Development Of New Genetic Tools Research Articles

New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

The emergence of antibiotic-resistant Klebsiella pneumoniae is a significant global health threat that has led to increased morbidity and mortality. This resistance also hinders basic research, as many strains are no longer susceptible to antibiotics commonly used in microbial genetics. Addressing this requires the development of new genetic tools with alternative selective markers. In this report, we introduce new allelic exchange vectors for use in drug-resistant strains. These vectors feature a conditional R6K origin of replication, an origin of transfer, SacB counter-selection, and alternative selectable markers. We validated the vectors by generating unmarked deletions in the K. pneumoniae KPPR1S bla (β-lactamase) and lacZ (β-galactosidase) genes. During this process, we defined optimized conditions for SacB-mediated allelic exchange in KPPR1S, significantly enhancing the efficiency of mutant generation. Furthermore, we demonstrated that lacZ is dispensable for virulence and that the lacZ mutant can serve as a surrogate for wild-type strains in competition assays using the Galleria mellonella infection model. Our findings provide new tools for the efficient genetic manipulation of K. pneumoniae and other drug-resistant bacteria.

Whole-genomic DNA amplifications from individually isolated sweet sorghum microspores.

Sorghum is a multi-use crop, the efficient breeding of which requires the development of new genetic tools. One such tool could be the genetic assessment of free microspores, which are released just after the tetrad stage of pollen development. Microspores are ideal for DNA isolation as they have underdeveloped cell walls and can be readily lysed as natural protoplasts. Four cultivars of Sorghum bicolor ('Achi Turi', 'Dale', 'Local', and 'Topper 76-6') were grown in a greenhouse until flowering (7.7-11.5 cm flag leaf internode length), after which 30 immature microspores were isolated from each line. Plant height, time to flowering, boot radius, and spikelet maturation were recorded for each cultivar. The exine development of the microspores was observed under an inverted Nikon microscope, and those with underdeveloped exine were subjected to whole-genome amplification and sequencing. Microspores in the early uninucleate to early binucleate stages had underdeveloped exine, and were therefore ideal for DNA extraction. High-quality DNA was obtained from these single-cell gametophytes. The average DNA concentration was 2902 ng/µL, with fragment sizes comparable to those obtained from leaf tissue extractions. Harvesting panicles with immature microspores means the entire gametic population is accessible for DNA analyses. This is the first amplification of whole-genome DNA fragments from sorghum single-cell microspores isolated during gametogenesis.

Transcriptome analysis reveals infection strategies employed by Fusarium graminearum as a root pathogen

The fungal pathogen Fusarium graminearum (Fg) infects both heads and roots of cereal crops causing several economically important diseases such as head blight, seedling blight, crown rot and root rot. Trichothecene mycotoxins such as deoxynivalenol (DON), a well-known virulence factor, produced by Fg during disease development is also an important health concern. Although how Fg infects above-ground tissues is relatively well studied, very little is known about molecular processes employed by the pathogen during below-ground infection. Also unknown is the role of DON during root infection. In the present study, we analyzed the transcriptome of Fg during root infection of the model cereal Brachypodium distachyon (Bd). We also compared our Fg transcriptome data obtained during Bd root infection with those reported during wheat head infection. These analyses suggested that both shared and unique infection strategies were employed by the pathogen during colonization of different host tissues. Several metabolite biosynthesis genes induced in Fg during root infection could be linked to phytohormone production, implying that the pathogen likely interferes with root specific defenses. In addition, to understand the role of DON in Fg root infection, we analyzed the transcriptome of the DON deficient Tri5 mutant. These analyses showed that the absence of DON had a significant effect on fungal transcriptional responses. Although DON was produced in infected roots, this mycotoxin did not act as a Fg virulence factor during root infection. Our results reveal new mechanistic insights into the below-ground strategies employed by Fg that may benefit the development of new genetic tools to combat this important cereal pathogen.

