Discovery of Sex-Determining Genes in Portunus trituberculatus: A Comparison of Male and Female Transcriptomes During Early Developmental Stages

Portunus trituberculatus is one of the main mariculture crabs of high economic value. To identify genes involved in sex determination, we first performed sex-specific transcriptome sequencing at six larval development stages using a DNA/RNA co-extraction method. A total of 907,952,938 and 828,774,880 reads were obtained from female and male crabs, respectively. 2,379 differentially expressed genes (DEGs) were found between females and males, and were mainly enriched in DNA replication, folate biosynthesis, and retinol metabolism pathways. Furthermore, transcription patterns of genes in the sex-determining region (SD) were analyzed based on the transcriptome data, and one Dmrt gene (PtDMY) was found to be exclusively expressed in males during early developmental stages. Notably, some known sex-related genes, including IAG, Dmrt11E, DmrtB1, and DmrtC2 were significantly down-regulated after knocking down PtDMY. Our results suggested that PtDMY is involved in sex determination and may be one of the key upstream regulators of the sex determination pathway. In addition, the massive volume of transcriptome data obtained in this study provided an important basis for the systematic study of sex determination mechanisms in P. trituberculatus.

Elucidation of the biosynthetic pathway of B-group vitamins via genome mining of food-derived \(\textit{Bacillus velezensis}\) VTX20

B-vitamins are micronutrients that play an important role in various cellular processes of organisms, which are only synthesized by plants, yeasts, and bacteria. Since animals and humans lack the ability to synthesize B-vitamins, supplements of vitamins from dietary and the B-vitamin producing bacteria are required. In this study, we, for the first time, shed some light on biosynthetic pathways involved in folate (vitamin B9), riboflavin (vitamin B2), and biotin (vitamin B7) production in Bacillus velezensis VTX20. The genome-wide comparison revealed that B. velezensis VTX20 shared high similarities with B. tequilensis KCTC 13622, B. subtilis 168, B. amyloliquefaciens DSM 7. Genomic analysis revealed the presence of a complete folate biosynthesis pathway in which some core components were not found in most Bacillus species. Moreover, strain VTX20 also had the metabolic pathways for riboflavin and biotin that are important probiotic traits. These results highlighted that B. velezensis VTX20 is a producer of B-vitamins, which can be applied further in the agricultural biotechnology industry.

Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds as well as heightened regulatory scrutiny. Of the known herbicide modes of action, 6-hydroxymethyl-7,8-dihydropterin synthase (DHPS) which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics - and while still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.6 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate asulam and the antibiotics sulfacetamide and sulfamethoxazole have herbicidal as well as antibacterial activity and explore the structural basis of their potency by modelling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and that pharmacokinetic or delivery differences between plants and microbes might be the best approaches to safeguard this mode of action.

Seeing the unseen: A trifoliate (MYB117) mutant allele fortifies folate and carotenoids in tomato fruits

Micronutrient deficiency also termed hidden hunger affects a large segment of the human population, particularly in developing and underdeveloped nations. Tomato the second most consumed vegetable crop in the world after potato can serve as a sustainable source to alleviate micronutrient deficiency. In tomato, the mutations in the R2R3-MYB117 transcription factor elicit trifoliate leaves and initiate axillary meristems; however, its effect on fruit metabolome remains unexplored. The fruits of a new trifoliate (tf) allele (tf-5) were firmer, had higher Brix, folate, and carotenoids. The transcriptome, proteome, and metabolome profiling of tf-5 reflected a broad-spectrum change in homeostasis. The tf-5 allele enhanced the fruit firmness by suppressing cell wall softening-related proteins. The tf-5 fruit displayed a substantial increase in aminome, particularly γ-aminobutyric acid, with a parallel reduction in aminoacyl t-RNA synthases. The increased lipoxygenases proteins and transcripts seemingly elevated jasmonic acid. In addition, increased abscisic acid hydrolases transcripts coupled with reduced precursor supply lowered abscisic acid. The upregulation of carotenoids was mediated by modulation of methylerythreitol and plastoquinone pathways along with an increase in carotenoids isomerization proteins. The upregulation of folate in tf-5 was connoted by the increase in precursor p-aminobenzoic acid and transcripts of several folate biosynthesis pathway genes. The reduction in pterin-6-carboxylate and γ-glutamyl hydrolase activity indicated that the diminished folate degradation also enriched folate levels. Our study delineates that introgression of the tf-5 can be used for the γ-aminobutyric acid, carotenoids, and folate fortification of tomato.

