Generating polyketide diversity in Dictyostelium: a Steely hybrid polyketide synthase produces alternate products at different developmental stages.

The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social amoeba Dictyostelium discoideum has a high genomic potential for producing polyketides to mediate its ecological interactions, including the unique 'Steely' enzymes, consisting of a fusion between a fatty acid synthase and a chalcone synthase. We report here that D. discoideum further increases its polyketide potential by using the StlB Steely enzyme, and a downstream chlorinating enzyme, to make both a chlorinated signal molecule, DIF-1, during its multi-cellular development, and a set of abundant polyketides in terminally differentiated stalk cells. We identify one of these as a chlorinated dibenzofuran with potent anti-bacterial activity. To do this, StlB switches expression from prespore to stalk cells in late development and is cleaved to release the chalcone synthase domain. Expression of this domain alone in StlB null cells allows synthesis of the stalk-associated, chlorinated polyketides. Thus, by altered expression and processing of StlB, cells make first a signal molecule, and then abundant secondary metabolites, which we speculate help to protect the mature spores from bacterial infection.

Simple and Fast One-Pot Cellulose Gel Preparation in Aqueous Pyrrolidinium Hydroxide Solution-Cellulose Solvent and Antibacterial Agent.

Cellulose is the main component of biomass and is the most abundant biopolymer on earth; it is a non-toxic, low-cost material that is biocompatible and biodegradable. Cellulose gels are receiving increasing attention as medical products, e.g., as wound dressings. However, the preparation of cellulose hydrogels employing unmodified cellulose is scarcely reported because of the cumbersome dissolution of cellulose. In previous studies, we developed the new promising cellulose solvent N-butyl-N-methylpyrrolidinium hydroxide in an aqueous solution, which can dissolve up to 20 wt% cellulose within a short time at room temperature. In this study, we employed this solvent system and investigated the gelation behavior of cellulose after crosslinker addition. The swelling behavior in water (swelling ratio, water uptake), the mechanical properties under compression, and the antibacterial activity against Escherichia coli and Bacillus subtilis were investigated. We have developed a simple and fast one-pot method for the preparation of cellulose gels, in which aqueous pyrrolidinium hydroxide solution was acting as the solvent and as an antibacterial reagent. The pyrrolidinium hydroxide content of the gels was controlled by adjustment of the water volume employed for swelling. Simple recovery of the solvent system was also possible, which makes this preparation method environmentally benign.

Small molecules and cell differentiation in Dictyostelium discoideum.

Dictyostelium is a microorganism found in soils that are known as the battle fields of chemical warfare. Genome analysis of Dictyostelium revealed that it has great potential for the production of small molecules, including secondary metabolites such as polyketides and terpenes.Polyketides are a large family of secondary metabolites which have a variety of structures. In accordance with their structural variety, polyketides have a plethora of biological activities, including antimicrobial, antifungal, and antitumor activities. Unsurprisingly, they have exceptional medical importance. Polyketides in nature work as protective compounds and /or function in pheromonal communication. Terpenes belong to another family of structurally diverse secondary metabolites which play roles in ecological interactions, including defence against predators and formation of mutually beneficial alliance with other organisms. Polyketides and terpenes work as intra- or inter-species signalling compounds, i.e. they play the role of a chemical language. However, in Dictyostelium, they work as paracrine signalling compounds which control the organism's multicellular morphogenesis. This review is primarily focused on the small molecules that regulate pattern formation in the slug stage of the organism and their biosynthetic pathways. Current in vivo understandings of polyketide DIF-1 induced cell differentiation and DIF-1-dependent/independent pathways are also discussed.

4-Methyl-5-Pentylbenzene-1,3-Diol Regulates Chemotactic Cell Aggregation and Spore Maturation Via Different Mechanisms in Dictyostelium discoideum.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.

Chemical compounds from Dictyostelium discoideum repel a plant-parasitic nematode and can protect roots.