The genetic architecture of target-site resistance to pyrethroid insecticides in the African malaria vectors Anopheles gambiae and Anopheles coluzzii.

Resistance to pyrethroid insecticides is a major concern for malaria vector control. Pyrethroids target the voltage‐gated sodium channel (VGSC), an essential component of the mosquito nervous system. Substitutions in the amino acid sequence can induce a resistance phenotype. We use whole‐genome sequence data from phase 2 of the Anopheles gambiae 1000 Genomes Project (Ag1000G) to provide a comprehensive account of genetic variation in the Vgsc gene across 13 African countries. In addition to known resistance alleles, we describe 20 other non‐synonymous nucleotide substitutions at appreciable population frequency and map these variants onto a protein model to investigate the likelihood of pyrethroid resistance phenotypes. Thirteen of these novel alleles were found to occur almost exclusively on haplotypes carrying the known L995F kdr (knock‐down resistance) allele and may enhance or compensate for the L995F resistance genotype. A novel mutation I1527T, adjacent to a predicted pyrethroid‐binding site, was found in tight linkage with V402L substitutions, similar to allele combinations associated with resistance in other insect species. We also analysed genetic backgrounds carrying resistance alleles, to determine which alleles have experienced recent positive selection, and describe ten distinct haplotype groups carrying known kdr alleles. Five of these groups are observed in more than one country, in one case separated by over 3000 km, providing new information about the potential for the geographical spread of resistance. Our results demonstrate that the molecular basis of target‐site pyrethroid resistance in malaria vectors is more complex than previously appreciated, and provide a foundation for the development of new genetic tools for insecticide resistance management.

Genetic Manipulation of Non-tuberculosis Mycobacteria.

Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms.

A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis.

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

Modulating Dopamine Signaling and Behavior with Chemogenetics: Concepts, Progress, and Challenges.

For more than 60 years, dopamine (DA) has been known as a critical modulatory neurotransmitter regulating locomotion, reward-based motivation, and endocrine functions. Disturbances in DA signaling have been linked to an array of different neurologic and psychiatric disorders, including Parkinson's disease, schizophrenia, and addiction, but the underlying pathologic mechanisms have never been fully elucidated. One major obstacle limiting interpretation of standard pharmacological and transgenic interventions is the complexity of the DA system, which only appears to widen as research progresses. Nonetheless, development of new genetic tools, such as chemogenetics, has led to an entirely new era for functional studies of neuronal signaling. By exploiting receptors that are engineered to respond selectively to an otherwise inert ligand, so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), chemogenetics enables pharmacological remote control of neuronal activity. Here we review the recent, extensive application of this technique to the DA field and how its use has advanced the study of the DA system and contributed to our general understanding of DA signaling and related behaviors. Moreover, we discuss the challenges and pitfalls associated with the chemogenetic technology, such as the metabolism of the DREADD ligand clozapine N-oxide (CNO) to the D2 receptor antagonist clozapine. We conclude that despite the recent concerns regarding CNO, the chemogenetic toolbox provides an exceptional approach to study neuronal function. The huge potential should promote continued investigations and additional refinements to further expound key mechanisms of DA signaling and circuitries in normal as well as maladaptive behaviors.

Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga Coccomyxa sp. strain KJ

BackgroundOxygen-evolving photosynthetic microorganisms, collectively termed as microalgae, are gaining attention as alternative fuel sources. The unicellular alga Coccomyxa sp. strain KJ that belongs to the class Trebouxiophyceae can grow rapidly in minimal mineral media and accumulate triacylglycerols at levels > 60% (w/w) of its dry weight under nitrogen depletion conditions. Thus, the strain can be a good candidate for biofuel production. Still, substantial improvements in lipid productivity and other traits of this strain are needed to meet commercial production requirements. Consequently, the development of new genetic tools including genome editing that are applicable to this strain is highly desired.ResultsIn this paper, we report successful genome editing of strain KJ by intracellular delivery of a ribonucleoprotein complex comprising recombinant Cas9 protein and guide RNA. For introduction of Cas9-guide RNA ribonucleoprotein into strain KJ cells, we used an electroporator with a short (2.5 ms) electric pulse at a high field strength (7500 V cm−1) followed by multiple 50-ms electric pulses at low field strength (250 V cm−1). Under these conditions, we successfully isolated several knockout lines of the FTSY gene of strain KJ, encoding a signal recognition particle-docking protein at a frequency of 0.01%.ConclusionsOur study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.

