Kinetic parameters of recombinant PHA synthases towards 3-hydroxybutyryl-CoA (3HBCoA) and 3-hydroxyoctanoyl-CoA (3HOCoA).

<p>The kinetic constants were calculated by nonlinear regression analysis, and the values are expressed as means ± standard deviations (n = 3). ND, not detectable.</p><p>Kinetic parameters of recombinant PHA synthases towards 3-hydroxybutyryl-CoA (3HBCoA) and 3-hydroxyoctanoyl-CoA (3HOCoA).</p

SDS-PAGE of purified PhaC1-His₆ and PhaC2-His₆.

<p>Lane 1, molecular mass standards (molecular mass are indicated on the left in kDa); lane 2, purified PhaC1-His₆; and lane 3, purified PhaC2- His₆.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Growth kinetics of <i>C</i>. <i>pinatubonensis</i> JMP134 and derived <i>phaC</i> mutant strains on MNP.

<p>All strains were grown in minimal salt medium containing 0.5 mM MNP. Their growth was evaluated as measurements of the optical density at 600 nm. N represents the OD₆₀₀ value; N₀ represents the initial OD₆₀₀ values. All points represent the mean values of triplicate trials with error bars denoting their standard deviations.</p

The gene clusters containing <i>phaC1</i> and <i>phaC2</i> in <i>C</i>. <i>pinatubonensis</i> JMP134 and <i>in silico</i> analysis of their products.

<p>(A) Organization of gene clusters containing <i>phaC1</i> and <i>phaC2</i> obtained from the reported genome sequence. The gene cluster containing <i>phaC1</i> begins at nucleotide position 1447474 and ends at 1451315 on the Watson strand in the genome of <i>C</i>. <i>pinatubonensis</i> JMP134 (NCBI Reference Sequence: NC_007347.1). The gene cluster containing <i>phaC2</i> begins at nucleotide position 2356098 and ends at 2361552 on the Crick strand in the genome. Open reading frames were annotated with a guide by BLAST results. PhaA: <i>β</i>-ketothiolase; PhaB: acetoacetyl-CoA reductase; PhaZ: PHA depolymerase; PhaP: phasin; PhaJ: <i>R</i>-specific enoyl-CoA hydratase; FadE: acyl-CoA dehydrogenase. (B) Amino acid sequence alignment of PhaC1 and PhaC2 from <i>C</i>. <i>pinatubonensis</i> JMP134, a typical class I PHA synthase PhaC1_<i>Re</i> from <i>R</i>. <i>eutropha</i> H16 and a typical class II PHA synthase PhaC1_<i>Pa</i> from <i>P</i>. <i>aeruginosa</i> PAO1. The boxed region indicates the signature motif of lipase sequence. The arrows indicate conserved residues involved in catalysis (catalytic triad). The triangle indicates a critical residue affecting substrate specificity. (C) Phylogenetic tree constructed using a multiple sequence alignment of several PHA synthases. Classes of different PHA synthases are indicated on the right side of the tree. The triangles indicate PHA synthases of unconfirmed function. The asterisks indicate PHA synthases from strain JMP134. All protein accession numbers are given in brackets.</p

Bacterial strains and plasmids used in this study.

<p>^a Cm^r, Spec^r, Tc^r, Kan^r: resistant to chloramphenicol, spectinomycin, tetracycline and kanamycin, respectively.</p><p>Bacterial strains and plasmids used in this study.</p

Transcriptional organization analysis of <i>phaZ</i>, <i>phaP</i>, <i>phaC2</i>, <i>phaJ</i> and <i>fadE</i> genes.

<p>Strain JMP134 was grown in MSM supplemented with 2% octanoate (w/v) at 30°C, and mRNA was extracted at the early stationary phase. The cDNA was synthesized with random primers. (A) Schematic of the gene cluster containing <i>phaC2</i>. The locations and number of nucleotides of DNA fragments amplified by PCR are represented by short solid lines below the relevant genes and designated RTZP, RTPC2, RTC2J and RTJE. (B) Electrophoresis of PCR products from transcriptional organization analysis of RTZP, RTPC2, RTC2J and RTJE. Lane M, molecular marker; +, presence of RT-PCR products; -, the corresponding negative controls with DNase-treated RNA samples.</p

Image1_Insulin-like peptide 8 (Ilp8) regulates female fecundity in flies.TIFF

Introduction: Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. Here we report the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster.Methods: Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Drosophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis.Results: Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 are specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly.Discussion: Our findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8.</p

Table1_Insulin-like peptide 8 (Ilp8) regulates female fecundity in flies.XLSX

Introduction: Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. Here we report the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster.Methods: Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Drosophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis.Results: Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 are specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly.Discussion: Our findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8.</p

Table2_Insulin-like peptide 8 (Ilp8) regulates female fecundity in flies.XLSX

Introduction: Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. Here we report the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster.Methods: Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Drosophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis.Results: Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 are specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly.Discussion: Our findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8.</p