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Nature Communications Feb 2024Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and...
Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.
Topics: Animals; Mice; Disease Models, Animal; Mutation; Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Protein Aggregates
PubMed: 38365903
DOI: 10.1038/s41467-024-45748-4 -
Frontiers in Microbiology 2021Disbalancing envelope stress responses was investigated as a strategy for sensitization of to antimicrobial agents. Seventeen isogenic strains were selected from the...
Disbalancing envelope stress responses was investigated as a strategy for sensitization of to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σ, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (Δ and Δ mutants), cefepime (Δ and Δ mutants) and ertapenem (Δ mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log (Δ mutant) and 0.9 log (Δ mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log (Δ mutant) and 4 log10 (Δ mutant) was shown after 4-6 h. For cefepime, 4.2 log (Δ mutant) and 2.6 log (Δ mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log (Δ mutant) was observed at 24 h. A deficient Psp envelope stress response increased susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.
PubMed: 33897667
DOI: 10.3389/fmicb.2021.653479 -
Rice (New York, N.Y.) Dec 2016Aluminum (Al) toxicity represents a major constraint for crop production on acid soils. Rice is a high Al-resistant plant species among small-grain cereals, but its...
BACKGROUND
Aluminum (Al) toxicity represents a major constraint for crop production on acid soils. Rice is a high Al-resistant plant species among small-grain cereals, but its molecular mechanisms of Al resistance are not fully understood. We adopted a forward genetic screen strategy to uncover the Al-resistance mechanisms in rice. In this study, we screened an ethylmethylsulfone (EMS)-mutagenized library to isolate and characterize mutants with altered sensitivity to Al in rice.
RESULTS
Treatment of an Al-intolerant indica variety Kasalath with 20 μM Al induced root swelling. This phenotype could be suppressed by the addition of aminoethoxyvinylglycine (AVG, an ethylene synthesis inhibitor), suggesting that increased production of ethylene is responsible for the root swelling under Al stress. By utilizing the root swelling as an indicator, we developed a highly effective method to screen Al-sensitive or -resistant mutants in rice. Through screening of ~5000 M2 lines, we identified 10 Al-sensitive mutants and one Al-resistant mutant ral1 (resistance to aluminum 1). ral1 mutant showed short root phenotype under normal growth condition, which was attributed to reduced cell elongation in the mutant. A dose-response experiment revealed that ral1 mutant was more resistant to Al than wild-type (WT) at all Al concentrations tested. The mutant was also more resistant to Al when grown in an acid soil. The mutant accumulated much lower Al in the root tips (0-1 cm) than WT. The mutant contained less Al in the cell wall of root tips than WT, whereas Al concentration in the cell sap was similar between WT and the mutant. In addition to Al, the mutant was also more resistant to Cd than WT. Quantitative RT-PCR analysis showed that the expression levels of known Al-resistance genes were not increased in the mutant compared to WT. Genetic analysis indicated that the Al-resistance phenotype in ral1 mutant was controlled by a single recessive gene mapped on the long arm of chromosome 6.
CONCLUSIONS
We have developed a highly efficient method for the screening of rice mutants with altered Al sensitivity. We identified a novel mutant ral1 resistant to Al by this screening. The increased resistance of ral1 to Al toxicity is caused by the reduced Al binding to the cell wall of root tips and the responsible gene is mapped on the long arm of chromosome 6.
PubMed: 27837430
DOI: 10.1186/s12284-016-0132-3 -
International Journal of Molecular... Aug 2022Induced mutation is useful for improving the disease resistance of various crops. Fusarium wilt and powdery mildew are two important diseases which severely influence...
Induced mutation is useful for improving the disease resistance of various crops. Fusarium wilt and powdery mildew are two important diseases which severely influence pea production worldwide. In this study, we first evaluated Fusarium wilt and powdery mildew resistance of mutants derived from two elite vegetable pea cultivars, Shijiadacaiwan 1 (SJ1) and Chengwan 8 (CW8), respectively. Nine SJ1 and five CW8 M mutants showed resistant variations in Fusarium wilt, and the same five CW8 mutants in powdery mildew. These resistant variations were confirmed in M and M mutants as well. Then, we investigated the genetic variations and relationships of mutant lines using simple sequence repeat (SSR) markers. Among the nine effective SSR markers, the genetic diversity index and polymorphism information content (PIC) values were averaged at 0.55 and 0.46, which revealed considerable genetic variations in the mutants. The phylogenetic tree and population structure analyses divided the M mutants into two major groups at 0.62 genetic similarity ( = 2), which clearly separated the mutants of the two cultivars and indicated that a great genetic difference existed between the two mutant populations. Further, the two genetic groups were divided into five subgroups at 0.86 genetic similarity ( = 5) and each subgroup associated with resistant phenotypes of the mutants. Finally, the homologous cDNA of five CW8 mutants that gained resistance to powdery mildew was amplified and cloned. A 129 bp fragment deletion was found in the gene, which was in accord with . The findings provide important information on disease resistant and molecular variations of pea mutants, which is useful for pea production, new cultivar breeding, and the identification of resistance genes.
Topics: Ascomycota; Disease Resistance; Pisum sativum; Phylogeny; Plant Breeding; Plant Diseases
PubMed: 35955926
DOI: 10.3390/ijms23158793 -
Plant Direct Oct 2023The functions of closely related Myb-like repressor and Myb-like activator proteins within the plant circadian oscillator have been well-studied as separate groups, but...
