Nostosin G and Spiroidesin B from the Cyanobacterium Dolichospermum sp. NIES-1697.

Chemical investigation of the cyanobacterium Dolichospermum sp. NIES-1697 afforded nostosin G (1), a linear tripeptide, spiroidesin B (2), and two known compounds, anabaenopeptins I (3) and J (4). Planar structures and absolute configurations for 1 and 2 were determined by 2D NMR, HRMS, Marfey's methodology, chiral-phase HPLC, and enzymatic degradation. Nostosin G (1) is a unique example of a linear peptide containing three subunits, 4-hydroxyphenyllactic acid (Hpla), homotyrosine (Hty), and argininal, with potent trypsin inhibitory properties. The biosynthetic gene clusters for nostosin G (1) and spiroidesin B (2) were investigated based on the genome sequence of Dolichospermum sp. NIES-1697.

Wewakazole B, a Cytotoxic Cyanobactin from the Cyanobacterium Moorea producens Collected in the Red Sea.

A mass spectrometry (MS)-guided isolation has led to the purification of a new cyanobactin, wewakazole B (1), along with the known compound curacin D from a Red Sea Moorea producens. The planar structure of 1 was elucidated using a combination of NMR and MS techniques. After ozonolysis and acid hydrolysis, the absolute configurations of the amino acid components of 1 were determined by chiral-phase LC-MS and HPLC analyses. Notably, compound 1 exhibited cytotoxic activity toward human MCF7 breast cancer cells (IC50 = 0.58 µM) and human H460 lung cancer cells (IC50 = 1.0 µM) and was also found to be inactive in a siderophore assay.

An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds.

The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.

cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica.

The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their Km values for Br(-) were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.

Efficacy of forming biofilms by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology and its molecular mechanisms.

In natural environments, bacteria often exist in close association with surfaces and interfaces. There they form "biofilms", multicellular aggregates held together by an extracellular matrix. The biofilms confer on the constituent cells high resistance to environmental stresses and diverse microenvironments that help generate cellular heterogeneity. Here we report on the ability of Pseudomonas stutzeri T102 biofilm-associated cells, as compared with that of planktonic cells, to degrade naphthalene and survive in petroleum-contaminated soils. In liquid culture system, T102 biofilm-associated cells did not degrade naphthalene during initial hours of incubation but then degraded it faster than planktonic cells, which degraded naphthalene at a nearly constant rate. This delayed but high degradation activity of the biofilms could be attributed to super-activated cells that were detached from the biofilms. When the fitness of T102 biofilm-associated cells was tested in natural petroleum-contaminated soils, they were capable of surviving for 10 wk; by then T102 planktonic cells were mostly extinct. Naphthalene degradation activity in the soils that had been inoculated with T102 biofilms was indeed higher than that observed in soils inoculated with T102 planktonic cells. These results suggest that inoculation of contaminated soils with P. stutzeri T102 biofilms should enable bioaugmentation to be a more durable and effective bioremediation technology than inoculation with planktonic cells.

A truncated form of SpoT, including the ACT domain, inhibits the production of cyclic lipopeptide arthrofactin, and is associated with moderate elevation of guanosine 3',5'-bispyrophosphate level in Pseudomonas sp. MIS38.

Arthrofactin is a biosurfactant produced by Pseudomonas sp. MIS38. We have reported that transposon insertion into spoT (spoT::Tn5) causes moderate accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) and abrogates arthrofactin production. To analyze the linkage of SpoT function and ablation of arthrofactin production, we examined the spoT::Tn5 mutation. The results showed that spoT::Tn5 is not a null mutation, but encodes separate segments of SpoT. Deletion of the 3' region of spoT increased the level of arthrofactin production, suggesting that the C-terminal region of SpoT plays a suppressive role. We evaluated the expression of a distinct segment of SpoT. Forced expression of the C-terminal region that contains the ACT domain resulted in the accumulation of ppGpp and abrogated arthrofactin production. Expression of the C-terminal segment also reduced MIS38 swarming and resulted in extensive biofilm formation, which constitutes the phenocopy of the spoT::Tn5 mutant.

Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna aoukikusa.