Resistance to coronavirus infection in amino peptidase N-deficient pigs

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.

Sensor-regulator and RNAi based bifunctional dynamic control network for engineered microbial synthesis

Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use. In this study, we present a sensor-regulator and RNAi-based bifunctional dynamic control network that can provide simultaneous upregulation and downregulation of cellular metabolism for engineered biosynthesis. The promoter-regulator-mediated upregulation function and its transduced downregulation function through RNAi are systematically verified and characterized. We apply this dynamic control network to regulate the phosphoenolpyruvate metabolic node in Escherichia coli and achieve autonomous distribution of carbon flux between its native metabolism and the engineered muconic acid biosynthetic pathway. This allows muconic acid biosynthesis to reach 1.8 g L−1. This study also suggests the circumstances where dynamic control approaches are likely to take effects.

Genomic Effects of the Vitamin D Receptor: Potentially the Link between Vitamin D, Immune Cells, and Multiple Sclerosis.

Vitamin D has a plethora of functions that are important for the maintenance of general health and in particular, the functional integrity of the immune system, such as promoting an anti-inflammatory cytokine profile and reducing the Treg/Th17 ratio. Multiple sclerosis (MS) is a chronic, inflammatory, and neurodegenerative central nervous system (CNS) disorder of probable autoimmune origin. MS is characterized by recurring or progressive demyelination and degeneration of the CNS due in part to a misguided immune response to as yet undefined (CNS) antigens, potentially including myelin basic protein and proteolipid protein. MS has also been shown to be associated significantly with environmental factors such as the lack of vitamin D. The role of vitamin D in the pathogenesis and progression of MS is complex. Recent genetic studies have shown that various common MS-associated risk-single-nucleotide polymorphisms (SNPs) are located within or in the vicinity of genes associated with the complex metabolism of vitamin D. The functional aspects of these genetic associations may be explained either by a direct SNP-associated loss- or gain-of-function in a vitamin D-associated gene or due to a change in the regulation of gene expression in certain immune cell types. The development of new genetic tools using next-generation sequencing: e.g., chromatin immunoprecipitation sequencing (ChIP-seq) and the accompanying rapid progress of epigenomics has made it possible to recognize that the association between vitamin D and MS could be based on the extensive and characteristic genomic binding of the vitamin D receptor (VDR). Therefore, it is important to analyze comprehensively the spatiotemporal VDR binding patterns that have been identified using ChIP-seq in multiple immune cell types to reveal an integral profile of genomic VDR interaction. In summary, the aim of this review is to connect genomic effects vitamin D has on immune cells with MS and thus, to contribute to a better understanding of the influence of vitamin D on the etiology and the pathogenesis of this complex autoimmune disease.

Magnetoreception-A sense without a receptor.

Evolution has equipped life on our planet with an array of extraordinary senses, but perhaps the least understood is magnetoreception. Despite compelling behavioral evidence that this sense exists, the cells, molecules, and mechanisms that mediate sensory transduction remain unknown. So how could animals detect magnetic fields? We introduce and discuss 3 concepts that attempt to address this question: (1) a mechanically sensitive magnetite-based magnetoreceptor, (2) a light-sensitive chemical-based mechanism, and (3) electromagnetic induction within accessory structures. In discussing the merits and issues with each of these ideas, we draw on existing precepts in sensory biology. We argue that solving this scientific mystery will require the development of new genetic tools in magnetosensitive species, coupled with an interdisciplinary approach that bridges physics, behavior, anatomy, physiology, molecular biology, and genetics.

Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii

Increasing interest in homoacetogenic bacteria for the production of biochemicals and biofuels requisites the development of new genetic tools for these atypical production organisms. An attractive host for the conversion of synthesis gas or electricity into multi-carbon compounds is Clostridium ljungdahlii. So far only limited achievements in modifying this organism towards the production of industrially relevant compounds have been made. Therefore, there is still a strong need for developing new and optimizing existing genetic tools to efficiently access its metabolism. Here, we report on the development of a stable and reproducible transformation protocol that is applicable to C. ljungdahlii and several other clostridial species. Further, we demonstrate the functionality of a temperature-sensitive origin of replication in combination with a fluorescence marker system as important tools for future genetic engineering of this host for microbial bioproduction.

Meiotic maps of sockeye salmon derived from massively parallel DNA sequencing

BackgroundMeiotic maps are a key tool for comparative genomics and association mapping studies. Next-generation sequencing and genotyping by sequencing are speeding the processes of SNP discovery and the development of new genetic tools, including meiotic maps for numerous species. Currently there are limited genetic resources for sockeye salmon, Oncorhynchus nerka. We develop the first dense meiotic map for sockeye salmon using a combination of novel SNPs found in restriction site associated DNA (RAD tags) and SNPs available from existing expressed sequence tag (EST) based assays.ResultsWe discovered and genotyped putative SNPs in 3,430 RAD tags. We removed paralogous sequence variants leaving 1,672 SNPs; these were combined with 53 EST-based SNP genotypes for linkage mapping. The map contained 29 male and female linkage groups, consistent with the haploid chromosome number expected for sockeye salmon. The female map contains 1,057 loci spanning 4,896 cM, and the male map contains 1,118 loci spanning 4,220 cM. Regions of conservation with rainbow trout and synteny between the RAD based rainbow trout map and the sockeye salmon map were established.ConclusionsUsing RAD sequencing and EST-based SNP assays we successfully generated the first high density linkage map for sockeye salmon.

Recent Development and Future Perspective of Antitubercular Therapy

Tuberculosis (TB) has remained an enemy of humankind before the beginning of recorded history. Although it is curable and preventable, TB claims the lives of more than 5,000 people every day. Despite of availability of effective chemotherapy and BCG vaccine in 21st century, TB remains one of the most deadly infectious diseases. The concomitant resurgence of TB with the Multi-drug resistant (MDR) or Extremely drug resistant (XDR)-TB and HIV/AIDS pandemic raises the threat posed by untreatable and fatal human TB, exposing the frailties of the current drug armatorium. No new drug is available acting through novel mechanism since last 40 years. The genome of Mycobacterium tuberculosis H37Rv was sequenced in 1998 and reannotated in 2002. This coupled with development of new genetic tools, have greatly contributed to the discovery of potential drug targets for new anti-TB agent. It is increasingly acknowledged that new drugs should not only be active against drug resistant TB, but should also kill persisters and shorten the lengthy TB treatment, which underlies the problem of drug resistance due to poor compliance to the length of therapy. This review article describes the current TB drugs, their merits and demerits as well as the new promising anti-TB agents classified on the basis of molecular framework. It is a comprehensive literature compilation on the present research paradigm of anti-TB drug discovery including advances in the new structural analogs reported in last decade.

The Krüppel-associated Box Repressor Domain Can Trigger de Novo Promoter Methylation during Mouse Early Embryogenesis

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.

Current Strategies for Identifying and Validating Targets for New Treatment-Shortening Drugs for TB

There is an urgent need for new drugs to treat tuberculosis. During the last forty years the only drugs to have been developed are variations on existing ones, but new drug candidates must offer improvements over existing agents. In particular, we require new drugs having novel mechanisms of action that are active against drug-resistant strains and also kill persistent bacilli, thus shortening the length of chemotherapy. Recent advances in our understanding of the biology of Mycobacterium tuberculosis, in particularly the availability of the genome sequence coupled with development of new genetic tools, have greatly contributed to the discovery of potential drug targets for new antituberculars. However, although many potential new drug targets have been identified, greater effort is required in target validation to show properly that they are essential for bacterial growth and survival. In this review, the current drug development pipeline and the strategies employed to identify and validate novel tuberculosis drug targets are presented.

