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Abstract For economic feasibility, sugarcane molasses (0.5%, w/v) containing K2HPO4 (0.26%, w/v) and mature coconut water, low value byproducts, were used in cultivation of Rhodococcus ruber S103 for inoculum production and immobilization, respectively. Physiological changes of S103 grown in low-cost media, including cell hydrophobicity, saturated/unsaturated ratio of cellular fatty acids and biofilm formation activity, enhanced stress tolerance and crude oil biodegradation in freshwater and even under high salinity (5%, w/v). Biobooms comprised of S103 immobilized on polyurethane foam (PUF) was achieved with high biomass content (1010 colony-forming units g−1 PUF) via a scale-up process in a 5-L modified fluidized-bed bioreactor within 3 days. In a 500-L mesocosm, natural freshwater was spiked with crude oil (72 g or 667 mg g−1 dry biobooms), and a simulated wave was applied. Biobooms could remove 100% of crude oil within only 3 days and simultaneously biodegraded 60% of the adsorbed oil after 7 days when compared to boom control with indigenous bacteria. In addition, biobooms had a long shelf-life (at least 100 days) with high biodegradation activity (85.2 ± 2.3%) after storage in 10% (w/v) skimmed milk at room temperature. This study demonstrates that the low-cost production of biobooms has potential for future commercial bioremediation.

In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 μm × 600 μm × 700 μm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.

Membrane vesicles (MVs) are released by various prokaryotes and play a role in the delivery of various cell-cell interaction factors. Recent studies have determined that these vesicles are capable of functioning as mediators of horizontal gene transfer. Outer membrane vesicles (OMVs) are a type of MV that is released by Gram-negative bacteria and primarily composed of outer membrane and periplasm components; however, it remains largely unknown why DNA is contained within OMVs. Our study aimed to understand the mechanism by which DNA that is localized in the cytoplasm is incorporated into OMVs in Gram-negative bacteria. We compared DNA associated with OMVs using Escherichia coli BW25113 cells harboring the non-conjugative, non-mobilized, and high-copy plasmid pUC19 and its hypervesiculating mutants that included ΔnlpI, ΔrseA, and ΔtolA. Plasmid copy per vesicle was increased in OMVs derived from ΔnlpI, in which peptidoglycan (PG) breakdown and synthesis are altered. When supplemented with 1% glycine to inhibit PG synthesis, both OMV formation and plasmid copy per vesicle were increased in the wild type. The bacterial membrane condition test indicated that membrane permeability was increased in the presence of glycine at the late exponential phase, in which cell lysis did not occur. Additionally, quick-freeze deep-etch and replica electron microscopy observations revealed that outer-inner membrane vesicles (O-IMVs) are formed in the presence of glycine. Thus, two proposed routes for DNA incorporation into OMVs under PG-damaged conditions are suggested. These routes include DNA leakage due to increased membrane permeation and O-IMV formation. Additionally, our findings contribute to a greater understanding of the vesicle-mediated horizontal gene transfer that occurs in nature and the utilization of MVs for DNA cargo.

Jasmonic acid (JA) and its biologically active form jasmonoyl-L-isoleucine (JA-Ile) regulate defense responses to various environmental stresses and developmental processes in plants. JA and JA-Ile are synthesized from α-linolenic acids derived from membrane lipids via 12-oxo-phytodienoic acid (OPDA). In the presence of JA-Ile, the COI1 receptor physically interacts with JAZ repressors, leading to their degradation, resulting in the transcription of JA-responsive genes by MYC transcription factors. Although the biosynthesis of JA-Ile is conserved in vascular plants, it is not recognized by COI1 in bryophytes and is not biologically active. In the liverwort Marchantia polymorpha, dinor-OPDA (dn-OPDA), a homolog of OPDA with two fewer carbons, and its isomer dn-iso-OPDA accumulate after wounding and are recognized by COI1 to activate downstream signaling. The moss Calohypnum plumiforme produces the antimicrobial-specialized metabolites, momilactones. It has been reported that JA and JA-Ile are not detected in C. plumiforme and that OPDA, but not JA, can induce momilactone accumulation and the expression of these biosynthetic genes, suggesting that OPDA or its derivative is a biologically active molecule in C. plumiforme that induces chemical defense. In the present study, we investigated the biological functions of OPDA and its derivatives in C. plumiforme. Searching for the components potentially involving oxylipin signaling from transcriptomic and genomic data revealed that two COI1, three JAZ, and two MYC genes were present. Quantification analyses revealed that OPDA and its isomer iso-OPDA accumulated in larger amounts than dn-OPDA and dn-iso-OPDA after wounding. Moreover, exogenously applied OPDA, dn-OPDA, or dn-iso-OPDA induced the transcription of JAZ genes. These results imply that OPDA, dn-OPDA, and/or their isomers potentially act as biologically active molecules to induce the signaling downstream of COI1-JAZ. Furthermore, co-immunoprecipitation analysis showed the physical interaction between JAZs and MYCs, indicating the functional conservation of JAZs in C. plumiforme with other plants. These results suggest that COI1-JAZ-MYC mediated signaling is conserved and functional in C. plumiforme.

