CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.

Ecology and Function of the Transmissible Locus of Stress Tolerance in Escherichia coli and Plant-Associated Enterobacteriaceae

The transmissible locus of stress tolerance (tLST) is a genomic island comprising 10 core genes that occurs in diverse Enterobacteriaceae and confers resistance to heat and chlorine. Experimentation described in the manuscript describes the physiological function of the core genes by characterization of the resistance of 13 single-knockout (KO) mutants and by characterization of protein and membrane oxidation in these strains after chlorine challenge.

Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice

Background Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. Methods In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. Results MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. Conclusion MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo. Graphic abstract

Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δβ-ca1 and Δβ-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δβ-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δβ-ca1ca5 plants normalized at 9,000 ppm CO2. Leaves of Δβ-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δβ-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δβ-ca1ca5 seedling germination and development is negatively affected at ambient CO2. Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the Δβ-ca1ca5 mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δβ-ca1 and Δβ-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.

Cell Types and Synapses Expressing the SNARE Complex Regulating Proteins Complexin 1 and Complexin 2 in Mammalian Retina

Complexins (Cplxs) 1 to 4 are components of the presynaptic compartment of chemical synapses where they regulate important steps in synaptic vesicle exocytosis. In the retina, all four Cplxs are present, and while we know a lot about Cplxs 3 and 4, little is known about Cplxs 1 and 2. Here, we performed in situ hybridization experiments and bioinformatics and exploited Cplx 1 and Cplx 2 single-knockout mice combined with immunocytochemistry and light microscopy to characterize in detail the cell type and synapse-specific distribution of Cplx 1 and Cplx 2. We found that Cplx 2 and not Cplx 1 is the main isoform expressed in normal and displaced amacrine cells and ganglion cells in mouse retinae and that amacrine cells seem to operate with a single Cplx isoform at their conventional chemical synapses. Surprising was the finding that retinal function, determined with electroretinographic recordings, was altered in Cplx 1 but not Cplx 2 single-knockout mice. In summary, the results provide an important basis for future studies on the function of Cplxs 1 and 2 in the processing of visual signals in the mammalian retina.

The G2A Receptor Deficiency Aggravates Atherosclerosis in Rats by Regulating Macrophages and Lipid Metabolism

The orphan G protein-coupled receptor G2A has been linked to atherosclerosis development. However, available data from mouse models are controversial. Rat G2A receptor bears more similarities with its human homolog. We proposed that the atherosclerosis model established from Ldlr–/– rat, which has been reported to share more similar phenotypes with the human disease, may help to further understand this lipid receptor. G2A deletion was found markedly aggravated in the lipid disorder in the rat model, which has not been reported in mouse studies. Examination of aortas revealed exacerbated atherosclerotic plaques in G2A deficient rats, together with increased oxidative stress and macrophage accumulation. In addition, consistently promoted migration and apoptosis were noticed in G2A deficient macrophages, even in macrophages from G2A single knockout rats. Further analysis found significantly declined phosphorylation of PI3 kinase (PI3K) and AKT, together with reduced downstream genes Bcl2 and Bcl-xl, suggesting possible involvement of PI3K/AKT pathway in G2A regulation to macrophage apoptosis. These data indicate that G2A modulates atherosclerosis by regulating lipid metabolism and macrophage migration and apoptosis. Our study provides a new understanding of the role of G2A in atherosclerosis, supporting it as a potential therapeutic target.

Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

Sperm membrane proteins DCST1 and DCST2 are required for the sperm-egg fusion process in mice and fish

The process of sperm-egg fusion is critical for successful fertilization, yet the underpinning mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) spermatozoa are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO spermatozoa can bind to the oolemma, they rarely fuse with oocytes, resulting in male sterility. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO spermatozoa are motile and can approach the egg, but rarely bind to the oolemma. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species highlighting their crucial role as conserved factors in fertilization.

Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins

The insecticidal Cry4Ba and Cry11Aa crystal proteins from Bacillus thuringiensis subsp. israelensis (Bti) are highly toxic to Ae. aegypti larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of Ae. aegypti to Bti toxicity in vivo. In this study, two Cry4Ba/Cry11Aa-binding Ae. aegypti GPI-APN isoforms (AeAPN1 and AeAPN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the AeAPN1/AeAPN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of AeAPN1 or AeAPN2, nor the simultaneous disruption of AeAPN1 and AeAPN2 resulted in significant changes in susceptibility of Ae. aegypti larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that AeAPN1 and AeAPN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae as their binding proteins.

Decreased content of ascorbic acid (vitamin C) in the brain of knockout mouse models of Na+,K+-ATPase-related neurologic disorders

Na+,K+-ATPase is a crucial protein responsible for maintaining the electrochemical gradients across the cell membrane. The Na+,K+-ATPase is comprised of catalytic α, β, and γ subunits. In adult brains, the α3 subunit, encoded by ATP1A3, is predominantly expressed in neurons, whereas the α2 subunit, encoded by ATP1A2, is expressed in glial cells. In foetal brains, the α2 is expressed in neurons as well. Mutations in α subunits cause a variety of neurologic disorders. Notably, the onset of symptoms in ATP1A2- and ATP1A3-related neurologic disorders is usually triggered by physiological or psychological stressors. To gain insight into the distinct roles of the α2 and α3 subunits in the developing foetal brain, whose developmental dysfunction may be a predisposing factor of neurologic disorders, we compared the phenotypes of mouse foetuses with double homozygous knockout of Atp1a2 and Atp1a3 (α2α3-dKO) to those with single knockout. The brain haemorrhage phenotype of α2α3-dKO was similar to that of homozygous knockout of the gene encoding ascorbic acid (ASC or vitamin C) transporter, SVCT2. The α2α3-dKO brain showed significantly decreased level of ASC compared with the wild-type (WT) and single knockout. We found that the ASC content in the basal ganglia and cerebellum was significantly lower in the adult Atp1a3 heterozygous knockout mouse (α3-HT) than in the WT. Interestingly, we observed a significant decrease in the ASC level in the basal ganglia and cerebellum of α3-HT in the peripartum period, during which mice are under physiological stress. These observations indicate that the α2 and α3 subunits independently contribute to the ASC level in the foetal brain and that the α3 subunit contributes to ASC transport in the adult basal ganglia and cerebellum. We propose that decreases in ASC levels may affect neural network development and are linked to the pathophysiology of ATP1A2- and ATP1A3-related neurologic disorders.