Characterization of two adenine nucleotide translocase paralogues in the stink bug, Plautia stali.

Adenine nucleotide translocase (ANT) is a nuclear-coded mitochondrial protein that exchanges ATP for ADP across the mitochondrial inner membrane. Most organisms possess several ANT paralogues, and functional differences among these paralogues remain largely unknown. In the present study, we identified ANT paralogue genes in hemipteran species: the stink bug, bean bug, pea aphid, and Japanese mealybug. The ANT paralogues of the stink bug, Plautia stali, are encoded by two genes, PsANTI1 and PsANTI2. PsANTI1 was constantly expressed at all developmental stages and in all tissues analyzed. In contrast, the expression levels of PsANTI2 were undetectable in first instar nymphs and adult antennae. Gene silencing of each paralogue in P. stali revealed that PsANTI1 plays an important role in homeostasis, whereas the depletion of PsANTI2 failed to result in lethality. Thus, we concluded that PsANTI1 is a good target gene for developing novel pesticides.

High-yield, zero-leakage expression system with a translational switch using site-specific unnatural amino Acid incorporation.

Synthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacterium Escherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system in E. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution.

BACKGROUND: Cationic antimicrobial peptides (CAMPs) are well recognized to be promising as novel antimicrobial and antitumor agents. To obtain novel skeletons of CAMPs, we propose a simple strategy using acid-amide substitution (i.e. Glu→Gln, Asp→Asn) to confer net positive charge to natural non-antimicrobial sequences that have structures distinct from known CAMPs. The potential of this strategy was verified by a trial study. METHODS: The pro-regions of nematode cecropin P1-P3 (P1P-P3P) were selected as parent sequences. P1P-P3P and their acid-amide-substituted mutants (NP1P-NP3P) were chemically synthesized. Bactericidal and membrane-disruptive activities of these peptides were evaluated. Conformational changes were estimated from far-ultraviolet circular dichroism (CD) spectra. RESULTS: NP1P-NP3P acquired potent bactericidal activities via membrane-disruption although P1P-P3P were not antimicrobial. Far-ultraviolet CD spectra of NP1P-NP3P were similar to those of their parent peptides P1P-P3P, suggesting that NP1P-NP3P acquire microbicidal activity without remarkable conformational changes. NP1P-NP3P killed bacteria in almost parallel fashion with their membrane-disruptive activities, suggesting that the mode of action of those peptides was membrane-disruption. Interestingly, membrane-disruptive activity of NP1P-NP3P were highly diversified against acidic liposomes, indicating that the acid-amide-substituted nematode cecropin pro-region was expected to be a unique and promising skeleton for novel synthetic CAMPs with diversified membrane-discriminative properties. CONCLUSIONS: The acid-amide substitution successfully generated some novel CAMPs in our trial study. These novel CAMPs were derived from natural non-antimicrobial sequences, and their sequences were completely distinct from any categories of known CAMPs, suggesting that such mutated natural sequences could be a promising source of novel skeletons of CAMPs.

An enhancer peptide for membrane-disrupting antimicrobial peptides.

BACKGROUND: NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu --> Gln, and Asp --> Asn). RESULTS: In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-alpha, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-alpha against Staphylococcus aureus), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at

Evolution of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides in nematodes: putative rearrangement of disulfide bonding patterns.

ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides are defensin-like cysteine-rich peptides that are widely distributed in the phylum Nematoda. In known members of ASABF-type antimicrobial peptides, an array consisting of eight cysteine residues is completely conserved. In this study, we report a novel member ASABF-6Cys-alpha, which contains only six cysteine residues, in the pig round worm A. suum. The two cysteine residues deleted in ASABF-6Cys-alpha were not identical to a pair of half-cystine forming a disulfide bridge in ASABF-alpha, suggesting a rearrangement of disulfide bonding patterns. Gene organization and phylogenetic analyses suggested that ASABF-6Cys-alpha was generated from an ancestral ASABF gene after the divergence of Ascaridida from Rhabditida. ASABF-6Cys-alpha transcripts dramatically increased after bacterial injection, suggesting that ASABF-6Cys-alpha may contribute to immunity in nematodes.

Anionic C-terminal proregion of nematode antimicrobial peptide cecropin P4 precursor inhibits antimicrobial activity of the mature peptide.

Recently, an anionic proregion was found to be conserved at the C terminus of the antimicrobial peptide, nematode cecropin. Our results suggest that the antimicrobial activity of mature peptide is suppressed by the proregion in its precursor and is released from inhibition after processing. Inhibition is not likely to be due to direct suppression of membrane disruption.

Thermococcus celericrescens sp. nov., a fast-growing and cell-fusing hyperthermophilic archaeon from a deep-sea hydrothermal vent.

