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Canadian Journal of Microbiology Nov 2023Biofilms are widely recognized as a prominent mode of microbial growth and strategy of antimicrobial tolerance in many environments. Characteristics that are often...
Biofilms are widely recognized as a prominent mode of microbial growth and strategy of antimicrobial tolerance in many environments. Characteristics that are often overlooked in biofilm investigations include the examination of metabolic pathways as the assumption might be that interference with central pathways such as glycolysis would only reduce growth and thus not be meaningful. Using the Keio collection of mutants, we investigated the influence of biofilm formation and planktonic growth in full-strength and diluted Luria-Bertani (LB) broths using strains with a disruption of glycolysis (Δ), the Entner-Doudoroff pathway (Δ), or the pentose phosphate pathway (Δ). Unexpectedly, in contrast to the Keio parent strain (BW25113), planktonic growth was enhanced in full strength and diluted LB broths in the metabolic mutants. Using a microtiter biofilm assay, the parent strain showed the highest crystal violet staining. However, when analyzed by culture assays, there was an increase in biofilm populations in the mutants in comparison to the parent strain. Fluorescence microscopy showed differences in colonization patterns in the strains. Given the availability of mutant collections in many model organisms, similar metabolic studies are warranted for biofilms, given their importance in nature.
Topics: Escherichia coli; Metabolic Networks and Pathways; Pentose Phosphate Pathway; Glycolysis; Biofilms
PubMed: 37728257
DOI: 10.1139/cjm-2023-0096 -
BMC Plant Biology Aug 2023Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic...
BACKGROUND
Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals.
RESULTS
In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019-2020 and 2020-2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173).
CONCLUSIONS
This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.
Topics: Starch; Triticum; Plant Proteins; Amylose; Amylopectin
PubMed: 37528349
DOI: 10.1186/s12870-023-04389-3 -
Nature Communications Apr 2024Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii...
Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant's growth and increases C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite's fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5'-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.
Topics: Toxoplasma; Diphosphates; Ribosemonophosphates; Glycolysis; Pentose Phosphate Pathway
PubMed: 38589375
DOI: 10.1038/s41467-024-47097-8 -
International Journal of Infectious... Aug 2024The prevalence of respiratory infectious diseases has changed in the post-COVID-19 epidemic era, and mycoplasma pneumoniae (MP) infection in children has attracted wide...
OBJECTIVES
The prevalence of respiratory infectious diseases has changed in the post-COVID-19 epidemic era, and mycoplasma pneumoniae (MP) infection in children has attracted wide attention.
METHODS
Children hospitalized for pneumonia in Wuhan, China, in 2023 were enrolled. Respiratory secretions were obtained for the targeted next-generation sequencing (tNGS) including mutation of MP. Pulmonary inflammation was divided into bronchopneumonia and pulmonary consolidation/atelectasis according to lung computed tomography imaging.
RESULTS
Of the 667 pediatric pneumonia, 478 were MP positive (72%). The positive rate of MP detected by tNGS increased from April, and MP had become the primary pathogen of pneumonia in children in 2023. The 23S rRNA mutations were all A2063G, accounting for 85% of detected MP. The clinical symptoms of the mutant and wild-type strains were similar, with half of them experiencing atelectasis and lung consolidation. Early bronchoscopic lavage combined with azithromycin in pediatric pulmonary consolidation was an effective therapy strategy, which could be an alternative selection to MP pneumonia treatment.
CONCLUSIONS
A2063G mutant strain MP was the primary pathogen of mycoplasma pneumoniae in children recently, which was often complicated by extra-pulmonary symptoms and complications.
Topics: Humans; Pneumonia, Mycoplasma; China; Mycoplasma pneumoniae; Female; Child; Male; Child, Preschool; Mutation; Infant; RNA, Ribosomal, 23S; Anti-Bacterial Agents; Azithromycin; COVID-19; High-Throughput Nucleotide Sequencing; Adolescent
PubMed: 38734057
DOI: 10.1016/j.ijid.2024.107074 -
Tuberculosis (Edinburgh, Scotland) Jul 2023Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the...
Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the operon encoding the primary FeS cluster biogenesis system. Previously, three independently isolated mutants (ΔRv1460stop_1.19, ΔRv1460stop _5.19 and ΔRv1460stop _5.20) harbouring the same deletion in sufR, displayed different growth kinetics in OADC supplemented 7H9 media. To investigate this discrepancy, we performed whole genome sequencing of the 3 mutants and the wild-type progenitor. Single nucleotide polymorphisms (SNPs) were identified in 3 genes in the ΔRv1460stop_1.19 mutant and one gene in the ΔRv1460stop_5.20 mutant. Phenotyping of the ΔRv1460stop_5.19 mutant, which had no additional SNPs, revealed increased susceptibility to clofazimine, DMNQ and menadione, while uptake and survival in THP-1 cells were not significantly different from the wild-type strain. Given that these results differ from those reported for other sufR deletion mutants (ΔSufR and MtbΔSufR), they suggest that the position of the sufR deletion and the genotype of the progenitor strain impact the resulting phenotype.
Topics: Mycobacterium tuberculosis; Iron-Sulfur Proteins; Bacterial Proteins; Genotype; Phenotype
PubMed: 37295353
DOI: 10.1016/j.tube.2023.102360 -
Frontiers in Plant Science 2024Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. genes encode MADS transcription factors, which are known...
Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model (Arabidopsis), where promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one gene, multiple gene duplication events have given rise to three paralogs within the genus in legumes: one Fabaceae group A gene, , and two tandemly repeated Fabaceae group B genes, and . Previously, we showed that has unique functions in floral promotion in Medicago. The retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single mutant and the Arabidopsis mutant; implicating multiple genes in critical overlapping roles in the transition to flowering in Medicago.
