Plasmid Integration Research Articles

Evidence of Sharing of Carbapenem-Resistant Klebsiella pneumoniae Strains Between Intensive Care Unit Patients and the Environment.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) has emerged as an important public health threat. Intestinal colonization with CR-KP increases the risk of infection and death, especially in intensive care unit patients. To clarify the source of colonizing bacteria is very important to prevent the spread of CR-KP, so the purpose of this study was to explore the relationship between the ward environment and intestinal colonization of CR-KP. In this study, 353 environmental swabs from ICU (Intensive Care Unit) wards and 241 anal swab samples from ICU patients were collected and screened on MacConkey plates containing 2 μg/mL ertapenem, and the origin and genotype of CR-KP were analyzed by PCR and sequencing. The sequence type of the strains was also obtained by multi-locus sequence type (MLST) analysis, and plasmid conjugation test was used to clarify whether CR-KP can promote the transmission of drug resistance genes through plasmid integration and rearrangement. A total of 20 CR-KP environmental strains and 7 intestinal strains were obtained, most of which were bla OXA-48 resistant genotypes. Four different STs were identified by multi-locus sequence type (MLST) analysis, among which the large logarithm was ST485 type, and PFGE clustering showed that the similarity between them was >85%. In the plasmid transcoupling assay, we report that one of the Klebsiella pneumoniae drug-resistant plasmids was successfully transferred to E. coli, indicating that it may promote the spread of drug-resistant genes through plasmid integration and rearrangement. Our research suggests that the environment may be a potential source of CR-KP and that there is a need for us to adopt more effective disinfection measures.

Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene blaNDM-1, with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes blaNDM-1 and blaSHV-12, with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the blaNDM-1 promoter region can silence the entire blaNDM-1-bleMBL-trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene blaKPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.

Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome.

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.

A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active β-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus.

ABSTRACTIn a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to β-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosome.IMPORTANCE The cell wall of bacteria, namely, peptidoglycan, is the target of several antibiotic classes such as β-lactams. Staphylococcus aureus is well known for its capacity to adapt to antibiotic stress and develop resistance strategies, namely, to β-lactams. In this context, the construction of cell wall mutants provides useful models to study the development of such resistance mechanisms. Here, we characterized a collection of independent mutants, impaired in the same peptidoglycan biosynthetic step, obtained through the insertion of a plasmid in the coding region of murF gene. S. aureus demonstrated the capacity to overcome the cell wall damage by amplifying the copy number of the inserted plasmid, through an undescribed mechanism that involves the Rho transcription termination factor.

Deagglomeration of DNA nanomedicine carriers using controlled ultrasonication

Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of ∼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

Giardia duodenalis carries out canonical homologous recombination and single-strand annealing

In the past decades, the ability of Giardia duodenalis to perform homologous recombination has been suggested, supported by the observations of genomic integration of foreign plasmids and the disruption of genes using CRISPR technology. Unfortunately, the direct study of a HR mechanism has not been addressed, which would be pertinent in a minimalist organism lacking fundamental DNA-repair elements and even complete pathways. In addition, the constant ploidy changes through the life cycle of this parasite highlight the conservation and relevance of homologous recombination in maintaining genomic stability. In this research, we analyzed different recombinable plasmid systems and their outcomes after G. duodenalis transfection, using this approach we determined genomic, intra-plasmid and inter-plasmid recombination, moreover, we examined the presence of the non-conservative single-strand annealing pathway. With the intention of corroborating that the observed processes were done by homologous recombination, we used a chemical inhibitor named Mirin, which specifically inhibits Mre11 3′- 5′ exonuclease activity, one of the first steps involved in homologous recombination and fundamental to success in repairing. Overall, these results describe the multiple recombinational substrates used by G. duodenalis to achieve HR and demonstrate the presence and use of single-strand annealing recombination.

Tandem integration of circular plasmid contributes significantly to the expanded mitochondrial genomes of the green-tide forming alga Ulva meridionalis (Ulvophyceae, Chlorophyta).