Slime mold species in the genus Dictyostelium are considered to have a close relationship with non-parasitic nematodes; they are sympatric in soils and can exhibit interspecific competition for food. We investigated whether this relationship extends to a plant-parasitic nematode that is active in the rhizosphere and has broad host specificity, damaging crops worldwide. Using a novel assay to examine the interaction between the cellular slime mold, Dictyostelium discoideum, and the plant-parasitic nematodes, Meloidogyne spp., we found that cellular slime molds can repel plant parasitic nematodes. Specifically, the repulsion activity was in response to chemical compounds released by cellular slime mold fruiting bodies. Under laboratory conditions, these soluble chemical extracts from fruiting bodies of D. discoideum showed repulsion activity strong enough to protect plant roots. The fruiting body cell extracts repelled but were not toxic to the plant-parasitic nematodes.

D-Serine Metabolism and Its Importance in Development of Dictyostelium discoideum.

In mammals, D-Ser is synthesized by serine racemase (SR) and degraded by D-amino acid oxidase (DAO). D-Ser acts as an endogenous ligand for N-methyl-D-aspartate (NMDA)- and δ2 glutamate receptors, and is involved in brain functions such as learning and memory. Although SR homologs are highly conserved in eukaryotes, little is known about the significance of D-Ser in non-mammals. In contrast to mammals, the slime mold Dictyostelium discoideum genome encodes SR, DAO, and additionally D-Ser specific degradation enzyme D-Ser dehydratase (DSD), but not NMDA- and δ2 glutamate receptors. Here, we studied the significances of D-Ser and DSD in D. discoideum. Enzymatic assays demonstrated that DSD is 460- and 1,700-fold more active than DAO and SR, respectively, in degrading D-Ser. Moreover, in dsd-null cells D-Ser degradation activity is completely abolished. In fact, while in wild-type D. discoideum intracellular D-Ser levels were considerably low, dsd-null cells accumulated D-Ser. These results indicated that DSD but not DAO is the primary enzyme responsible for D-Ser decomposition in D. discoideum. We found that dsd-null cells exhibit delay in development and arrest at the early culmination stage. The efficiency of spore formation was considerably reduced in the mutant cells. These phenotypes were further pronounced by exogenous D-Ser but rescued by plasmid-borne expression of dsd. qRT-PCR analysis demonstrated that mRNA expression of key genes in the cAMP signaling relay is perturbed in the dsd knockout. Our data indicate novel roles for D-Ser and/or DSD in the regulation of cAMP signaling in the development processes of D. discoideum.

Effects of deletion of the receptor CrlA on Dictyostelium aggregation and MPBD-mediated responses are strain dependent and not evident in strain Ax2.

The polyketide MPBD (4-methyl-5-pentylbenzene-1, 3-diol) is produced by the polyketide synthase SteelyA (StlA) in Dictyostelium discoideum. MPBD is required for appropriate expression of cAMP signalling genes involved in cell aggregation and additionally induces the spore maturation at the fruiting body stage. The MPBD signalling pathway for regulation of cell aggregation is unknown, but MPBD effects on sporulation were reported to be mediated by the G-protein coupled receptor CrlA in D. discoideum KAx3. In this study, we deleted the crlA gene from the same parental strain (Ax2) that was used to generate the MPBD-less mutant. We found that unlike the MPBD-less mutant, Ax2-derived crlA- mutants exhibited normal cell aggregation, indicating that in Ax2 MPBD effects on early development do not require CrlA. We also found that the Ax2/crlA- mutant formed normal spores in fruiting bodies. When transformed with PkaC, both Ax2 and Ax2/crlA- similarly responded to MPBD in vitro with spore encapsulation. Our data make it doubtful that CrlA acts as the receptor for MPBD signalling during the development of D. discoideum Ax2.

Stalk cell differentiation without polyketides in the cellular slime mold.

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

Synthesis and SAR of 4-methyl-5-pentylbenzene-1,3-diol (MPBD), produced by Dictyostelium discoideum.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD) is a secondary metabolite of SteelyA polyketide synthase, which controls cell aggregation and spore maturation of Dictyostelium discoideum. In this study, chemical synthesis of MPBD and its derivatives was achieved. Structure-activity relationship (SAR) studies for antimicrobial activities against Escherichia coli and Bacillus subtilis were also conducted.

