Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita.     

Observations on the production of purple pigments in the egg capsules,hypobranchial and reproductive glands from seven species of Muricidae (Gastropoda: Mollusca)

Summary

Tyrian purple is a well-known colourant that can be obtained from the hypobranchial glands of muricids. Here we investigate the presence of purple and related pigments in the hypobranchial glands, reproductive glands and egg capsules of three Rapaninae, three Ocenebrinae and one Muricinae species. Observations on the dissected specimens revealed the presence of purple pigmentation in the hypobranchial glands of all species. All of the Rapaninae and the Muricinae, but only some species of Ocenebrinae, appear to transfer the pigment precursors to their egg capsules. This provides evidence that the precursors of Tyrian purple are not produced for the sole purpose of protecting the egg masses as has been previously suggested. In all the subfamilies, the hypobranchial and reproductive (capsule and prostate) glands lie adjacent to one another. Colour changes in the reproductive glands, indicating the presence of dye precursors, were observed in two species of Rapaninae. In Dicathais orbita, colour changes could be seen in both the capsule glands of females and prostrate glands of males. The final colour was more red in the reproductive glands than the purple observed in the hypobranchial glands. Preliminary observations of detached hypobranchial gland sections in both D. orbita and Pterynotus triformis indicate a different suite of colour reactions occur when compared to sections that are intact and attached to the reproductive glands. This demonstrates that the reproductive glands can influence the chemical conversions of dye precursors synthesized by these species. Further studies on the secondary metabolism occurring within the hypobranchial glands and reproductive organs of Muricidae are ongoing, including histological sectioning and confirmation of the chemistry behind the colours observed and functional analysis of Tyrian purple precursors.     

Characterization of a novel gastropod toxin (6-bromo-2-mercaptotryptamine) that inhibits shaker K channel activity

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6-6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.     

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.     

Cloning,differential tissue expression of a novel hcApo gene,and its correlation with total carotenoid content in purple and white inner-shell color pearl mussel Hyriopsis cumingii

As a molecular carrier and storage protein, apolipoprotein (Apo) mediates the intracellular uptake of lipids, proteins, vitamins and carotenoids. In this study, we identified a novel Apo gene, designated hcApo, from the freshwater pearl mussel Hyriopsis cumingii. The complete hcApo cDNA consists of 4104 nucleotides with an open reading frame encoding 1155 amino acid residues. The hcApo protein contains a conserved lipoprotein N-terminal domain (LPD-N) that is a characteristic of the large lipid transfer protein (LLTP) superfamily. The hcApo mRNA is constitutively expressed in a wide range of tissues with the highest expression level in the liver. Moreover, differential expression analysis revealed that the hcApo gene is more highly expressed in the liver, kidney, mantle and gill of purple line mussels compared to white line mussels. In situ hybridization investigations of the precise expression site of hcApo mRNA in the mantle showed that hcApo mRNA is specifically expressed in the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold of the mantle, as well as throughout the outer epithelium of the outer fold and ventral mantle. Another very important finding is that significantly positive correlation existed between the hcApo gene expression level and the total carotenoid content in purple line mussels. These findings may provide a better understanding of the roles of hcApo in the molecular mechanisms of shell formation and coloring of H. cumingii.     

The transport of phenylacetic acid across the peroxisomal membrane is mediated by the PaaT protein in Penicillium chrysogenum

Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl–CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT–DsRed and EGFP–SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.     

Identification of a biosynthetic gene cluster in rice for momilactones

Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.     

MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic ΔmucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.     

Cross-kingdom vitamin B5 biosynthesis and cyst nematode susceptibility

Vitamin deficiencies are known to cause disorders in human beings. Siddique et al. discovered that vitamin B5 biosynthesis in cyst nematodes requires steps in their host plants. Disruption of an Arabidopsis thaliana ‘susceptibility gene’, which is involved in the production of vitamin B5 precursors, results in reduced parasitism.     

RNA interference of pheromone biosynthesis-activating neuropeptide receptor suppresses mating behavior by inhibiting sex pheromone production in Plutella xylostella (L.)

Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. We cloned a PBAN receptor (Plx-PBANr) gene from the female pheromone gland of the diamondback moth, Plutella xylostella (L.). Plx-PBANr encodes 338 amino acids and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains, indicating a typical G protein-coupled receptor. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. Taken together, the identified PBAN receptor is functional in PBAN signaling via calcium secondary messenger, which leads to activation of pheromone biosynthesis and male attraction.     

Genes involved in formation and attachment of a two-carbon chain as a component of eurekanate, a branched-chain sugar moiety of avilamycin A

Eurekanate belongs to the important class of branched-chain carbohydrates present in a wide variety of natural sources. It is a component of avilamycin A, a potent inhibitor of bacterial protein synthesis targeting the 50S ribosomal subunit. The present work provides experimental proof for the function of two genes of the avilamycin biosynthetic gene cluster, aviB1 and aviO2, that are both involved in avilamycin structure modification. The functions of both genes were identified by gene inactivation experiments and nuclear magnetic resonance analyses of extracts produced by the mutants. We suggest that both AviO2 and AviB1 are involved in the biosynthesis of eurekanate within avilamycin biosynthesis. Moreover, two other genes (aviO1 and aviO3) have been inactivated, resulting in a breakdown of avilamycin production in the mutants ITO1 and ITO3, which clearly shows the essential role of both enzymes in avilamycin biosynthesis. The exact functions of both aviO1 and aviO3 remained unknown.     

Genes Involved in the Biosynthesis of Photosynthetic Pigments in the Purple Sulfur Photosynthetic Bacterium Thiocapsa roseopersicina

A pigment mutant strain of the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina BBS was isolated by plasposon mutagenesis. Nineteen open reading frame, most of which are thought to be genes involved in the biosynthesis of carotenoids, bacteriochlorophyll, and the photosynthetic reaction center, were identified surrounding the plasposon in a 22-kb-long chromosomal locus. The general arrangement of the photosynthetic genes was similar to that in other purple photosynthetic bacteria; however, the locations of a few genes occurring in this region were unusual. Most of the gene products showed the highest similarity to the corresponding proteins in Rubrivivax gelatinosus. The plasposon was inserted into the crtD gene, likely inactivating crtC as well, and the carotenoid composition of the mutant strain corresponded to the aborted spirilloxanthin pathway. Homologous and heterologous complementation experiments indicated a conserved function of CrtC and CrtD in the purple photosynthetic bacteria. The crtDC and crtE genes were shown to be regulated by oxygen, and a role of CrtJ in aerobic repression was suggested.     

Genetic evidence that Arabidopsis ALTERED ROOT ARCHITECTURE encodes a putative dehydrogenase involved in homoserine biosynthesis

Key message

Genetic and molecular analysis of an Arabidopsis root development mutant identified a putative dehydrogenase gene involved in homoserine biosynthesis.

Abstract

In higher plants, homoserine (Hse) is derived from aspartate (Asp) and is an important intermediate for production of methionine (Met), threonine (Thr), and isoleucine (Ile). In Arabidopsis, six enzymes involved in the biosynthesis of Hse from Asp have been well characterized. It is not known, however, whether there exist other enzymes involved in this process. In this work, we characterized an Arabidopsis mutant, ara (a ltered r oot a rchitecture), with a short primary root and an increased number of lateral roots. Genetic and molecular analysis indicated that the ARA gene encodes a protein with a D-isomer specific 2-hydroxyacid dehydrogenase domain. ARA is expressed in all plant organs and is localized in the cell periphery. The ara mutant phenotypes can be rescued by exogenously applied Hse, Met, Ile and 2-oxobutanoate. Based on the results presented here, we propose that the ARA protein may be a dehydrogenase involved in homoserine biosynthesis.     

Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens

The production of the blue pigment indigoidine has been achieved in the entomopathogenic bacterium Photorhabdus luminescens by a promoter exchange and in Escherichia coli following heterologous expression of the biosynthesis gene indC. Moreover, genes involved in the regulation of this previously “silent” biosynthesis gene cluster have been identified in P. luminescens.