AbstractMomilactone B is a natural product with dual biological activities, including antimicrobial and allelopathic properties, and plays a major role in plant chemical defense against competitive plants and pathogens. The pharmacological effects of momilactone B on mammalian cells have also been reported. However, little is known about the molecular and cellular mechanisms underlying its broad bioactivity. In this study, the genetic determinants of momilactone B sensitivity in yeast were explored to gain insight into its mode of action. We screened fission yeast mutants resistant to momilactone B from a pooled culture containing genome-wide gene-overexpressing strains in a drug-hypersensitive genetic background. Overexpression of pmd1bfr1pap1arp9SPAC9E9.06c

Plasmids can provide advantageous traits to host bacteria, although they may impose a fitness cost. Chromosome-encoded factors are important for regulating the expression of genes on plasmids, and host chromosomes may differ in terms of their interactions with a given plasmid. Accordingly, differences in fitness cost loading and compensatory co-evolution may occur for various host chromosome/plasmid combinations. However, the mechanisms of compensatory evolution are highly divergent and require further insights. Here, we reveal novel evolutionally mechanisms of Pseudomonas putida KT2440 to improve the fitness cost imposed by the incompatibility P-1 (IncP-1) multidrug resistance plasmid RP4. A mixed culture of RP4-harboring and -free KT2440 cells was serially transferred every 24 h under non-selective conditions. Initially, the proportion of RP4-harboring cells decreased rapidly, but it immediately recovered, suggesting that the fitness of RP4-harboring strains improved during cultivation. Larger-sized colonies appeared during 144-h mixed culture, and evolved strains isolated from larger-sized colonies showed higher growth rates and fitness than those of the ancestral strain. Whole-genome sequencing revealed that evolved strains had one of two mutations in the same intergenic region of the chromosome. Based on the research of another group, this region is predicted to contain a stress-inducible small RNA (sRNA). Identification of the transcriptional start site in this sRNA indicated that one mutation occurred within the sRNA region, whereas the other was in its promoter region. Quantitative reverse-transcription PCR showed that the expression of this sRNA was strongly induced by RP4 carriage in the ancestral strain but repressed in the evolved strains. When the sRNA region was overexpressed in the RP4-free strain, the fitness decreased, and the colony size became smaller. Using transcriptome analysis, we also showed that the genes involved in amino acid metabolism and stress responses were differentially transcribed by overexpression of the sRNA region. These results indicate that the RP4-inducible chromosomal sRNA was responsible for the fitness cost of RP4 on KT2440 cells, where this sRNA is of key importance in host evolution toward rapid amelioration of the cost.

H-NS family proteins regulate the expression of many genes by preferably binding to AT-rich genomic regions and altering DNA topology. They are found in both bacterial chromosomes and plasmids, and plasmid-encoded H-NS family proteins have sometimes been suggested to act as a molecular backup of the chromosomally encoded ones. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1, which belongs to incompatibility P-7 group. We have investigated the function of Pmr in Pseudomonas putida KT2440, where two H-NS family proteins (TurA and TurB) encoded on the chromosome are expressed predominantly. Previous transcriptome analyses suggested that TurA, TurB, and Pmr cooperatively regulate numerous genes, but the differentially transcribed genes in KT2440ΔturA(pCAR1), KT2440ΔturB(pCAR1), and KT2440(pCAR1Δpmr) compared with those in KT2440(pCAR1) were somewhat different. Here, we performed RNA sequencing analyses to compare the differentially transcribed genes after the deletion of turA or turB in KT2440, and turA, turB or pmr in KT2440(pCAR1). Three pCAR1-free strains (KT2440, KT2440ΔturA, KT2440ΔturB) and four pCAR1-harboring strains [KT2440(pCAR1), KT2440ΔturA(pCAR1), KT2440ΔturB(pCAR1), KT2440(pCAR1Δpmr)], grown until the log and stationary phases, were used. In KT2440, TurA was the major H-NS family protein regulating a large number and wide range of genes, and both TurA and TurB were suggested to functionally compensate each other, particularly during the stationary phase. In KT2440(pCAR1), the numbers of differentially transcribed genes after the deletion of turA or turB drastically increased compared to those in KT2440. Notably, more than half of the differentially transcribed genes in KT2440ΔturA and KT2440ΔturB did not overlap with those in KT2440ΔturA(pCAR1) and KT2440ΔturB(pCAR1). This dynamic change could be explained by the acquisition of pCAR1 itself and the expression of Pmr. After pCAR1 was transferred into the host, TurA and TurB could be detached from the chromosome of KT2440 and they could newly bind to pCAR1. Moreover, Pmr could reconstitute the chromosome-binding heteromeric oligomers which were formed by TurA and TurB. Our study revealed that horizontal transfer of a plasmid changes the transcriptional network of the chromosomally encoded H-NS family proteins.