relA Inactivation Converts Sulfonamides Into Bactericidal Compounds

Folates are required for the de novo biosynthesis of purines, thymine, methionine, glycine, and pantothenic acid, key metabolites that bacterial cells cannot survive without. Sulfonamides, which inhibit bacterial folate biosynthesis and are generally considered as bacteriostats, have been extensively used as broad-spectrum antimicrobials for decades. Here we show that, deleting relA in Escherichia coli and other bacterial species converted sulfamethoxazole from a bacteriostat into a bactericide. Not as previously assumed, the bactericidal effect of SMX was not caused by thymine deficiency. When E. coli ∆relA was treated with SMX, reactive oxygen species and ferrous ion accumulated inside the bacterial cells, which caused extensive DNA double-strand breaks without the involvement of incomplete base excision repair. In addition, sulfamethoxazole showed bactericidal effect against E. coli O157 ∆relA in mice, suggesting the possibility of designing new potentiators for sulfonamides targeting RelA. Thus, our study uncovered the previously unknown bactericidal effects of sulfonamides, which advances our understanding of their mechanisms of action, and will facilitate the designing of new potentiators for them.

Potentially Probiotic Lactiplantibacillus Plantarum Strains From Indian Foods: Isolation, Characterization and Comparative Genomics

L. plantarum is one of the most diverse species of lactic acid bacteria found in various habitats. Here we report the isolation of two distinct strains of L. plantarum from Indian foods, one each from dhokla batter and jaggery, and analysis of their probiotic potential, technical properties, and genomic features. Both the strains were bile and acid tolerant, utilized various sugars, adhered to intestinal epithelial cells, produced exopolysaccharides, were susceptible for tetracycline, erythromycin, and chloramphenicol, did not cause hemolysis, and exhibited antimicrobial activity against a few pathogenic bacteria. The genetic determinants of bile tolerance, cell-adhesion, bacteriocins production, riboflavin and folate biosynthesis, plant polyphenols utilization, and exopolysaccharide production were found in both the strains. One of the strains contained a large number of unique genes while the other had a simultaneous presence of glucansucrase and fructansucrase genes which is a rare trait in L. plantarum. Comparative genome analysis of 149 L. plantarum strains highlighted high variation in the cell-adhesion and sugar metabolism genes while the genomic regions for some other properties were relatively conserved. This work highlights the unique properties of our strains along with the probiotic and technically important genomic features of a large number of L. plantarum strains.

Sulfamethoxazole-Altered Transcriptomein Green Alga Raphidocelis subcapitata Suggests Inhibition of Translation and DNA Damage Repair

Occurrence of sulfonamide antibiotics has been reported in surface waters with the exposures ranging from < 1 ng L–1 to approximately 11 μg L–1, which may exert adverse effects on non-target algal species, inhibiting algal growth and further hindering the delivery of several ecosystem services. Yet the molecular mechanisms of sulfonamide in algae remain undetermined. The aims of the present work are: (1) to test the hypothesis whether sulfamethoxazole (SMX) inhibits the folate biosynthesis in a model green alga Raphidocelis subcapitata; and (2) to explore the effects of SMX at an environmentally relevant concentration on algal health. Here, transcriptomic analysis was applied to investigate the changes at the molecular levels in R. subcapitata treated with SMX at the concentrations of 5 and 300 μg L–1. After 7-day exposure, the algal density in the 5 μg L–1 group was not different from that in the controls, whereas a marked reduction of 63% in the high SMX group was identified. Using the adj p < 0.05 and absolute log2 fold change > 1 as a cutoff, we identified 1 (0 up- and 1 downregulated) and 1,103 (696 up- and 407 downregulated) differentially expressed genes (DEGs) in the 5 and 300 μg L–1 treatment groups, respectively. This result suggested that SMX at an environmentally relevant exposure may not damage algal health. In the 300 μg L–1 group, DEGs were primarily enriched in the DNA replication and repair, photosynthesis, and translation pathways. Particularly, the downregulation of base and nucleotide excision repair pathways suggested that SMX may be genotoxic and cause DNA damage in alga. However, the folate biosynthesis pathway was not enriched, suggesting that SMX does not necessarily inhibit the algal growth via its mode of action in bacteria. Taken together, this study revealed the molecular mechanism of action of SMX in algal growth inhibition.

Quantitative proteomic and metabolomic profiling reveals altered mitochondrial metabolism and folate biosynthesis pathways in the aging Drosophila eye

Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease, that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. Here, we profiled the aging proteome and metabolome of the Drosophila eye, and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Notably, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. Our integration of the proteomic, metabolomic and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. We propose that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases.