Purification,Characterization, and Sequencing of a Novel Type of Antimicrobial Peptides,Fa-AMP1 and Fa-AMP2, from Seeds of Buckwheat (Fagopyrum esculentum Moench.)

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 μg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.     

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI–TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0–8.9 μg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.     

Isolation and Some Properties of the Antimicrobial Peptide (Pa-AMP) from the Seeds of Pokeweed (Phytolacca americana).

An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 μg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria.     

Amino acid sequence and antimicrobial activity of chitin-binding peptides, Pp-AMP 1 and Pp-AMP 2, from Japanese bamboo shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Enzymatic resolution of methyl dl-β-acetylthioisobutyrate and dl-β-acetylthioisobutyramide using a stereoselective esterase from Pseudomonas putida IFO12996

Esterase (PpEST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-β-acetylthioisobutyrate (DL-MATI) and dl-β-acetylthioisobutyramide (DL-ATIA) to give d-β-acetylthioisobutyric acid (DAT). DAT is a key intermediate for the synthesis of a series of angiotensin converting enzyme inhibitors. To use enzyme for the DAT production, the PpEST gene of P. putida IFO12996 was cloned and expressed in Escherichia coli. PpEST with a molecular weight of 33 kDa could hydrolyze DL-MATI and DL-ATIA to give DAT with enantiometric excess value (e.e. value) about 97% and enantioselectivity value (E-value) >150, respectively. The kinetic constants of PpEST for DL-MATI and DL-ATIA were examined and they showed that DL-ATIA was a poorer substrate than DL-MATI for PpEST. However, DL-ATIA was 20-fold more soluble in water than DL-MATI, it was more stable than DL-MATI and it did not show substrate inhibition of the PpEST up to 780 mM. This result suggested that PpEST is an esterase but with amidase activity, which can kinetically resolve DL-ATIA to yield DAT and DL-ATIA is a better choice than DL-MATI for industrial production of DAT by the enzymatic resolution method.     

Expression and characterization of (<Emphasis Type="Italic">R</Emphasis>)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4 _Pa from Pseudomonas aeruginosa, termed phaJ4 _Pp, was identified. The gene products of phaJ1 _Pp, which was identified previously, and phaJ4 _Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4_Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4_Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1_Pp was suggested to be different from that of PhaJ1_Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity.     

Phytochemical Variations and Enhanced Efficiency of Antioxidant and Antimicrobial Ingredients in Salvia officinalis as Inoculated with Different Rhizobacteria

Plants produce a variety of secondary metabolites to improve their performance upon exposure to pathogens, pests, herbivores, or environmental stresses. Secondary metabolism in plants is, therefore, highly regulated by presence of biotic or abiotic elicitors in the environment. The present research was undertaken to characterize plant growth‐promoting attributes of four plant growth‐promoting rhizobacteria (PGPR) including two Pseudomonas fluorescens (Pf Ap1, Pf Ap18) and two P. putida (Pp Ap9, Pp Ap14) strains, and to determine their role (individually or in consortium) on growth of Salvia officialis, and biosynthesis of secondary metabolites such as essential oils (EOs), total phenolics, and flavonoids. The antioxidant and antibacterial properties of the extracts and EOs obtained from the inoculated plants were also investigated. The PGPR inoculum was applied to soil, cuttings, and foliage. Results indicated that different PGPR strains varied in their efficiency for production of auxin, siderophore, 1‐aminocyclopropane‐1‐carboxylate deaminase, and phosphate solubilization. All individually inoculated plants had significantly higher shoot and root biomass, leaf P content, EOs yield, total phenolics, and flavonoids content compared to uninoculated control plants. The major constituents of EOs, cis‐thujene, camphor, and 1,8‐cineol, increased following inoculation with reference PGPRs. Although the extract from all inoculated plants had improved antioxidant activity, it was remarkable for the Pf Ap18 strain, which had the lowest IC₅₀ value across treatments. Antibacterial assay of various EOs and their major constituents against pathogenic bacteria showed that the highest activity was observed against Staphylococcus aureus using EOs of Pp Ap14 source. Based on our findings, we suggest that individual inoculation with effective PGPR strains can substantially improve plant growth and secondary metabolism in S. officinalis plants.     

Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.     

Computational investigation of the histidine ammonia-lyase reaction: a modified loop conformation and the role of the zinc(II) ion

Possible reaction intermediates of the histidine ammonia-lyase (HAL) reaction were investigated within the tightly closed active site of HAL from Pseudomonas putida (PpHAL). The closed structure of PpHAL was derived from the crystal structure of PpHAL inhibited with l-cysteine, in which the 39–80 loop including the catalytically essential Tyr53 was replaced. This modified loop with closed conformation was modeled using the structure of phenylalanine ammonia-lyase from Anabaena variabilis (AvPAL) with a tightly closed active site as a template. Three hypothetical structures of the covalently bound intermediate in the PpHAL active site were investigated by conformational analysis. The distances between the acidic pro-S β-hydrogen of the ligand and the appropriate oxygen atoms of Tyr53, Ty280 and Glu414 − which may act as enzymic bases − in the conformations of the three hypothetical intermediate structures were analyzed together with the substrate and product arrangements. The calculations indicated that the most plausible HAL reaction pathway involves the N-MIO intermediate structure in which the L-histidine substrate is covalently bound to the N-3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic group of the apoenzyme via the amino group. Density functional theory (DFT) calculations − on a truncated model of the N-MIO intermediate containing a Zn²⁺ ion coordinated to the imidazole ring of the ligand and to His83, Met382 and a water molecule − indicated that Zn-complex formation plays a role in the reactivity and substrate specificity of HAL.     

Enhanced resistance to the poplar fungal pathogen, Septoria musiva, in hybrid poplar clones transformed with genes encoding antimicrobial peptides

Plasmids, pCA1 and pCWEA1, carrying antimicrobial peptide gene(s), Ac-AMP1.2 and ESF12, were used to transform hybrid poplar clones Ogy and NM6. Peptide Ac-AMP1.2 is an analog of Ac-AMP1 which is one of the smallest chitin-binding proteins. Synthetic peptide ESF12 mimics the amphipathic -helix found in magainins. Transgene mRNA was detected in the transformed plants. When evaluated for resistance to hybrid poplar pathogen Septoria musiva with an in vitro leaf disk assay, the transformed Ogy plants showed significantly increased pathogen resistance as compared to the untransformed Ogy.     

Engineered Escherichia coli for Short-Chain-Length Medium-Chain-Length Polyhydroxyalkanoate Copolymer Biosynthesis from Glycerol and Dodecanoate

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of β-ketothiolase (PhaA _Re ) and NADPH-dependent acetoacetyl-CoA reductase (PhaB _Re ) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaC _Re ), Aeromonas hydrophila (PhaC _Ah ), and Pseudomonas putida (PhaC2 _Pp ) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1 _Pp and PhaJ4 _Pp ), and A. hydrophila (PhaJ _Ah ). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C₄ to C₁₀. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

Functional characterization of the recombinant antimicrobial peptide Trx-<Emphasis Type="Italic">Ace</Emphasis>-AMP1 and its application on the control of tomato early blight disease

Ace-AMP1 is a potent antifungal peptide found in onion (Allium cepa) seeds with sequence similarity to plant lipid transfer proteins. Transgenic plants over-expressing Ace-AMP1 gene have enhanced disease resistance to some fungal pathogens. However, mass production in heterologous systems and in vitro application of this peptide have not been reported. In this study, Ace-AMP1 was highly expressed in a prokaryotic Escherichia coli system as a fusion protein. The purified protein inhibited the growth of many plant fungal pathogens, especially Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, and Verticillium dahliae. The inhibitory effect was accompanied by hyphal hyperbranching for V. dahliae but not for F. oxysporum f. sp. vasinfectum and A. solani, suggesting that the mode of action of Ace-AMP1 on different fungi might be different. Application of Ace-AMP1 on tomato leaves showed that the recombinant protein conferred strong resistance to the tomato pathogen A. solani and could be used as an effective fungicide.     