PubMed: 38469328
DOI: 10.3389/fpls.2024.1357924 -
ACS Pharmacology & Translational Science Jul 2023Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel...
Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds and were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ∼5 μM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.
PubMed: 37470015
DOI: 10.1021/acsptsci.3c00056 -
International Journal of Molecular... Mar 2024Anthracnose, induced by , poses a substantial economic threat to rubber tree yields and various other tropical crops. Ede1, an endocytic scaffolding protein, plays a...
Anthracnose, induced by , poses a substantial economic threat to rubber tree yields and various other tropical crops. Ede1, an endocytic scaffolding protein, plays a crucial role in endocytic site initiation and maturation in yeast. Metacaspases, sharing structural similarities with caspase family proteases, are essential for maintaining cell fitness. To enhance our understanding of the growth and virulence of , we identified a homologue of Ede1 (CgEde1) in . The knockout of led to impairments in vegetative growth, conidiation, and pathogenicity. Furthermore, we characterized a weakly interacted partner of CgEde1 and CgMca (orthologue of metacaspase). Notably, both the single mutant Δ and the double mutant Δ/Δ exhibited severe defects in conidiation and germination. Polarity establishment and pathogenicity were also disrupted in these mutants. Moreover, a significantly insoluble protein accumulation was observed in Δ and Δ/Δ strains. These findings elucidate the mechanism by which CgEde1 and CgMca regulates the growth and pathogenicity of . Their regulation involves influencing conidiation, polarity establishment, and maintaining cell fitness, providing valuable insights into the intricate interplay between CgEde1 and CgMca in .
Topics: Virulence; Fungal Proteins; Colletotrichum; Plant Diseases
PubMed: 38474190
DOI: 10.3390/ijms25052943 -
Plant Direct Jul 2023The developmental genetics of reproductive structure control in maize must consider both the staminate florets of the tassel and the pistillate florets of the ear...
The developmental genetics of reproductive structure control in maize must consider both the staminate florets of the tassel and the pistillate florets of the ear synflorescences. Pistil abortion takes place in the tassel florets, and stamen arrest is affected in ear florets to give rise to the monoecious nature of maize. Gibberellin (GA) deficiency results in increased tillering, a dwarfed plant syndrome, and the retention of anthers in the ear florets of maize. The mutant results in suppression of silks in the ear. We demonstrate in this study that jasmonic acid (JA) and GA act independently and show additive phenotypes resulting in androecious double mutant plants. The persistence of pistils in the tassel can be induced by multiple mechanisms, including JA deficiency, GA excess, genetic control of floral determinacy, and organ identity. The mutant can suppress both silks in the ear and the silks in the tassel of JA-deficient and AP2 transcription factor mutants. We previously demonstrated that GA production was required for brassinosteroid (BR) deficiency to affect persistence of pistils in the tassel. We find that BR deficiency affects pistil persistence by an independent mechanism from the mutant and JA pathway. The mutant did not prevent the formation of pistils in the tassel by in double mutants. In addition, we demonstrate that there is more to the mutant than just a suppression of pistil growth. We document novel phenotypes of mutants including weakly penetrant ear fasciation and anther persistence in the ear florets. Thus, the JA/AP2 mechanism of pistil retention in the tassel and silk growth in the ear are similarly sensitive to loss of the SILKLESS1 protein, while the BR/GA mechanism is not.
PubMed: 37440932
DOI: 10.1002/pld3.501 -
Frontiers in Plant Science 2023Effective chromosome synapsis and crossover formation during meiosis are essential for fertility, especially in grain crops such as wheat. These processes function most...
Effective chromosome synapsis and crossover formation during meiosis are essential for fertility, especially in grain crops such as wheat. These processes function most efficiently in wheat at temperatures between 17-23 °C, although the genetic mechanisms for such temperature dependence are unknown. In a previously identified mutant of the hexaploid wheat reference variety 'Chinese Spring' lacking the long arm of chromosome 5D, exposure to low temperatures during meiosis resulted in asynapsis and crossover failure. In a second mutant (), containing a 4 Mb deletion in chromosome 5DL, exposure to 13 °C led to similarly high levels of asynapsis and univalence. Moreover, exposure to 30 °C led to a significant, but less extreme effect on crossovers. Previously, we proposed that, of 41 genes deleted in this 4 Mb region, the major meiotic gene was the most likely candidate for preservation of synapsis and crossovers at low (and possibly high) temperatures. In the current study, using RNA-guided Cas9, we developed a new Chinese Spring CRISPR mutant, containing a 39 bp deletion in the 5D copy of , representing the first reported CRISPR-Cas9 targeted mutagenesis in Chinese Spring, and the first CRISPR mutant for in wheat. In controlled environment experiments, wild-type Chinese Spring, CRISPR and backcrossed mutants were exposed to either high or low temperatures during the temperature-sensitive period from premeiotic interphase to early meiosis I. After 6-7 days at 13 °C, crossovers decreased by over 95% in the mutants, when compared with wild-type plants grown under the same conditions. After 24 hours at 30 °C, mutants exhibited a reduced number of crossovers and increased univalence, although these differences were less marked than at 13 °C. Similar results were obtained for mutants, although their scores were more variable, possibly reflecting higher levels of background mutation. These experiments confirm our previous hypothesis that is responsible for preservation of normal crossover formation at low and, to a certain extent, high temperatures. Given that reductions in crossovers have significant effects on grain yield, these results have important implications for wheat breeding, particularly in the face of climate change.
PubMed: 37615022
DOI: 10.3389/fpls.2023.1208285