Comparative mitogenomics of Ulva species have revealed remarkable variations in genome size due to the integration of exogenous DNA fragments, the proliferation of group I/II introns, and the change of repeat sequences. The genus Ulva is a species-rich taxonomic group, containing a variety of green-tide forming algae. In this study, five complete mitogenomes of the green-tide forming macroalga, Ulva meridionalis R. Horimoto and S. Shimada, were assembled and compared with the available ulvophyceae mtDNAs. The main circular mitogenomes of U. meridionalis ranged from 82.94 to 111.49 kb in size, and its 111.49-kb mitogenome was the largest Ulva mitogenome sequenced so far. The expansion of U. meridionalis mitogenomes is mainly due to the tandem integration of a 5.36-kb mitochondrial circular plasmid (pUme), as well as the proliferation of introns. An intact DNA-directed RNA polymerase gene (rpo) was present in pUme of U. meridionalis and was then detected in two putative plasmids (pUmu1 and pUmu2) found in Ulva mutabilis. The observed integration of the circular plasmid into U. meridionalis mitogenomes seems to occur via homologous recombination, and is a more recent evolutionary event. Many highly homologous sequences of these three putative plasmids can be detected in the other Ulva mtDNAs sequenced thus far, indicating the integration of different mitochondrial plasmid DNA into the mitogenomes is a common phenomenon in the evolution of Ulva mitogenomes. The random incidence of destruction of plasmid-derived rpos and open reading frames (orfs) suggests that their existence is not the original characteristic of Ulva mitogenomes and there is no selective pressure to maintain their integrity. The frequent integration and rapid divergence of plasmid-derived sequences is one of the most important evolutionary forces to shape the diversity of Ulva mitogenomes.

Abstract P1138: Combination Of Pluripotency And Cardiac Transcription Factors Reprogram Skin Fibroblasts Into Cardiomyocytes

Direct reprogramming of autologous somatic cells into cardiomyocytes is a novel approach which is being employed for cardiac regeneration. Multiple studies have combined different cardiac-specific factors that could directly reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, this approach needs heart biopsy for the isolation of autologous fibroblasts which involves extensive surgical procedures that require precision. In this study, we aimed to develop an approach which can directly reprogram somatic cells into cardiomyocyte-like cells using a combination of pluripotency and cardiac transcription factors. Skin fibroblasts were isolated from rat neonatal pups and were induced into cardiomyocyte-like cells using non-viral integration of cardiac transcription factors (GATA4, Mef2c and Nkx2.5) and OKSIM plasmid carrying the iPSC factors, Sox2, Oct4, Klf4 and cMyc. After 72 h, cells were analyzed for the expression of cardiac markers by qPCR and immunocytochemistry. Gene expression analysis showed significantly higher expression of cardiac markers GATA4, cMHC, Mef2c, cTnT, cTnI, and Nkx2.5 Immunostaining confirmed the expression of cardiac proteins GATA4, cMHC, cTnT, cTnI, and Nkx2.5. These results imply that the transfected cells started differentiating towards cardiac lineage. Transfected cells were also transplanted in rat myocardial infarction (MI) model immediately after ligation of left ventricle descending (LAD) artery. After 2 and 4 weeks of cell transplantation, the animals were assessed for cardiac function via echocardiography. They showed significant improvement in the cardiac function as compared to MI and non-transfected groups. After 4 weeks, the hearts were harvested, and histological analysis was performed for the assessment of fibrosis and left ventricular wall thickness. Rats transplanted with transfected cells showed significantly reduced fibrosis and increased wall thickness as compared to MI and non-transfected groups. In conclusion, transient and combined expression of cardiac transcription and iPSC factors in neonatal somatic cells leads to the transdifferentiation of skin cells into myogenic lineage. This approach can be useful for future therapeutic application for cardiovascular diseases.

Synthetic Saccharomyces cerevisiae tolerate and degrade highly pollutant complex hydrocarbon mixture