Abstract Background H-NS family proteins are nucleoid-associated proteins that form oligomers on DNA and function as global regulators. They are found in both bacterial chromosomes and plasmids, and were suggested to be candidate effectors of the interaction between them. TurA and TurB are the predominantly expressed H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440, while Pmr is encoded on the carbazole-degradative incompatibility group P-7 plasmid pCAR1. Previous transcriptome analyses suggested that they function cooperatively, but play different roles in the global transcriptional network. In addition to differences in protein interaction and DNA-binding functions, cell expression levels are important in clarifying the detailed underlying mechanisms. Here, we determined the precise protein amounts of TurA, TurB, and Pmr in KT2440 in the presence and absence of pCAR1. Results The intracellular amounts of TurA and TurB in KT2440 and KT2440(pCAR1) were determined by quantitative western blot analysis using specific antibodies. The amount of TurA decreased from the log phase (~80,000 monomers per cell) to the stationary phase (~20,000 monomers per cell), while TurB was only detectable upon entry into the stationary phase (maximum 6000 monomers per cell). Protein amounts were not affected by pCAR1 carriage. KT2440(pCAR1pmrHis), where histidine-tagged Pmr is expressed under its original promotor, was used to determine the intracellular amount of Pmr, which was constant (~30,000 monomers per cell) during cell growth. Quantitative reverse transcription PCR demonstrated that the transcriptional levels of turA and turB were consistent with protein expression, though the transcriptional and translational profiles of Pmr differed. Conclusion The amount of TurB increases as TurA decreases, and the amount of Pmr does not affect the amounts of TurA and TurB. This is consistent with our previous observation that TurA and TurB play complementary roles, whereas Pmr works relatively independently. This study provides insight into the molecular mechanisms underlying reconstitution of the transcriptional network in KT2440 by pCAR1 carriage.

Streptomyces are a group of Gram-positive bacteria belonging to the Actinobacteria class, which are among the most important bacteria for producing secondary bioactive metabolites such as antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. Recently, we have isolated the Streptomyces sp. GMR22 from Cajuput rhizospheric soil at Wanagama Forest, Indonesia. GMR22 produced secondary metabolite that inhibited Candida albicans with IC50 of 62,5 μg/mL. The objective of this work was to reveal the novel secondary metabolites from GMR22 by genome mining approach. The antiSMASH 3.0 was used to predict gene clusters that encode the biosynthetic pathways of secondary metabolites in the genome of GMR22, and their core chemical structures. The pylogenomic analysis showed that GMR22 was closely related to Streptomyces bingchenggensis BCW1, as well as to the large genome size (9.5-12.7Mbp) groups of Streptomyces. AntiSMASH 3.0 analysis revealed that the genome of Streptomyces sp. GMR22 harbored at least 63 gene clusters that encode the biosynthetic pathways of secondary metabolites. It was the highest number of gene clusters had been observed among the members of Streptomyces groups, with PKS was predicted as the major groups of the identified gene cluster products. The results suggested that GMR22 could be a strong potential candidate of secondary bioactive metabolites source.