The Lactic Acid Bacteria Genome Project

ABSTRACT: In 2002, the Joint Genome Institute and the Lactic Acid Bacteria Genome Consortium (LABGC) released draft genome sequences for 11 food fermentation‐related bacteria including: Lactococcus lactis ssp. cremoris, Lactobacillus gasseri, Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbrueckii ssp. bul‐garicus, Streptococcus thermophilus, Leuconostoc mesenteroides, Pediococcus pentosaceus, Oenococcus oeni, Bifidobacterium longum, and Brevibacterium linens. Public availability of these aggregate sequence data will allow researchers to examine these microbes, in the context of food and beverage production, in new detail. Sequence annotation of these strains is ongoing and comparisons between the lactic acid bacteria will shed light on those genetic programs conserved across a range of fermentation niches. Future functional genomic approaches (transcriptomics, proteomics, and metabolomics) will help define the molecular nature of metabolic networks within these microbes. This knowledge, aided by the development of new genetic tools, will better enable the selection and/or tailoring of strains for various fermentation or probiotic rationales.

SCE Jumping: Genetic Tool for Allelic Exchange in Bacteria

As more microbial genome sequence information becomes available, the field of bacterial pathogenesis would benefit from the development of new genetic tools designed to facilitate gene function studies on a genomic scale. The complete DNA sequence of the bacterium Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major opportunistic human pathogen. Here, I describe the development of a new gene replacement scheme termed "SCE jumping" in P. aeruginosa. The system uses the yeast I-SceI homing endonuclease in conjunction with in vitro mariner-transposon mutagenesis to generate mutations within targeted regions of the chromosome for genetic footprinting. Use of SCE jumping for generating transposon insertion mutants is anticipated to be widely applicable to other bacterial organisms. This allelic exchange strategy is discussed in context with other methods of gene replacement strategies available in P. aeruginosa. Development of SCE jumping provides an excellent example of the power of importing systems from unrelated organisms to circumvent practical challenges in molecular genetics.

Selection of an Escherichia coli host that expresses mutant forms of Mycobacterium tuberculosis 2- trans enoyl-ACP(CoA) reductase and 3-ketoacyl-ACP(CoA) reductase enzymes

Tuberculosis (TB) still remains a worldwide health concern. Efforts to understand the complex biology of Mycobacterium tuberculosis, the causative agent of TB, are important for new antitubercular drug development. Despite the completion of the genome sequence and the development of new genetic tools to manipulate this organism, the availability of sufficient amounts of mycobacterial proteins still remains an essential and laborious step to study the biochemical features of this pathogen. The T7-RNA polymerase-based pET system has been largely employed to express mycobacterial proteins in Escherichia coli, but it presents some limitations. To overcome problems with unstable expression of an M. tuberculosis inhA-encoded enoyl reductase mutant protein and lack of expression of two mabA-encoded ketoacyl reductase mutants, a sub-population of E. coli BL21(DE3) host cells was selected from a small-opaque colony. This empirically selected host, named BL21(DE3)NH, allowed stable expression of these mutant proteins. Although the mechanism that led the BL21(DE3)NH host to express the recombinant mutant proteins remains unknown, the persistent phenotype points to a stable genetic switch. This genetic alteration resulted in a tight control of the highly processive T7 RNA polymerase. Moreover, the absolute requirement for IPTG to obtain protein expression in the BL21(DE3)NH host cells suggests that no inherent defect in the transcriptional activity of the T7 promoter is present. Empirical host selection requires no further genetic manipulation of recombinant plasmids and may represent a means of obtaining tailor-made E. coli strains that overcome toxic effects associated with heterologous protein expression.