Inducible Promoter Research Articles

A Tunable and Expandable Transactivation System in Probiotic Yeast Saccharomyces boulardii.

Precise transcriptional modulation is a key requirement for developing synthetic probiotics with predictably tunable functionalities. In this study, an expandable and tunable transactivation system was constructed and validated in probiotic yeast Saccharomyces boulardii. The use of nuclease-null Cas9 and scaffold RNA (scRNA) directed regulation enabled transactivation under the control of a synthetic promoter in S.boulardii. A synthetic promoter consisting of the scRNA target sequence and the core GAL7 promoter region restricted interference from the native galactose regulon. The system was readily expanded by introducing new target sequences to the promoter and scRNA. Complementarity between the promoter and scRNA, and binding specificity between scRNA and transcriptional activator, served as two layers of orthogonality of the transactivation. In addition, activator expression under the control of an inducible promoter enabled control of the transactivation via chemical inducer. The described system has the potential to enable engineering of probiotic yeast to more precisely perform therapeutic functions.

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells.

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (PPDH, PPYK, PFBA, PPGM, PGLK, PTRI, PGPI, PADH1, PTEF1 and PGCW14) were obtained with the activity range of 13%–130% of the common promoter PGAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which PPDH and PGCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced PICL1, rhamnose-induced PLRA3 and PLRA4, and a bidirectional promoter (PMal-PPer) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter PLRA3 by 4.7–10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens.

Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/P hpdH ) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%–100%) to that of the widely used strong promoters such as P mxaF and P L/O4 . The HpdR/P hpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.

Development of a Highly Efficient Copper-Inducible GAL Regulation System (CuIGR) in Saccharomyces cerevisiae.

Dynamic regulation of gene expression to decouple growth and production has been proven to be effective for improving the biosynthetic efficiency of microbial cell factories. However, the number of efficient regulatory systems available for regulation of Saccharomyces cerevisiae is limited. In the present study, a novel copper-inducible gene expression system (CuIGR) composed of the copper-induced transcriptional activator Gal4 and the copper-inhibited repressor Gal80 was constructed in S.cerevisiae. When Gal80 was fused with a N-degron tag (K15), the resulting CuIGR4 system exhibited the most stringent regulation of gene expression driven by GAL1/2/7/10 promoters. As compared to the native Cu2+-inducible CUP1 promoter, the CuIGR4 system amplified the response to copper by as much as 2.7 folds, resulting in 72-fold induction of EGFP expression and a 33-fold change in lycopene production (3-100 mg/L) with addition of 20 μM copper. This newly developed copper-inducible system provides a powerful tool for gene expression control in S.cerevisiae, which is expected to be widely applicable in the regulation of yeast cell factories for enhanced biosynthesis of valuable products.

Chemical biology-whole genome engineering datasets predict new antibacterial combinations.

Trimethoprim and sulfamethoxazole are used commonly together as cotrimoxazole for the treatment of urinary tract and other infections. The evolution of resistance to these and other antibacterials threatens therapeutic options for clinicians. We generated and analysed a chemical-biology-whole-genome data set to predict new targets for antibacterial combinations with trimethoprim and sulfamethoxazole. For this we used a large transposon mutant library in Escherichia coli BW25113 where an outward-transcribing inducible promoter was engineered into one end of the transposon. This approach allows regulated expression of adjacent genes in addition to gene inactivation at transposon insertion sites, a methodology that has been called TraDIS-Xpress. These chemical genomic data sets identified mechanisms for both reduced and increased susceptibility to trimethoprim and sulfamethoxazole. The data identified that over-expression of FolA reduced trimethoprim susceptibility, a known mechanism for reduced susceptibility. In addition, transposon insertions into the genes tdk, deoR, ybbC, hha, ldcA, wbbK and waaS increased susceptibility to trimethoprim and likewise for rsmH, fadR, ddlB, nlpI and prc with sulfamethoxazole, while insertions in ispD, uspC, minC, minD, yebK, truD and umpG increased susceptibility to both these antibiotics. Two of these genes’ products, Tdk and IspD, are inhibited by AZT and fosmidomycin respectively, antibiotics that are known to synergise with trimethoprim. Thus, the data identified two known targets and several new target candidates for the development of co-drugs that synergise with trimethoprim, sulfamethoxazole or cotrimoxazole. We demonstrate that the TraDIS-Xpress technology can be used to generate information-rich chemical-genomic data sets that can be used for antibacterial development.