Phenol-degrading bacteria were isolated from the rhizosphere of duckweed (Lemna aoukikusa) using an enrichment culture method. One of the isolates, P23, exhibited an excellent ability to degrade phenol and attach to a solid surface under laboratory conditions. Phylogenetic analysis revealed that P23 belongs to the genera Acinetobacter and has the highest similarity to Acinetobacter calcoaceticus. P23 rapidly colonized on the surface of sterilized duckweed roots and formed biofilms, indicating that the conditions provided by the root system of duckweed are favorable to P23. A long-term performance test (160 h) showed that continuous removal of phenol can be attributed to the beneficial symbiotic interaction between duckweed and P23. P23 is the first growth-promoting bacterium identified from Lemna aoukikusa. The results in this study suggest the potential usefulness of dominating a particular bacterium in the rhizosphere of duckweeds to achieve efficient and sustainable bioremediation of polluted water.

Identification and characterization of the genes responsible for the production of the cyclic lipopeptide arthrofactin by Pseudomonas sp. MIS38.

Pseudomonas sp. MIS38 produces an effective biosurfactant named arthrofactin, which is a cyclic lipopeptide synthesized by a mega complex composed of three nonribosomal peptide synthetases. In order to gain insight into the control mechanism of arthrofactin production, a Tn5 mutant library was constructed and screened for arthrofactin-deficient mutants. Along with a number of mutations that occurred in the arthrofactin synthetase operon, three other mutants harbored distinct Tn5 insertions in the genes encoding SyrF-like protein (arfF), heat shock protein (htpG), and (p)ppGpp synthetase/hydrolase (spoT). Epistasis analyses revealed that spoT functions early in the arthrofactin production pathway. We also found that spoT affects MIS38 swarming, biofilm formation, and the cell morphology.

The role of urease activity on biofilm formation by Staphylococcus sp. T-02 isolated from the toilet bowl.

Urolith, which consists of dirty yellow-colored attachments on the toilet bowl, is associated with a variety of odorous chemicals, including ammonia, and causes disadvantages in daily life. Although largely it is derived from microorganisms, little is known about the microbial processes underlying the formation of urolith. In order to gain insight into the types and the activities of microorganisms present in urolith, culturable bacteria were isolated, identified, and physiologically characterized. One of the isolates exhibited higher ability to produce ammonia when it was grown in artificial urine medium. Phylogenetic and physiological analyses indicated that this strain (T-02) belonged to a new group of Staphylococcus species, showing combined phenotypes as between S. lentus and S. xylosus. T-02 exhibited high urease activity and was capable of growing in the urinary condition by forming robust biofilms. The results of this study indicate that T-02 has successfully adapted itself to the environment of urolith.

Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants.

Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

Biofilm formation and proteolytic activities of Pseudoalteromonas bacteria that were isolated from fish farm sediments.

In order to save natural resources and supply good fishes, it is important to improve fish-farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish-farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture-based methods for the identification and characterization of biofilm-forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm-dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane.

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.

Functional analysis of a pyoverdine synthetase from Pseudomonas sp. MIS38.

Fluorescent Pseudomonas sp. MIS38 produces a cyclic lipopeptide, arthrofactin. Arthrofactin is synthesized by a unique nonribosomal peptide synthetase (NRPS) with dual C/E-domains. In this study, another class of cyclic peptide, pyoverdine, was isolated from MIS38, viz., Pvd38. The main fraction of Pvd38 had an m/z value of 1,064.57 and contained Ala, Glu, Gly, (OHOrn), Ser, and Thr at a ratio of 2:1:1:(1):1:1 in the peptide part, suggesting a new structure compound. A gene encoding NRPS for the chromophore part of Pvd38 was identified, and we found that it contained a conventional E-domain. Gene disruption completely impaired the production of Pvd38, demonstrating that the synthetase is functional. This observation allows us to conclude that different NRPS systems with dual C/E-domains (in arthrofactin synthetase) and a conventional E-domain (in pyoverdine synthetase) are both functional in MIS38.

In vivo characterization of tandem C-terminal thioesterase domains in arthrofactin synthetase.

Macrocyclization of a peptide or a lipopeptide occurs at the last step of synthesis and is usually catalyzed by a single C-terminal thioesterase (Te) domain. Arthrofactin synthetase (Arf) from Pseudomonas sp. MIS38 represents a novel type of nonribosomal peptide synthetase that contains unique tandem C-terminal Te domains, ArfC_Te1 and ArfC_Te2. In order to analyze their function in vivo, site-directed mutagenesis was introduced at the putative active-site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were absent in ArfC_Te1:S89A, ArfC_Te1:S89T, and ArfC_Te1:E26G/F27A mutants, and the production of arthrofactin by ArfC_Te2:S92A, ArfC_Te2:S92A/D118A, and ArfCDeltaTe2 was reduced by 95 % without an alteration of the cyclic lipoundecapeptide structure. These results suggest that Ser89 in ArfC_Te1 is essential for the completion of macrocyclization and the release of product. Glu26 and Phe27 residues are also part of the active site of ArfC_Te1. ArfC_Te2 might have been added during the evolution of Arf in order to improve macrocyclization efficiency.