Role of the ACL2 locus in flower stalk elongation in Arabidopsis thaliana.

The acaulis2 (acl2) mutant of Arabidopsis thaliana shows a defect in flower stalk elongation. We identified the mutation point of acl2 by map-based cloning. The ACL2 locus is located within an approximately 320-kb region at around 100 map units on chromosome 1. One nucleotide substitution was detected in this region in the acl2 mutant, but no significant open reading frames were found around this mutation point. When wild-type DNA fragments containing the mutation point were introduced into acl2 mutant plants, some transgenic plants partially or almost completely recovered from the defect in flower stalk elongation. 3'-RACE experiments showed that bidirectional transcripts containing the acl2 mutation point were expressed, and the Plant MPSS database revealed that several small RNAs were produced from this region. Microarray analysis showed that transcription of many genes is activated in flower stalks of acl2 mutant plants. Overexpression of some of these genes caused a dwarf phenotype in wild-type plants. These results suggest the following novel mechanism for control of the elongation of flower stalks. Bidirectional non-coding RNAs are transcribed from the ACL2 locus, and small RNAs are generated from them in flower stalks. These small RNAs repress the transcription of a set of genes whose expression represses flower stalk elongation, and flower stalks are therefore fully elongated.

AtRBP1, which encodes an RNA-binding protein containing RNA-recognition motifs, regulates root growth in Arabidopsis thaliana.

AtRBP1 is an RNA-binding protein containing RNA-recognition motifs in Arabidopsis thaliana, homologues of which are not observed in metazoa. Transgenic plants expressing artificial microRNAs for repressing AtRBP1 expression displayed a stunted primary root phenotype during germination. Transgenic plants overexpressing AtRBP1 also displayed the same phenotype. Tight regulation of the AtRBP1 transcript may be required for normal root growth. An in vitro binding assay showed that AtRBP1 preferentially binds to sequences containing UUAGG, GUAGG and/or UUAGU. In vivo selection of RNAs bound to AtRBP1 suggests that transcripts of At3g06780, At4g15910, At5g11760 and At5g07350 are target RNAs of AtRBP1.

The hybrid type polyketide synthase SteelyA is required for cAMP signalling in early Dictyostelium development.

BACKGROUND: In our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development. PRINCIPAL FINDINGS: Our stlA null mutant showed aggregation delay and abnormally small aggregation territories. Chemotaxis analysis revealed defective cAMP chemotaxis in the stlA null mutant. cAMP chemotaxis was restored by MPBD addition during early stage development. Assay for cAMP relay response revealed that the stlA null mutant had lower cAMP accumulation during aggregation, suggesting lower ACA activity than the wild type strain. Exogenous cAMP pulses rescued the aggregation defect of the stlA null strain in the absence of MPBD. Expression analysis of cAMP signalling genes revealed lower expression levels in the stlA null mutant during aggregation. CONCLUSION: Our data indicate a regulatory function by SteelyA on cAMP signalling during aggregation and show that SteelyA is indispensable for full activation of ACA.

Overexpression of the TIR-X gene results in a dwarf phenotype and activation of defense-related gene expression in Arabidopsis thaliana.

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28°C than at 22°C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.

Role of fatty acid synthase in the development of Dictyostelium discoideum.

Fatty acids are fundamental cellular components, and provide essential building blocks for membrane biosynthesis. Although the use of gene knockout mutants is a robust method for examining the function of specific cellular metabolic networks, fatty acid synthase knockout mutants are extremely difficult to isolate. In the Dictyostelium discoideum genome, we found two putative fatty acid synthase genes, and we created a knockout mutant for one of them to examine the physiological consequences. In this study, we found that a continuous fatty acid supply was necessary for normal development, and the fatty acid synthase knockout mutant showed severe developmental delay. This developmental defect was corrected in chimeras composed of wild type cells and knockout mutant cells (3:7, respectively). The knockout mutant also showed aberrant expression of fatty acid biosynthesis genes. These results showed that D. discoideum needs correct fatty acid synthesis for normal development.