Fungal laccase (Lac) has become a very useful biocatalyst in different industries, bio-refineries and, most importantly, bioremediation. Many reports have also linked hydrocarbon tolerance and degradation by various microorganisms with Lac secretion. In this study, Trametes trogii Lac (Ttlcc1) was engineered into Saccharomyces cerevisiae strain CEN.PK2–1 C under the constitutive GPD promoter (pGPD) for multi-fold synthesis with efficient hydrocarbon tolerance and degradation. Protein expression in heterologous hosts is strictly strain-specific, it can also be influenced by the synthetic design and culture conditions. We compared synthetic designs with different shuttle vectors for the yeast strains and investigated the best culture conditions by varying the pH, temperature, carbon, nitrogen sources, and CuSO4 amount. Two S. cerevisiae strains were built in this study: byMM935 and byMM938. They carry the transcription unit pGPD-Ttlcc1-CYC1t either inside the pRSII406 integrative plasmid (byMM935) or the pRSII426 multicopy plasmid (byMM938). The performance of these two synthetic strains were studied by comparing them to the wild-type strain (byMM584). Both byMM935 and byMM938 showed significant response to different carbon sources (glucose, galactose, lactose, maltose, and sucrose), nitrogen sources (NH4Cl, NH4NO3, KNO3, malt extract, peptone, and yeast extract), and solid state fermentation of different plant biomasses (bagasse, banana peels, corn cob, mandarin peels, and peanut shells). They performed best in optimized growth conditions with specific carbon and nitrogen sources, and a preferred pH in the range 3.5–4.5, temperature between 30 and 40 0C, and 1 mM CuSO4. In optimized yeast-growth medium, strain byMM935 showed the highest laccase activities of 1.621 ± 0.063 U/mL at 64 h, whereas byMM938 gave its highest activity (1.417 ± 0.055 U/mL) at 48 h. In this work, we established, by using Bushnell Hass synthetic medium, that the new Ttlcc1-yeast strains tolerated extreme pH and complex hydrocarbon mixture (CHM) toxicity. They degraded 60–90% of the key components in CHM within 48 h, including poly-cyclic aromatic hydrocarbons, alkyl indenes, alkyl tetralines, alkyl benzenes, alkyl biphenyls, and BTEX (Benzene, Toluene, Ethylbenzene, and Xylenes). This is the first report on the hydrocarbon degradation potential of a Ttlcc1-yeast. Compared to the native organism, such synthetic strains are better suited for meeting growing demands and have potentials for application in large-scale in situ bioremediation of hydrocarbon-polluted sites.

A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.

Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus.

Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 103 in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 105 in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species.

Antimicrobial resistance determinants in silage

Animal products may play a role in developing and spreading antimicrobial resistance in several ways. On the one hand, residues of antibiotics not adequately used in animal farming can enter the human body via food. However, resistant bacteria may also be present in animal products, which can transfer the antimicrobial resistance genes (ARG) to the bacteria in the consumer’s body by horizontal gene transfer. As previous studies have shown that fermented foods have a meaningful ARG content, it is indicated that such genes may also be present in silage used as mass feed in the cattle sector. In our study, we aspired to answer what ARGs occur in silage and what mobility characteristics they have? For this purpose, we have analyzed bioinformatically 52 freely available deep sequenced silage samples from shotgun metagenome next-generation sequencing. A total of 16 perfect matched ARGs occurred 54 times in the samples. More than half of these ARGs are mobile because they can be linked to integrative mobile genetic elements, prophages or plasmids. Our results point to a neglected but substantial ARG source in the food chain.

Target lines for recombinase-mediated gene stacking in soybean.

Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking. For introgression of new transgenic traits to field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an in planta transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration. Here, we describe 5 soybean target lines from the screening of 118 Agrobacterium-mediated transgenic plants that were positive for gus expression. Each of the 5 target lines has a single copy of the transgenic DNA with precise DNA sequences of the recombinase recognition sites, located at least 1kb away from the nearest coding region, not close to the centromere, and showed good expression of the reporter gene. We tested Bxb1 integrase-mediated integration of a gfp-containing plasmid into each of these lines and showed precise site-specific integration in bombarded calluses. For plant regeneration, we used embryonic axes of mature soybean seeds to conduct a new set of biolistic transformation with a DsRed-containing plasmid. Three integration events were regenerated into whole plants, demonstrating the principle that target lines can serve as foundation lines for the stacking of DNA to predefined locations in the soybean genome.

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB′-EX1 is a nonreplicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB′-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and in the presence of isopropyl-1-thio-β-d-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB′-EX1 occurred, simultaneously. Through this procedure, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

LC_Glucose-Inhibited Division Protein Is Required for Motility, Biofilm Formation, and Stress Response in Lysobacter capsici X2-3.

Glucose-inhibited division protein (GidA) plays a critical role in the growth, stress response, and virulence of bacteria. However, how gidA may affect plant growth-promoting bacteria (PGPB) is still not clear. Our study aimed to describe the regulatory function of the gidA gene in Lysobacter capsici, which produces a variety of lytic enzymes and novel antibiotics. Here, we generated an LC_GidA mutant, MT16, and an LC_GidA complemented strain, Com-16, by plasmid integration. The deletion of LC_GidA resulted in an attenuation of the bacterial growth rate, motility, and biofilm formation of L. capsici. Root colonization assays demonstrated that the LC_GidA mutant showed reduced colonization of wheat roots. In addition, disruption of LC_GidA showed a clear diminution of survival in the presence of high temperature, high salt, and different pH conditions. The downregulated expression of genes related to DNA replication, cell division, motility, and biofilm formation was further validated by real-time quantitative PCR (RT–qPCR). Together, understanding the regulatory function of GidA is helpful for improving the biocontrol of crop diseases and has strong potential for biological applications.

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu.

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.