A fast-growing and cell-fusing hyperthermophilic archaeon was isolated from a hydrothermal vent at Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean. Strain TS2(T) is an irregular, motile coccus that is generally 0.7-1.5 microm in diameter and possesses a polar tuft of flagella. In the mid-exponential phase of growth, cells that appeared black under phase-contrast microscopy fused at room temperature in the presence of a DNA-intercalating dye, as previously observed in Thermococcus coalescens. Cell fusion was not observed in later growth phases. Transmission electron microscopy revealed that the cells in the mid-exponential phase had a 5 nm-thick, electron-dense cell envelope that appeared to associate loosely with the cytoplasmic membrane. As the growth stage progressed, a surface layer developed on the membrane under the envelope and the envelope eventually peeled off. These observations suggest that the surface layer prevents the fusion of cells. Cells of strain TS2(T) grew at 50-85 degrees C, pH 5.6-8.3 and at NaCl concentrations of 1.0 to 4.5 %, with optimal growth occurring at 80 degrees C, pH 7.0 and 3.0 % NaCl. Under optimal growth conditions, strain TS2(T) grew very fast with an apparent doubling time of 20 min. It is suggested that the biosynthesis of the surface layer cannot catch up with cell multiplication in the mid-exponential phase and thus cells without the surface layer are generated. Strain TS2(T) was an anaerobic chemo-organotroph that grew on either yeast extract or tryptone as the sole growth substrate. The genomic DNA G+C content was 54.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate belongs to the genus Thermococcus. However, no significant DNA-DNA hybridization was observed between the genomic DNA of strain TS2(T) and phylogenetically related Thermococcus species. On the basis of this evidence, strain TS2(T) is proposed to represent a novel species, Thermococcus celericrescens sp. nov., a name chosen to reflect the fast growth of the strain. The type strain is TS2(T) (=NBRC 101555(T)=JCM 13640(T)=DSM 17994(T)).

Activation of nematode G protein GOA-1 by the human muscarinic acetylcholine receptor M2 subtype. Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals.

Signal transduction mediated by heterotrimeric G proteins regulates a wide variety of physiological functions. We are interested in the manipulation of G-protein-mediating signal transduction using G-protein-coupled receptors, which are derived from evolutionarily distant organisms and recognize unique ligands. As a model, we tested the functionally coupling GOA-1, G alpha(i/o) ortholog in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M2 subtype (M2), which is one of the mammalian G alpha(i/o)-coupled receptors. GOA-1 and M2 were prepared as a fusion protein using a baculovirus expression system. The affinity of the fusion protein for GDP was decreased by addition of a muscarinic agonist, carbamylcholine and the guanosine 5'-[3-O-thio]triphosphate ([35S]GTPgammaS) binding was increased with an increase in the carbamylcholine concentrations in a dose-dependent manner. These effects evoked by carbamylcholine were completely abolished by a full antagonist, atropine. In addition, the affinity for carbamylcholine decreased under the presence of GTP as reported for M2-G alpha(i/o) coupling. These results indicate that the M2 activates GOA-1 as well as G alpha(i/o).

Thermococcus coalescens sp. nov., a cell-fusing hyperthermophilic archaeon from Suiyo Seamount.

A cell-fusing hyperthermophilic archaeon was isolated from hydrothermal fluid obtained from Suiyo Seamount of the Izu-Bonin Arc. The isolate, TS1(T), is an irregular coccus, usually 0.5-2 microm in diameter and motile with a polar tuft of flagella. Cells in the exponential phase of growth fused at room temperature in the presence of DNA-intercalating dye to become as large as 5 microm in diameter. Fused cells showed dark spots that moved along in the cytoplasm. Large cells with a similar appearance were also observed upon culture at 87 degrees C, suggesting the occurrence of similar cell fusions during growth. Transmission electron microscopy revealed that cells in the exponential phase possessed a thin and electron-lucent cell envelope that could be lost subsequently during culture. The fragile cell envelope must be related to cell fusion. The cells grew at 57-90 degrees C, pH 5.2-8.7 and at NaCl concentrations of 1.5-4.5 %, with the optima being 87 degrees C, pH 6.5 and 2.5 % NaCl. The isolate was an anaerobic chemo-organotroph that grew on either yeast extract or tryptone as the sole growth substrate. The genomic DNA G+C content was 53.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate was closely related to Thermococcus species. However, no significant DNA-DNA hybridization was observed between genomic DNA of strain TS1(T) and phylogenetically related Thermococcus species. We propose that isolate TS1(T) represents a novel species, Thermococcus coalescens sp. nov., with the name reflecting the cell fusion activity observed in the strain. The type strain is TS1(T) (=JCM 12540T=DSM 16538T).