Project description:Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in Escherichia coli. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of P. aeruginosa were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor fpvA gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I fpvA gene, and the inactivation of type I fpvA in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I fpvA in trans restored sensitivity to S2. Some S2-resistant type I fpvA-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I fpvA sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I fpvA genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I fpvA genes were introduced into strain 7NSK2 carrying mutations in type II fpvA (encoding the type II pyoverdine receptor) and fpvB (encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.
Project description:Ornamental kale is a popular decorative plant. We identified a peculiar bicolor leaf double haploid line, with green margins and red centers. The development of bicolor leaves can be divided into three stages: S1, S2, and S3. To probe the reason for bicolor formation, we analyzed the anthocyanin and chlorophyll contents, detected the changes in indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin 3 (GA3), sugar, and starch contents, and identified the differentially expressed genes (DEGs) using RNA-seq. Results showed that the bicolor leaf phenotype is gradually formed with anthocyanin degrading and chlorophyll accumulation. Anthocyanin content is lower in the green margin (S3_S) than in the red center (S3_C) part at S3. IAA content was positively correlated with anthocyanin content during the bicolor leaf development. During anthocyanin degrading from S1 to S2, cinnamate-4-hydroxylase (C4H) and transport inhibitor response 1 (TIR1) were downregulated, while lateral organ boundaries domain 39 (LBD39) was upregulated. Two peroxidases, two β-glucosidases (BGLU), LBD39, LBD37, detoxifying efflux carrier 35 (DTX35), three no apical meristem (NAC) transcription factors (TFs), and 15 WRKY DNA-binding protein (WRKY) TFs were downregulated in S3_S vs. S3_C. The bicolor phenotype was mainly linked to anthocyanin degrading and chlorophyll accumulation, and that anthocyanin degrading resulted from reduced anthocyanin biosynthesis and increased anthocyanin degradation.
Project description:Degradation of toxins by microorganisms is a promising approach for detoxification of agricultural products. Here, a bacterial strain, Sphingomonas S3-4, that has the ability to degrade the mycotoxin deoxynivalenol (DON) was isolated from wheat fields. Incubation of Fusarium-infected wheat grains with S3-4 completely eliminated DON. In S3-4 DON is catabolized into compounds with no detectable phytotoxicity, 3-oxo-DON and 3-epi-DON, via two sequential reactions. Comparative analysis of genome sequences from two DON-degrading strains, S3-4 and Devosia D17, and one non-DON-degrading strain, Sphingobium S26, combined with functional screening of a S3-4 genomic BAC library led to the discovery that a novel aldo/keto reductase superfamily member, AKR18A1, is responsible for oxidation of DON into 3-oxo-DON. DON-degrading activity is completely abolished in a mutant S3-4 strain where the AKR18A1 gene is disrupted. Recombinant AKR18A1 protein expressed in Escherichia coli catalyzed the reversible oxidation/reduction of DON at a wide range of pH values (7.5 to 11) and temperatures (10 to 50?°C). The S3-4 strain and recombinant AKR18A1 also catabolized zearalenone and the aldehydes glyoxal and methyglyoxal. The S3-4 strain and the AKR18A1 gene are promising agents for the control of Fusarium pathogens and detoxification of mycotoxins in plants and in food/feed products.
Project description:S-type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor-binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore-forming activity. Pyocins S2, S3, S4, and S5 recognize TonB-dependent ferri-siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P. aeruginosa cystic fibrosis (CF) clinical isolate, CF_PA39. Similarly to pyocins S1 and S2, the S6 toxin-immunity gene tandem was recruited to the genomic region encoding exotoxin A. The pyocin S6 receptor-binding and translocation domains are identical to those of pyocin S1, whereas the killing domain is similar to the 16S ribonuclease domain of Escherichia coli colicin E3. The cytotoxic activity was abolished in pyocin S6 forms with a mutation in the colicin E3-equivalent catalytic motif. The CF_PA39 S6 immunity gene displays a higher expression level than the gene encoding the killing protein, the latter being only detected when bacteria are grown under iron-limiting conditions. In the S1-pyocinogenic strain P. aeruginosa ATCC 25324 and pyocin S2 producer P. aeruginosa PAO1, a remnant of the pyocin S6 killing domain and an intact S6-type immunity gene are located downstream of their respective pyocin operons. Strain PAO1 is insensitive for pyocin S6, and its S6-type immunity gene provides protection against pyocin S6 activity. Purified pyocin S6 inhibits one-fifth of 110 P. aeruginosa CF clinical isolates tested, showing clearer inhibition zones when the target cells are grown under iron limitation. In this panel, about half of the CF clinical isolates were found to host the S6 genes. The pyocin S6 locus is also present in the genome of some non-CF clinical isolates.
Project description:The diversity of a highly variable RNA plant virus was considered to determine the range of virulence substitutions, the evolutionary pathways to virulence, and whether intraspecific diversity modulates virulence pathways and propensity. In all, 114 isolates representative of the genetic and geographic diversity of Rice yellow mottle virus (RYMV) in Africa were inoculated to several cultivars with eIF(iso)4G-mediated Rymv1-2 resistance. Altogether, 41 virulent variants generated from ten wild isolates were analyzed. Nonconservative amino acid replacements at five positions located within a stretch of 15 codons in the central region of the 79-aa-long protein VPg were associated with virulence. Virulence substitutions were fixed predominantly at codon 48 in most strains, whatever the host genetic background or the experimental conditions. There were one major and two isolate-specific mutational pathways conferring virulence at codon 48. In the prevalent mutational pathway I, arginine (AGA) was successively displaced by glycine (GGA) and glutamic acid (GAA). Substitutions in the other virulence codons were displaced when E48 was fixed. In the isolate-specific mutational pathway II, isoleucine (ATA) emerged and often later coexisted with valine (GTA). In mutational pathway III, arginine, with the specific S2/S3 strain codon usage AGG, was displaced by tryptophane (TGG). Mutational pathway I never arose in the widely spread West African S2/S3 strain because G48 was not infectious in the S2/S3 genetic context. Strain S2/S3 least frequently overcame resistance, whereas two geographically localized variants of the strain S4 had a high propensity to virulence. Codons 49 and 26 of the VPg, under diversifying selection, are candidate positions in modulating the genetic barriers to virulence. The theme and variations in the evolutionary pathways to virulence of RYMV illustrates the extent of parallel evolution within a highly variable RNA plant virus species.
Project description:Polylactic acid is a plastic polymer widely used in different applications from printing filaments for 3D printer to mulching films in agriculture, packaging materials, etc. Here, we report the production of poly-D-lactic acid (PDLA) in an engineered yeast strain of Yarrowia lipolytica. Firstly, the pathway for lactic acid consumption in this yeast was identified and interrupted. Then, the heterologous pathway for PDLA production, which contains a propionyl-CoA transferase (PCT) converting lactic acid into lactyl-CoA, and an evolved polyhydroxyalkanoic acid (PHA) synthase polymerizing lactyl-CoA, was introduced into the engineered strain. Among the different PCT proteins that were expressed in Y. lipolytica, the Clostridium propionicum PCT exhibited the highest efficiency in conversion of D-lactic acid to D-lactyl-CoA. We further evaluated the lactyl-CoA and PDLA productions by expressing this PCT and a variant of Pseudomonas aeruginosa PHA synthase at different subcellular localizations. The best PDLA production was obtained by expressing the PCT in the cytosol and the variant of PHA synthase in peroxisome. PDLA homopolymer accumulation in the cell reached 26 mg/g-DCW, and the molecular weights of the polymer (Mw = 50.5 × 103 g/mol and Mn = 12.5 × 103 g/mol) were among the highest reported for an in vivo production.
Project description:Members of the genus Acanthamoeba are widely distributed throughout the world, and some of them are considered pathogenic, as they are capable of causing corneal and central nervous system diseases. In this study, we isolated Acanthamoeba strains from soil and tap water in Yanji, China.We identified four strains of Acanthamoeba (CJY/S1, CJY/S2, CJY/S3, and CJY/W1) using mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis. Nuclear 18S rDNA sequences were used for phylogenetic analysis and species identification.Genotypic characterization of the isolates showed that they belonged to genotypes T4 (CJY/S1 and CJY/S2), T5 (CJY/S3), and T16 (CJY/W1). Sequence differences between CJY/S1 and Acanthamoeba castellanii Neff, CJY/S2 and Acanthamoeba KA/E7, and CJY/S3 and Acanthamoeba lenticulata 68-2 were 0.31, 0.2, and 0.26%, respectively. 18S ribosomal deoxyribonucleic acid (rDNA) of CJY/W1 had 99% sequence identity to that of Acanthamoeba sp. U/H-C1. Strains CJY/S1 and CJY/S2, isolated from soil, had similar mtDNA RFLP patterns, whereas strain CJY/W1, isolated from tap water, displayed a different pattern.To the best of our knowledge, this is the first report on the identification of genotypes T4, T5, and T16 from environmental sources in Yanji, China.
Project description:The genome of Sphingobacterium sp. strain PM2-P1-29 was sequenced. The bacterium contains a physiologically active tet(X) gene, encoding a tetracycline-degrading monooxygenase. To our knowledge, this is the only bacterium naturally harboring tet(X) for which tetracycline degradation has been demonstrated.
Project description:The voltage-sensing domain of voltage-gated ion channels is characterized by specific, conserved, charged residues. Positively charged residues on segment S4 are the main contributors to voltage-sensing and negatively charged residues on the S2 and S3 segments are believed to participate to the process. However, their function in the voltage sensor is not well understood. To probe the role of three acidic residues in NaChBac (D-58 and E-68 in S2, and D-91 in S3), we employed site-directed mutagenesis to substitute native acidic residues with cysteine (neutral), lysine (positive charge), or either aspartate or glutamate (negative charge). We used a combination of the patch-clamp technique to record Na+ currents and molecular modeling to visualize interacting amino acid residues. We suggest that the acidic residues on the S2 and S3 segments form specific interactions with adjacent amino acids in the voltage-sensor domain. The main interactions in NaChBac are D-58 (S2) with A-97-G-98 (S3) and R-120 (S4), E-68 (S2) with R-129 (L4-5), and D-91 (S3) with R-72 (S2). Changing these acidic residues modified the interactions, which in turn altered the sensitivity of the voltage sensor.