The study of biological activity of mandelic acid and its alkali metal salts in wastewaters

In the present work we compared the biological activity of mandelic acid (MA) and its Li, Na, K, Rb and Cs salts. The study also investigated the effect of raw wastewaters (RW) and treated wastewaters (TW), comparable to microbial medium (MM) on the biological activity of the tested chemical compounds used in concentrations of 5; 2.5; 1.25; 0.625; 0.3125 mg/ml. In the present experiment the evaluation of the following parameters was performed: E. coli (ATCC 25922) cells viability, growth inhibition of E. coli (ATCC 25922), the inhibition of GFP protein, genotoxicity and ROS generation. Our results showed that three main factors differentiated the antibacterial activity of MA and its Li, Na, K, Rb and Cs salts: study environment (MM, RW, TW), metal forming salt of mandelic acid and concentration of tested compounds. Additionally, raw and treated wastewater, compared to microbial medium, changes the antimicrobial activity of MA and its salts in relation to the E. coli strain. We also detected that both MA and its salts affect the GFP protein and the induction of the recA promoter (genotoxicity test). The activity of the tested salts in relation to these two parameters is strictly dependent on the type of salt-forming metal and the concentration used. The analysis of ROS synthesis suggests that in the majority of the studied mandelic acid salts, oxidative stress is the dominant mechanism of cytotoxicity and genotoxicity. We also showed that both raw wastewaters (RW) and treated wastewaters (TW), compared to microbial medium (MM), change significantly the activity of MA and its salts.

Independent control of mean and noise by convolution of gene expression distributions

Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from individual promoters. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.

Poplar Allene Oxide Synthase 1 Gene Promoter Drives Rapid and Localized Expression by Wounding

Promoters play critical roles in controlling the transcription of genes and are important as tools to drive heterologous expression for biotechnological applications. In addition to core transcription factor-binding motifs that assist in the binding of RNA polymerases, there are specific nucleotide sequences in a promoter region to allow regulation of gene expression. The allene oxide synthase (AOS) gene family are cytochrome P450s that are responsive to a variety of environmental stress, making them good candidates for the discovery of inducible promoters. Populus AOS homologs separate phylogenetically into two clades. Based on the 19 promoter motifs with significant abundance differences between the two clades, Clade I AOS genes are likely more responsive to hormones, salt, and pathogen, whereas clade II homologs are likely inducible by water stress. In this study, an upstream promoter from a Clade I poplar AOS encoding gene (AOS1) was cloned and used to drive the expression of a ß-glucuronidase (GUS) gene in Arabidopsis. AOS is an essential enzyme in the lipoxygenase pathway that is responsible for the production of many non-volatile oxylipins in plants, including the jasmonates, which are regulatory phytohormones coordinating a variety of biological and stress response functions. Consistent with AOS transcript expression patterns, we found that the poplar AOS1 promoter drives rapid and localized expression by wounding. The study provides insight on the responsive elements in the poplar AOS promoters, but more importantly identifies a strong wound-inducible and localized promoter for future applications.

ABA-INSENSITIVE 3 with or without FUSCA3 highly up-regulates lipid droplet proteins and activates oil accumulation.

