, we prepared their pure products and further confirmed our inference by Oxford Cup and MIC assays.In summary, the good biocontrol effect of B. subtilis JCL16 against fish Nocardia is likely due to the synergistic strategy among surfactin, bacilysin and subtilosin A. Here we propose a synergistic mechanism among them, which provides some insight into the powerful biocontrol mechanisms of Bacillus.

Invention sharing is the mother of developmental biology.

Invention sharing is the mother of developmental biology.

Stable transfection system for Babesia sp. Xinjiang

BackgroundStable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers.MethodsWe evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites.ResultsTransfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months.ConclusionsIn our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj.Graphical abstract

Gene knockout revealed the role of gene feoA in cell growth and division of Lactobacillus delbrueckii subsp. bulgaricus.

Gene feoA plays an important role in cell growth because of its function of transport Fe2+ which is a necessary element for cells. In this study, the recombinant plasmid pUC19-feoA-Tet was successfully constructed using the inserted gene inactivation method. Using the homologous recombination technique, the tet gene was used as a resistance screening marker to knock out the feoA gene of Lactobacillus delbrueckii subsp. bulgaricus 34.5 (strain 34.5). Comparative analysis of growth curves revealed the growth changes in the absence of feoA gene in strain 34.5. The results showed that the growth of the bacteria was prolonged by 2h and could be restored in the stationary phase. To further study whether feoA is related to the cell division of strain 34.5, the qPCR experiment was carried out. The results showed that, compared with the wild-type strain, the expression of genes related to cell division in the mutant strain was up-regulated in the pre-log phase, down-regulated in the late-log phase, and returned to the original level in the stationary phase. These findings provide ideas for Lactobacillus delbrueckii subsp. bulgaricus to control division and cell cycle.

Antifungal activity of biosynthesized silver nanoparticles from Candida albicans on the strain lacking the CNP41 gene

The upsurge of immunocompromised patients has led to extensive study of fungal infections with Candida albicans being the frontline model of pathogenic yeast in humans. In the quest to find novel antifungal agents, this study reports the potential usage of wild-type C. albicans strain C86 to biosynthesise silver nanoparticles by microwave assisted technique. Visual colour change and UV-spectrophotometer were used for primary detection of silver nanoparticles. Additionally, the FTIR peaks confirm the particles’ formation and surface characterisation techniques such as FESEM and EDX suggests that the silver nanoparticles were sized in the range of 30–70 nm. Furthermore, pioneering work of homologous recombination technique was systematically employed to delete uncharacterized gene orf19.3120 (CNP41) in the C86 strain creating the deletion strain C403 of C. albicans. To amalgamate the two significant findings, biosynthesized silver nanoparticles were subjected to antifungal studies by disk diffusion assay on the strain C403 that lacks the gene orf19.3120 (CNP41) of C. albicans. As a synergetic approach, combinational effect was studied by incorporating antifungal drug fluconazole. Both individual and enhanced combinational antifungal effects of silver nanoparticles and fluconazole were observed on genetically modified C403 strain with 40% increase in fold area compared to wild-type C86 strain. This can be attributed to the synergetic effect of the bonding reaction between fluconazole and AgNPs. Taken together, this first-ever interdisciplinary study strongly suggests that the CNP41 gene could play a vital role in drug resistance in this fungal pathogen.

Effects of Lmo2672 Deficiency on Environmental Adaptability, Biofilm Formation, and Motility of Listeria monocytogenes

Background: Listeria monocytogenes is a food-borne pathogen with strong environmental adaptability. It can survive at high temperatures and in acidic high-salt and other unfavorable stressful environments and consequently form a biofilm. Objectives: This study aimed to explore the roles of lmo2672 on the environmental adaptability, biofilm formation, and motility of L. monocytogenes. Methods: We analyzed the molecular characteristics of lmo2672 protein, constructed a lmo2672 gene deletion strain of L. monocytogenes strain (L. monocytogenes-Δlmo2672), using the homologous recombination technique and compared the environmental adaptability, biofilm formation, and motility of L. monocytogenes-Δlmo2672 with its parental strain L. monocytogenes EGD-e. The impacts of lmo2672 gene deficiency on the transcription of genes associated with flagella formation and the environmental adaptability of L. monocytogenes were also determined. Results: The results showed that the lmo2672 contains two helix-turn-helix (HTH) DNA-binding domains. Compared to L. monocytogenes EGD-e, L. monocytogenes-Δlmo2672 grew significantly slower at different temperatures (37°C and 42°C) under different osmotic pressures (5% and 8% NaCl) and in different media containing 0.3% bile salts, 5 mM H2O2, or 1% Triton X-100 (P < 0.05). Moreover, LM-Δlmo2672 showed a significant decrease in biofilm formation (P < 0.01) and motility (P < 0.05) at 48 h of culture. Furthermore, the transcriptional levels of regulatory gene prfA and flagella-related genes motA, fliP and fliE significantly decreased in LM-Δlmo2672 (P < 0.05). Conclusions: In general, these findings indicated that lmo2672 played critical regulatory roles in the environmental adaptability, biofilm formation, and motility of L. monocytogenes, thus providing new insights into the regulatory mechanism of lmo2672 in L. monocytogenes.

A T3SS Regulator Mutant of Vibrio alginolyticus Affects Antibiotic Susceptibilities and Provides Significant Protection to Danio rerio as a Live Attenuated Vaccine.

Vibrio alginolyticus is a major cause of Vibriosis in farmed marine aquatic animals and has caused large economic losses to the Asian aquaculture industry in recent years. Therefore, it is necessary to control V. alginolyticus effectively. The virulence mechanism of V. alginolyticus, the Type III secretion system (T3SS), is closely related to its pathogenicity. In this study, the T3SS gene tyeA was cloned from V. alginolyticus wild-type strain HY9901 and the results showed that the deduced amino acid sequence of V. alginolyticus tyeA shared 75–83% homology with other Vibrio spp. The mutant strain HY9901ΔtyeA was constructed by Overlap-PCR and homologous recombination techniques. The HY9901ΔtyeA mutant exhibited an attenuated swarming phenotype and an ~40-fold reduction in virulence to zebrafish. However, the HY9901ΔtyeA mutant showed no difference in growth, biofilm formation and ECPase activity. Antibiotic susceptibility test was observed that wild and mutant strains were extremely susceptible to Amikacin, Minocycline, Gentamicin, Cefperazone; and resistant to oxacillin, clindamycin, ceftazidime. In contrast wild strains are sensitive to tetracycline, chloramphenicol, kanamycin, doxycycline, while mutant strains are resistant to them. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes, the results showed that compared with HY9901 wild type, ΔtyeA had increased expression of vscL, vscK, vscO, vopS, vopN, vscN, and hop. Following vaccination with the mutant strain, zebrafish had significantly higher survival than controls following infection with the wild-type HY9901 (71.2% relative percent survival; RPS). Analysis of immune gene expression by qPCR showed that vaccination with HY9901ΔtyeA increased the expression of IgM, IL-1β, IL-6, and TNF-α in zebrafish. This study provides evidence of protective efficacy of a live attenuated vaccine targeting the T3SS of V. alginolyticus which may be facilitated by up-regulated pro-inflammatory and immunoglobulin-related genes.

Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System.

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek’s disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

The nuclear localization signal-mediated nuclear targeting of herpes simplex virus 1 early protein UL2 is important for efficient viral production.

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and 12RRR14 was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

Overexpression of acetyl-CoA carboxylase in Aspergillus terreus to increase lovastatin production

The present work describes the application of homologous recombination techniques in a wild-type Aspergillus terreus (ATCC 20542) strain to increase the flow of precursors towards the lovastatin biosynthesis pathway. A new strain was generated to overexpress acetyl-CoA carboxylase (ACCase) by replacing the native ACCase promoter with a strong constitutive PadhA promoter from Aspergillus nidulans. Glycerol and a mixture of lactose and glycerol were used independently as the carbon feedstock to determine the degree of response by the A. terreus strains towards the production of acetyl-CoA, and malonyl-CoA. The new strain increased the levels of malonyl-CoA and acetyl-CoA by 240% and 14%, respectively, compared to the wild-type strain. As a result, lovastatin production was increased by 40% and (+)-geodin was decreased by 31% using the new strain. This study shows for the first time that the metabolism of Aspergillus terreus can be manipulated to attain higher levels of precursors and valuable secondary metabolites.