Resistance to Botrytis cinerea in scented geranium transformed with a gene encoding the antimicrobial protein Ace-AMP1

Scented geranium (Pelargonium sp. `Fren-sham') was transformed with a gene encoding an antimicrobial protein (Ace-AMP1) from onion through an Agrobacterium-mediated transformation system. The binary vector pFAJ3033 contained the coding region of the Ace-AMP1 preproprotein-encoding cDNA. Transformants were verified by polymerase chain reaction and Southern blot analysis. Transgenic plants expressing high levels of Ace-AMP1 were identified by immunoblots and those plants were shown to have increased resistance to Botrytis cinerea leaf infection. Received: 23 July 1998 / Revision received: 24 November 1998 / Accepted: 5 December 1998     

Developmental regulation of cytokinin, spore germination inhibitor discadenine and related enzymes in Dictyostelium discoideum

The amounts of discadenine (spore germination inhibitor of Dictyostelium discoideum) and its precursor, N⁶-Δ²-isopentenyladenine (i⁶Ade) in cells of D. discoideum were measured at various stages of differentiation. The activities of the enzymes involved in the biosynthesis of these bases, i.e., discadenine synthetase and 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase (5′-AMP isopentenyltransferase) in the cells were also measured. During differentiation, discadenine appeared in the cells at the stage of culmination before i⁶Ade was detected. The activity of 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase in cell extracts increased after the beginning of culmination, much later than the increase in discadenine synthetase activity. The order of appearance of these bases and enzymes is apparently the reverse of that expected from the biosynthetic route. The implications of these findings are discussed.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Synergistic action of co-expressed xylanase/laccase mixtures against milled sugar cane bagasse

The primary plant cell wall is composed of cellulose, hemicellulose, lignin and protein in a stable matrix. The concomitant depolymerization of lignin by laccase and of hemicelluloses by xylanase can improve lignocellulose degradation in the production of second generation biofuels. A thermophilic variant of xylanase A (XynAG3) and the thermostable laccase (CotA), both from Bacillus subtilis, were produced in co-transformed Pichia pastoris strain GS115. Mobility changes in SDS-PAGE after Endo H digestion indicated that both enzymes were glycosylated. The maximum catalytic activity of the XynAG3_Pp and the CotA_Pp was observed at 58 °C and 75 °C, respectively, and both enzymes presented high activity at pH 5.0. The half-life at 60 °C of XynAG3_Pp and CotA_Pp was 150 min and 540 min, respectively. The relative levels of CotA_Pp and XynAG3_Pp in culture broths were altered by the concentration of methanol used for induction, and CotA_Pp:XynAG3_Pp ratios of 1:1.5 and 1:2 were evaluated against milled sugar-cane bagasse. The highest activity was observed at a 1:2 ratio of CotA_Pp:XynAG3_Pp, and was 44% higher as compared to the sum of the activities of the individual enzymes in the same assay conditions. These results demonstrate the synergistic action between an endoxylanase and a laccase against the natural lignocellulosic substrate.     

Effects of thyrotropin,prostaglandin E1 and iodide on cyclic 3′,5′-AMP concentration in dog thyroid slices

The kinetics and concentration effect on the relationship of thyrotropin (TSH) action on cyclic 3′,5′-AMP concentration has been studied in dog thyroid slices in vitro. TSH markedly increased cyclic 3′,5′-AMP level after 5 min, the effect reached a plateau after 10–60 min and slowly declined afterwards. TSH enhanced in parallel the cyclic 3′,5′-AMP level and the binding of iodide to proteins. For this latter effect of TSH, the four criteria of the validity of the Sutherland model for a hormonal action are therefore fulfilled. The effect of TSH on cyclic 3′,5′-AMP concentration in thyroid did not require the presence of a methylxanthine inhibitor of cyclic 3′,5′-AMP phosphodiesterase in the medium. Prostaglandin E1 increased cyclic 3′,5′-AMP levels in control and stimulated slices. The omission of Ca²⁺ in the incubation medium decreased the action of TSH but partial replacement of Na⁺ by K⁺ had little effect. Iodide, 1 μM to 100 μM, inhibited the action of TSH. This inhibitory effect was relieved by NaClO₄, methimazole and propylthiouracil (1 mM). The possible role of this inhibitory effect in an intracellular regulatory mechanism is discussed.