ABA-INSENSITIVE 3 (ABI3) has long been known for activation of storage protein accumulation. A role of ABI3 on oil accumulation was previously suggested based on a decrease of oil content in seeds of abi3 mutant. However, this conclusion could not exclude possibilities of indirect or pleiotropic effects, such as through mutual regulatory interactions with FUSCA3 (FUS3), an activator of oil accumulation. To identify that ABI3 functions independent of the effects of related seed transcription factors, we expressed ABI3 under the control of an inducible promoter in tobacco BY2 cells and Arabidopsis rosette leaves. Inducible expression of ABI3 activated oil accumulation in these non-seed cells, demonstrating a general role of ABI3 in regulation of oil biosynthesis. Further expressing ABI3 in rosette leaves of fus3 knockout mutant still caused up to 3-fold greater triacylglycerol accumulation, indicating ABI3 can activate lipid accumulation independently of FUS3. Transcriptome analysis revealed that LIPID DROPLET PROTEIN (LDP) genes, including OLEOSINs and CALEOSINs, were up-regulated up to 1000-fold by ABI3 in the absence of FUS3, while the expression of WRINKLED1 was doubled. Taken together, our results provide genetic evidence that ABI3 activates oil accumulation with or without FUS3, most likely through up-regulating LDPs and WRINKLED1.

Pathogen-induced expression of a blight tolerance transgene inAmerican chestnut.

American chestnut (Castanea dentata) is a susceptible host of the invasive necrotrophic fungus Cryphonectria parasitica, which causes chestnut blight disease. The fungal pathogen attacks chestnut stems by invading wounded tissue and secreting oxalate. This process leads to the death of infected host cells and the formation of cankers, eventually girdling stems and killing the tree above the infections. To reduce damage caused by fungal oxalate, American chestnut has been genetically engineered to express a wheat oxalate oxidase (OxO). This enzyme degrades the oxalate produced by the pathogen and confers elevated tolerance to Cryphonectria parasitica infection. We report new lines of transgenic American chestnut that have been developed with the win3.12 inducible promoter from poplar (Populus deltoides) driving OxO expression. This promoter is responsive to both wounding and pathogen infection, with a low level of baseline expression. Targeted expression of OxO to wounded and infected tissue is sought as an alternative to constitutive expression for potential metabolic resource conservation and transgene stability over the long lifetime of a tree and over successive generations of breeding. Transgenic Castanea dentata lines harbouring the win3.12‐OxO construct were evaluated for transgene expression patterns and tolerance to chestnut blight infection. OxO transcript levels were low in uninfected plants, but robust infection‐induced expression levels were observed, with one transgenic line reaching levels comparable to those of previously characterized CaMV35S‐OxO lines. In chestnut blight infection bioassays, win3.12‐OxO lines showed elevated disease tolerance similar to blight‐resistant Chinese chestnut (Castanea mollissima) controls.

A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application.

Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (P tac or P trc ) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolR q cassette and the myo-inositol-inducible promoter P iolT1 . The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemA M and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

Mds1 CreERT2-Based Lineage-Tracing Reveals Increasing Contributions of HSCs to Fetal Hematopoiesis and to Adult Tissue-Resident Macrophages in the Marrow

The ontogeny of the hematopoietic system consists of two broad programs. The first, an HSC-independent program, consists of overlapping waves of primitive, erythro-myeloid (EMP), and some lymphoid progenitors. HSC-independent hematopoiesis is required for normal fetal development, and provides self-renewing tissue-resident macrophage populations that persist in the adult. This is followed by the emergence of an HSC-dependent program that arises from arterial vessels within the body of the embryo. The overlapping emergence and lineage output of HSC-independent and HSC-derived hematopoiesis raises important questions regarding the identity and potential functional differences of their mature progeny. However, the transition from HSC-independent to HSC-derived hematopoiesis in the murine fetus remains incompletely characterized, particularly since the maturing erythroid, megakaryocytic and myeloid progeny of EMP and HSCs are currently not easily distinguishable. Additionally, lineage-tracing approaches have been challenging because they have relied largely on the temporal induction of promoters that are expressed both in HSC-independent progenitors and in HSCs, which have significant temporal overlap in their developmental emergence and result in incomplete or in mixed labeling. To help resolve this question, we have developed Mds1 CreERT2 mice, utilizing the first transcription start site of MECOM gene, which is expressed in HSC and emerging HSC (Yuasa et al., 2005 EMBO; Hou et al. 2020 Cell Research; Zhu et al. 2020, Blood). When mated with Rosa-YFP reporter mice and induced at E9.5 with tamoxifen, this construct lineage-traces pre-HSCs present in the E11.5 AGM region, as well as HSCs in the fetal liver and adult marrow. Importantly, no labeling of primitive erythroid cells, primitive macrophage-derived microglia, EMP, or EMP-derived cells in the E11.5 or E12.5 fetal liver was detected with tamoxifen induction at either E9.5 or E8.5. Analysis of E9.5 tamoxifen-treated Mds1 CreERT2Rosa26 LSL-YFP embryos indicates that HSCs have begun to generate small numbers of differentiating erythroid, myeloid and lymphoid progeny in the liver between E12.5 and E14.5. By E16.5, a significant proportion of differentiating erythroid, myeloid and B-cell lineage cells in the liver are HSC-derived, and HSCs have now begun to contribute erythroid and myeloid cells to the rapidly expanding pool of circulating blood cells. In the adult, we found increasing contributions of HSCs to macrophages in liver, lung and kidney. Interestingly, the majority of F4/80+ cells in the adult bone marrow and spleen were also lineage-traced in these mice. Thus, HSCs ultimately provide the majority of adult marrow macrophages that go on to self-maintain in the adult marrow (Hashimoto et al., 2013, Immunity). The Mds1 CreERT2 mouse model will serve as a useful to deconvolute the complexity of hematopoiesis as it unfolds in the embryo and functions postnatally. DisclosuresPalis: Rubius Therapeutics: Consultancy.

Generation of an NFκB-Driven Alpharetroviral "All-in-One" Vector Construct as a Potent Tool for CAR NK Cell Therapy.

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen—so-called “T cells redirected for universal cytokine-mediated killing” (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.

Sense and sensibility: of synthetic biology and the redesign of bioreporter circuits.

Sense and sensibility: of synthetic biology and the redesign of bioreporter circuits.

A dual prokaryotic (E. coli) expression system (pdMAX).

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

Overexpression approaches to advance understanding of Candida albicans

Candida albicans is an opportunistic fungal pathogen that is responsible for infections linked to high mortality. Loss‐of‐function approaches, taking advantage of gene knockouts or inducible down‐regulation, have been successfully used in this species in order to understand gene function. However, overexpression of a gene provides an alternative, powerful tool to elucidate gene function and identify novel phenotypes. Notably, overexpression can identify pathway components that might remain undetected using loss‐of‐function approaches. Several repressible or inducible promoters have been developed which allow to shut off or turn on the expression of a gene in C. albicans upon growth in the presence of a repressor or inducer. In this review, we summarize recent overexpression approaches used to study different aspects of C. albicans biology, including morphogenesis, biofilm formation, drug tolerance, and commensalism.

Lactose Inducible Expression of Transcription Factor Gene SEF1 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin (vitamin B2) is required for synthesis of the flavin coenzymes: riboflavin-5’-phosphate (flavin mononucleotide) and flavin adenine dinucleotide. Riboflavin is important biotechnological commodity with annual market around 250 million US dollars. It is mostly used as component of feed premixes for animals (80%), in food industry as food colorant, in medicine and component of multivitamin mixtures and as drug for treatment of some diseases. Over the past two decades, the microbial production of riboflavin by fermentation completely replaces the chemical synthetic route. The main producers of riboflavin in industry are engineered strains of the bacterium Bacillus subtilis and of the mycelial fungus Ashbya gossypii. Flavinogenic yeast Candida famata has great biosynthetic potential. Using combination of classical selection and metabolic engineering (overexpression of SEF1, RIB1 and RIB7 genes coding the positive regulator, the first and the last structural enzymes of riboflavin synthesis) resulted in the construction of genetically stable strain of C. famata that produces 16 gram of riboflavin per liter in bioreactor. However, the productivity of riboflavin biosynthesis remains still insufficient for industrial production of this vitamin. Studies of transcriptional regulation of genes involved in riboflavin synthesis and using of strong promoters of C. famata for construction of efficient producers of vitamin B2 are areas of both scientific and industrial interest. Aim. The aim of the current work was to improve riboflavin oversynthesis by the available C. famata strains in synthetic and natural lactose-containing media. Methods. The plasmid DNA isolation, restriction, ligation, electrophoresis in agarose gel, electrotransformation, and PCR were carried out by the standard methods. Riboflavin was assayed fluorometrically using solution of synthetic riboflavin as a standard. The cultivation of yeasts was carried out in YNB or YPD media containing different source of carbon and on whey. Results. The strains of C. famata expressed additional copy of central regulatory gene SEF1 under control of the promoter of LAC4 gene (coding for β–galactosidase) C. famata were constructed. The influence of SEF1 gene expression under control of lactose inducible promoter of CfLAC4 gene on riboflavin production was studied. It was shown that the C. famata strains containing “pLAC4_cf-SEF1_cf” expression cassette revealed 1.6-2.1-fold increase in riboflavin yield on lactose when compared to the parental strain. The riboflavin production constructed strains on whey reached 1.69 gram per liter in flask batch culture. Conclusions. The constructed strains containing additional copy of SEF1 gene under the control of LAC4 promoter is a perfect platform for development of industrial riboflavin production on by-product of dairy industry, whey.