Steely enzymes are involved in prestalk and prespore pattern formation.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of SteelyA enzyme, controls Dictyostelium spore maturation. Since the expression of stlA split the in early and terminal stages, we cannot exclude the possibility that MPBD regulates spore differentiation from the early stage by creating a bias between the cells. 1-(3,5-Dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-on (DIF-1), a product of SteelyB, was identified as the major stalk cell inducer by in vitro assay, but in vivo assay revealed that DIF-1 induces only prestalkB (pstB) and prestalkO (pstO) cells and, that the major prestalkA (pstA) cells differentiated without DIF-1. In order to determine mechanism of polyketide regulated pattern formation, we examined the spatial expression patterns of prestalk and prespore markers in stlA and stlB knockout mutants. We found that MPBD regulates spore maturation at the culmination stage. We also found that the stlA and stlB double-knockout mutant lost pstA marker gene expression.

Identification of des-methyl-DIF-1 methyltransferase in Dictyostelium purpureum.

The signalling molecule 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one (DIF-1) is required for differentiation and pattern formation in Dictyostelium discoideum development. DIF-1 is synthesized by three enzymes, a hybrid polyketide synthase, a flavin-dependent halogenase, and a des-methyl-DIF-1 methyltransferase. The genome data on the related species D. purpureum are now public. Using this genome information, des-methyl-DIF-1 methyltransferase of D. purpureum was identified, and was named Dp dmtA. Overexpression of Dp dmtA complemented the defects in basal disc formation and lower cup formation in a dmtA knock-out mutant of D. discoideum. This indicates that Dp dmtA has the same function as D. discoideum dmtA and compensates for loss of the dmtA gene in the D. discoideum dmtA mutant. The materials released in the medium by D. purpureum contained stalk-inducing activity with the same retention time as that of DIF-1 in HPLC fractionation. This indicates that the stalk-inducing signal of DIF-1 and des-methyl-DIF-1 methyltransferase are conserved in D. purpureum.

Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum.

BACKGROUND: The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum. RESULTS: We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict. CONCLUSIONS: The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

Dictyostelium hybrid polyketide synthase, SteelyA, produces 4-methyl-5-pentylbenzene-1,3-diol and induces spore maturation.

The genome of Dictyostelium contains two novel hybrid-type polyketide synthases (PKSs) known as 'Steely'; the Steely enzyme is formed by the fusion of type I and type III PKSs. One of these enzymes, SteelyB, is known to be responsible for the production of the stalk cell-inducing factor DIF-1 in vivo. On the other hand, the product(s) and expression pattern of SteelyA are not clearly understood, because there are two different reports associated with the in vitro products of SteelyA and its expression pattern. To solve this problem, we first examined the expression pattern using two different primer sets and found that it was quite similar to that shown in the dictyExpress database. stlA expression peaked at approximately 3 h and declined, but showed a small peak around the end of development. Next, we examined the in vivo product of SteelyA using a stlA null mutant and found that the mutant lacked 4-methyl-5-pentylbenzene-1,3-diol (MPBD). This null mutant showed aberrant, glassy sori, and most of the cells in the sori remained amoeba-like without a cell wall. This defect was restored by adding 200 nM of MPBD to the agar. These results indicate that SteelyA produces MPBD in vivo and induces spore maturation.

Overexpression of the NAC transcription factor family gene ANAC036 results in a dwarf phenotype in Arabidopsis thaliana.

NAC proteins comprise one of the largest families of transcription factors in the plant genome. They are known to be involved in various aspects of plant development, but the functions of most of them have not yet been determined. ANAC036, a member of the Arabidopsis NAC transcription factor family, contains unique sequences that are conserved among various NAC proteins found in other plant species. Expression analysis of the ANAC036 gene indicated that this gene was strongly expressed in leaves. Transgenic plants overexpressing the ANAC036 gene showed a semidwarf phenotype. The lengths of leaf blades, petioles and stems of these plants were smaller than those in wild-type plants. Microscopy revealed that cell sizes in leaves and stems of these plants were smaller than those in wild-type plants. These findings suggested that ANAC036 and its orthologues are involved in the growth of leaf cells.