Common mechanisms regulating expression of rice aleurone genes that contribute to the primary response for gibberellin.

During germination of cereal seeds, aleurone cells respond to gibberellins (GA) by synthesizing and secreting hydrolytic enzymes that mobilize the reserved nutrients. It has been shown that products of early GA response genes, like a transcription factor GAMyb, act as key molecules leading to this regulation. Pivotal roles of GAMyb on expression of hydrolase genes have been well documented, whereas regulation of GAMyb expression itself remains obscure. In order to understand virtual mechanisms of the GA-mediated expression of genes, it is important to know how GA control expression of early GA response genes. Using an aleurone transient expression system of rice (Oryza sativa L.), we examined GA responsive domains of early GA response genes in the aleurone, such as GAMyb and OsDof3. The upstream promoter could not confer GA response. Extensive analyses revealed the presence of enhancer-like activities in a large first intron. In Arabidopsis, intron enhancers have been identified in MADS-box homeotic genes, AGAMOUS (AG) and FLOWERING LOCUS C (FLC), in which large introns should not only confer proper gene expressions, but also associate with gene silencing by covalent modifications of both DNA and histone. These evidences prompt us to assign that chromatin-based control might be important for initial GA action. Based on this assumption, we have identified DNA methylation of the GAMyb locus in germinated rice seeds.

Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetases.

Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are l-peptidyl donors, d-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from l to d-form of incorporating amino acid acceptor occurs during or after peptide bond formation. l-peptidyl donors and d-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process.

Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy1A gene in the rice aleurone.

In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules. Previous analyses suggest that the key events controlling the GA-regulated gene expression in the aleurone are formation of active transcription machinery referred to as the GA responsive complex, followed by recruiting GAMYB. In general, bipartite promoter contexts composed of the GA-responsive element and the pyrimidine box are observed within the regulatory regions of cereal GA-responsive genes. Protein factors that recognize each promoter sequence were identified and distinct effects on the GA-mediated activation of gene expression have been also investigated; however, the connection and intercalation between two promoter motifs remain obscure. In this study, I have evaluated cooperative function of GAMYB and a pyrimidine box-binding protein OsDOF3 that influenced the promoter activity of the most predominant GA-responsive gene (RAmy1A) of rice (Oryza sativa). Transient expression of OsDOF3 in the germinated aleurone prolonged GAMYB function on the reporter expression in the absence of GA. The synergistic effect required a set of DNA bindings of two proteins on the RAmy1A promoter region. The yeast two-hybrid assay showed the physical interaction of GAMYB and OsDOF3 in yeast cells, indicating that the association of GAMYB and OsDOF3 may be a functional unit in transcription regulation. The results showed the accessory function of OsDOF3 responsible for a dosage-dependent mediation of GA signaling that leads to high-level expression of physiological target genes.

Cloning and characterization of cDNA of the GPI-anchored purple acid phosphatase and its root tissue distribution in Spirodela oligorrhiza.

A cDNA clone of the glycosylphosphatidylinositol (GPI)-anchored purple acid phosphatase (PAP) has been obtained by a combination of cDNA library screening and 5' rapid amplification of cDNA ends from Spirodela oligorrhiza plants grown under phosphate-deficient (-P) conditions. The open reading frame of the S. oligorrhiza PAP cDNA consists of 1 365 bp encoding a 455 amino acid protein. Its deduced amino acid sequence shows 82 and 80% similarity to Arabidopsis thaliana and Phaseolus vulgaris PAP, respectively. The amino acid residue, Ala439, followed by two more small amino acid residues, Asp and Ser, is predicted to be the GPI-anchoring (omega) site. The absence of a dibasic motif upstream of the putative omega site suggests that the PAP is a cell wall protein. This presumption is supported by the finding that PAP was released by digestion of the cell wall fraction with cellulase. The GPI anchor is speculated to be a signal for transporting PAP to the cell wall. Immunohistochemical results using -P plant roots demonstrate that PAP is preferentially distributed in the outermost cortical cells of roots but not in the epidermis, suggesting its role in acquiring inorganic phosphate under phosphate-deficient conditions. Northern blot analysis using the S. oligorrhiza PAP cDNA as a probe demonstrates that expression of the PAP gene increased during growth of -P plants and this time-dependent occurrence in mRNA levels of the PAP in -P plants was also observed in their protein and activity levels.