An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. coli

CRISPR-interference (CRISPRi) is a powerful genetic tool with wide application in bacteria. In this work a single plasmid system is presented which expresses deactivated Cas9 and a short guide RNA sequence from separate inducible promoters, enabling up to 40-fold repression of target genes with minimal leaky repression in the uninduced state. The plasmid is designed for rapid spacer insertion with easy screening for correct clones, and a simple one-day protocol with >99.9% assembly efficiency is described. This plasmid is made available to the research community with a view to facilitating wider use of CRISPRi in bacterial systems.

Cross-Activation of Two Nitrogenase Gene Clusters by CnfR1 or CnfR2 in the Cyanobacterium Anabaena variabilis.

ABSTRACTIn Anabaena variabilis, the nif1 genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the nif2 genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the cnfR2 promoter or from the Co2+-inducible coaT promoter, activated the expression of both nifB1 and nifB2. Activation of nifB2, but not nifB1, by CnfR1 required NtcA. Thus, expression of the nif1 system requires no heterocyst-specific factor other than CnfR1. In contrast, CnfR2, when it was expressed in heterocysts under the control of the cnfR1 promoter or from the coaT promoter, did not activate the expression of nifB1 or nifB2. Thus, activation of the nif2 system in anaerobic vegetative cells by CnfR2 requires additional factors absent in heterocysts. CnfR2 made from the coaT promoter activated nifB2 expression in anaerobic vegetative cells grown with fixed nitrogen; however, oxygen inhibited CnfR2 activation of nifB2 expression. In contrast, activation of nifB1 and nifB2 by CnfR1 was unaffected by oxygen. CnfR1, which does not activate the nifB2 promoter in heterocysts, activated the expression of the entire nif2 gene cluster from a nifB2::nifB1::nifB2 hybrid promoter in heterocysts, producing functional Nif2 nitrogenase in heterocysts. However, activity was poor compared to the normal Nif1 nitrogenase. Expression of the nif2 cluster in anaerobic vegetative cells of Nostoc sp. PCC 7120, a strain lacking the nif2 nitrogenase, resulted in expression of the nif2 genes but weak nitrogenase activity.IMPORTANCE Cyanobacterial nitrogen fixation is important in the global nitrogen cycle, in oceanic productivity, and in many plant and fungal symbioses. While the proteins that mediate nitrogen fixation have been well characterized, the regulation of this complex and expensive process is poorly understood in cyanobacteria. Using a genetic approach, we have characterized unique and overlapping functions for two homologous transcriptional activators CnfR1 and CnfR2 that activate two distinct nitrogenases in a single organism. We found that CnfR1 is promiscuous in its ability to activate both nitrogenase systems, whereas CnfR2 depends on additional cellular factors; thus, it activates only one nitrogenase system.