The functions of closely related Myb-like repressor and Myb-like activator proteins within the plant circadian oscillator have been well-studied as separate groups, but the genetic interactions between them are less clear. We hypothesized that these repressors and activators would interact additively to regulate both circadian and growth phenotypes. We used CRISPR-Cas9 to generate new mutant alleles and performed physiological and molecular characterization of plant mutants for five of these core Myb-like clock factors compared with a repressor mutant and an activator mutant. We first examined circadian clock function in plants likely null for both the repressor proteins, () and (), and the activator proteins, (), (), and (). The triple mutant has a long period and flowers late, while quintuple mutants, similarly to mutants, have poor circadian rhythms and flower early. This suggests that and are epistatic to , , and for circadian clock and flowering time function. We next examined hypocotyl elongation and rosette leaf size in these mutants. The mutants have growth phenotypes intermediate between and mutants, suggesting that , , , , and interact additively to regulate growth. Together, our data suggest that these five Myb-like factors interact differently in regulation of the circadian clock versus growth. More generally, the near-norm al seedling phenotypes observed in the largely arrhythmic quintuple mutant demonstrate that circadian-regulated output processes, like control of hypocotyl elongation, do not always depend upon rhythmic oscillator function.
PubMed: 37811362
DOI: 10.1002/pld3.533 -
Frontiers in Plant Science 2023has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes,...
has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes, genetic mutant resources are very useful and necessary tools. Fast Neutron (FN) mutagenesis is effective in inducing deletion mutations in genomes of diverse species. Through this method, we have generated a large mutant resource in . This mutant resources have been used to screen for different mutant using a forward genetics methods. We have isolated and identified a large amount of symbiotic nitrogen fixation (SNF) deficiency mutants. Here, we describe the detail procedures that are being used to characterize symbiotic mutants in . In recent years, whole genome sequencing has been used to speed up and scale up the deletion identification in the mutant. Using this method, we have successfully isolated a SNF defective mutant FN007 and identified that it has a large segment deletion on chromosome 3. The causal deletion in the mutant was confirmed by tail PCR amplication and sequencing. Our results illustrate the utility of whole genome sequencing analysis in the characterization of FN induced deletion mutants for gene discovery and functional studies in the . It is expected to improve our understanding of molecular mechanisms underlying symbiotic nitrogen fixation in legume plants to a great extent.
PubMed: 37457346
DOI: 10.3389/fpls.2023.1209664 -
Infection and Immunity Jan 2022A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The...
A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a mutant of Salmonella enterica serovar Typhimurium 14028s ( Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and mutant Typhimurium. CFA levels in the mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. Typhimurium mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. Typhimurium lacking was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.
Topics: Animals; Bacterial Physiological Phenomena; Bacterial Proteins; Biosynthetic Pathways; Cyclopropanes; Disease Models, Animal; Fatty Acids; Hydrogen-Ion Concentration; Macrophages; Mice; Microbial Viability; Mutation; Oxidative Stress; Salmonella Infections; Salmonella typhimurium; Virulence
PubMed: 34662213
DOI: 10.1128/IAI.00479-21 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588 -
Probiotics and Antimicrobial Proteins Feb 2023The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans....
The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans. The absence of specific biotherapeutics requires new strategies to prevent the spread and prophylaxis of the novel virus and its variants. The SARS-CoV-2 virus shows pathogenesis by entering the host cells via spike protein and Angiotensin-Converting Enzyme 2 receptor protein. Thus, the present study aims to compute the binding energies between a wide range of bacteriocins with receptor-binding domain (RBD) on spike proteins of wild type (WT) and beta variant (lineage B.1.351). Molecular docking analyses were performed to evaluate binding energies. Upon achieving the best bio-peptides with the highest docking scores, further molecular dynamics (MD) simulations were performed to validate the structure and interaction stability. Protein-protein docking of the chosen 22 biopeptides with WT-RBD showed docking scores lower than -7.9 kcal/mol. Pediocin PA-1 and salivaricin P showed the lowest (best) docking scores of - 12 kcal/mol. Pediocin PA-1, salivaricin B, and salivaricin P showed a remarkable increase in the double mutant's predicted binding affinity with -13.8 kcal/mol, -13.0 kcal/mol, and -12.5 kcal/mol, respectively. Also, a better predicted binding affinity of pediocin PA-1 and salivaricin B against triple mutant was observed compared to the WT. Thus, pediocin PA-1 binds stronger to mutants of the RBD, particularly to double and triple mutants. Salivaricin B showed a better predicted binding affinity towards triple mutant compared to WT, showing that it might be another bacteriocin with potential activity against the SARS-CoV-2 beta variant. Overall, pediocin PA-1, salivaricin P, and salivaricin B are the most promising candidates for inhibiting SARS-CoV-2 (including lineage B.1.351) entrance into the human cells. These bacteriocins derived from lactic acid bacteria hold promising potential for paving an alternative way for treatment and prophylaxis of WT and beta variants.
Topics: Humans; Bacteriocins; SARS-CoV-2; Lactobacillales; COVID-19; Molecular Docking Simulation; Pandemics
PubMed: 34837166
DOI: 10.1007/s12602-021-09879-0 -
BMC Biology Dec 2020It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for...
BACKGROUND
It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low.
RESULTS
We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results.
CONCLUSION
This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable "tragedy of the commons" case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar "tragedy of the commons-like" cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.
Topics: Escherichia coli; Microbial Interactions; Mutation; Nutrients
PubMed: 33317515
DOI: 10.1186/s12915-020-00913-1