ABSTRACT Understanding the mechanisms underlying plasmid behavior under conditions of various environments is important to predict the fate of plasmids in nature. Most previous studies on plasmid transfer employed two strains: one as a donor and the other as a recipient. However, in natural environments, there are usually different recipient cells available to which plasmid can be transferred. In this study, to reveal the underlying mechanisms, we assessed the transferability of plasmids from one donor strain to either of two recipient candidates as the most simplified model. We used Pseudomonas putida KT2440 and Pseudomonas resinovorans CA10dm4 as model hosts and pCAR1 (IncP-7), NAH7 (IncP-9), pB10 (IncP-1β), and R388 (IncW) as model plasmids. As expected, in most cases these plasmids were generally transferred more frequently to a recipient of the same species than to a recipient of a different one under conditions of liquid and filter mating, although NAH7 was transferred from P. resinovorans more frequently to P. putida than to P. resinovorans during filter mating. With the exception of pCAR1, which was less affected, the coexistence of other recipients enhanced the preferences of conjugative transfer to the same species. In particular, preferences corresponding to transfer from P. putida to a different recipient (P. resinovorans) were reduced by the presence of a coexisting same recipient (P. putida) during transfer of NAH7 in liquid and transfer of R388 in filter mating. We determined that large cell aggregates and substances secreted into culture supernatant were not responsible for this phenomenon. Overall, the results of this study suggest the existence of unknown factors determining optimal plasmid transfer to native recipients. IMPORTANCE Most previous studies on plasmid conjugal transfer employed experimental setups with two strains: one as a donor and the other as a recipient. However, the results obtained sometimes failed to agree with observations obtained under natural environmental conditions or in a model microcosm using natural soil and water samples. Therefore, we consider that there is a “gap” in our understanding of plasmid behavior in the context of bacterial consortia that exist under the actual environmental conditions. In this study, we clearly showed that the conjugation selectivity of a plasmid can be affected by the recipient candidates existing around the donor strain by the use of a simplified experimental setup with one strain as the donor and two strains as recipients. These phenomena could not be explained by factors known to affect plasmid transfer as suggested by previous studies. Therefore, we suggest the presence of novel elements regulating plasmid transfer within consortia.

Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplification and restriction fragment analysis of NRPS genes with HaeIII from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces. Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development.

Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are the major diterpenoid phytoalexins in rice and are synthesised from geranylgeranyl diphosphate, which is derived from the methylerythritol phosphate (MEP) pathway. The hyperaccumulation of momilactones and phytocassanes due to the hyperinductive expression of the relevant biosynthetic genes and the MEP pathway gene OsDXS3 in OsTGAP1-overexpressing (OsTGAP1ox) rice cells has previously been shown to be stimulated by the chitin oligosaccharide elicitor. In this study, to clarify the mechanisms of the elicitor-stimulated coordinated hyperinduction of these phytoalexin biosynthetic genes in OsTGAP1ox cells, transcriptome analysis and chromatin immunoprecipitation with next-generation sequencing were performed, resulting in the identification of 122 OsTGAP1 target genes. Transcriptome analysis revealed that nearly all of the momilactone and phytocassane biosynthetic genes, which are clustered on chromosomes 4 and 2, respectively, and the MEP pathway genes were hyperinductively expressed in the elicitor-stimulated OsTGAP1ox cells. Unexpectedly, none of the clustered genes was included among the OsTGAP1 target genes, suggesting that OsTGAP1 did not directly regulate the expression of these biosynthetic genes through binding to each promoter region. Interestingly, however, several OsTGAP1-binding regions were found in the intergenic regions among and near the cluster regions. Concerning the MEP pathway genes, only OsDXS3, which encodes a key enzyme of the MEP pathway, possessed an OsTGAP1-binding region in its upstream region. A subsequent transactivation assay further confirmed the direct regulation of OsDXS3 expression by OsTGAP1, but other MEP pathway genes were not included among the OsTGAP1 target genes. Collectively, these results suggest that OsTGAP1 participates in the enhanced accumulation of diterpenoid phytoalexins, primarily through mechanisms other than the direct transcriptional regulation of the genes involved in the biosynthetic pathway of these phytoalexins.

Enterobacterial H-NS-like proteins and Pseudomonas MvaT-like proteins share low homology at the amino acid sequence level, but both can function as xenogeneic silencers and are included in the H-NS family of proteins. H-NS family members have dimerization/oligomerization and DNA-binding domains connected by a flexible linker and form large nucleoprotein complexes using both domains. Pmr, an MvaT-like protein encoded on the IncP-7 carbazole-degradative plasmid pCAR1, is a key regulator of an interaction between pCAR1 and its host Pseudomonas putida KT2440. KT2440 has two transcribed genes that encode the MvaT-like proteins TurA and TurB. Our previous transcriptome analyses suggested that the functions of Pmr, TurA and TurB are non-equivalent, although the detailed underlying mechanisms remain unclear. In this study, we focused on the protein-protein interactions of Pmr, and assessed the homo-oligomerization capacity of various substituted and truncated Pmr derivatives by protein-protein cross-linking analysis. Six of the seven residues identified as important for homo-oligomerization in Pmr were located near the N-terminus, and the putative flexible linker or the region near that was not involved in homo-oligomerization, suggesting that Pmr homo-oligomerization is different from that of enterobacterial H-NS and that the functional mechanism differs between H-NS-like and MvaT-like proteins. In addition, we assessed homo- and hetero-oligomerization of Pmr by surface plasmon resonance analysis and found that the coupling ratio of TurB-Pmr oligomers is smaller than that of Pmr-Pmr or TurA-Pmr oligomers. These results raised the possibility that composition of the hetero-oligomers of Pmr, TurA, and TurB could explain why the different gene sets were affected by either pmr, turA, or turB disruption in our previous studies.

WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 μm × 600 μm × 700 μm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples. [ABSTRACT FROM AUTHOR]

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the “dehydrogenase momilactone A synthase” gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique. [ABSTRACT FROM AUTHOR]

In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta. [ABSTRACT FROM AUTHOR]

Phytocassanes and momilactones are known as major diterpenoid phytoalexins (DPs), characterized by abundant production and antimicrobial activity, and their biosynthetic genes are clustered in rice genomes. The basic leucine zipper transcription factor OsTGAP1 is known to act as a regulator of the coordinated production of DPs in cultured rice cells, but in planta functions of OsTGAP1 remain largely unknown. Here, we present evidence on the biological function of OsTGAP1 in planta. In wild-type plants, OsTGAP1 is abundantly expressed in roots compared with that in shoots. Moreover, the inductive expression of OsTGAP1 under jasmonic acid (JA) treatment was only observed in a root-specific manner consistent with the JA-inducible expressions of DP biosynthetic genes in roots. In reverse genetic approaches on OsTGAP1-overexpressing and OsTGAP1-knockdown plants, expressions of the biosynthetic genes relevant for DP accumulation were found to be remarkably increased and decreased, respectively. Reporter analysis in planta revealed that OsTGAP1 activated the promoters of OsDXS3 and momilactone biosynthetic gene OsKSL4, presumably through binding to the TGACGT motif. Furthermore, cocultivation experiments with barnyard grass suggested that the allelopathic effect of knockdown and overexpression of OsTGAP1 was significantly changed compared with the controls. These results demonstrate that OsTGAP1 positively regulates DP accumulation via the transcriptional regulation of DP biosynthetic genes in rice roots, and this is indispensable for maintaining allelopathic interactions with paddy weeds by regulating the production of specialized metabolites like momilactones. [ABSTRACT FROM AUTHOR]

Streptomyces are a group of Gram-positive bacteria belonging to the Actinobacteria class, which are among the most important bacteria for producing secondary bioactive metabolites such as antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. Recently, we have isolated the Streptomyces sp. GMR22 from Cajuput rhizospheric soil at Wanagama Forest, Indonesia. GMR22 produced a secondary metabolite that inhibited Candida albicans with IC50 of 62.5µg/mL. The objective of this work was to reveal the novel secondary metabolites from GMR22 by genome mining approach. The antiSMASH 3.0 was used to predict gene clusters that encode the biosynthetic pathways of secondary metabolites in the genome of GMR22, and their core chemical structures. The phylogenomic analysis showed that GMR22 was closely related to Streptomyces bingchenggensis BCW1, as well as to the large genome size (9.5-12.7Mbp) groups of Streptomyces. AntiSMASH 3.0 analysis revealed that the genome of Streptomyces sp. GMR22 harbored at least 63 gene clusters that encode the biosynthetic pathways of secondary metabolites. It was the highest number of gene clusters had been observed among the members of Streptomyces groups, with polyketide synthetase (PKS) was predicted as the major groups of the identified gene cluster products. The results suggested that GMR22 could be a strong potential candidate for secondary bioactive metabolites source. [ABSTRACT FROM AUTHOR]

Background: Shewanella oneidensis MR-1 is capable of reducing extracellular electron acceptors, such as metals and electrodes, through the Mtr respiratory pathway, which consists of the outer membrane cytochromes OmcA and MtrC and associated proteins MtrA and MtrB. These proteins are encoded in the mtr gene cluster (omcA-mtrCAB) in the MR-1 chromosome. Results: Here, we investigated the transcriptional mechanisms for the mtr genes and demonstrated that omcA and mtrC are transcribed from two upstream promoters, PomcA and PmtrC, respectively. In vivo transcription and in vitro electrophoretic mobility shift assays revealed that a cAMP receptor protein (CRP) positively regulates the expression of the mtr genes by binding to the upstream regions of PomcA and PmtrC. However, the expression of omcA and mtrC was differentially regulated in response to culture conditions; specifically, the expression from PmtrC was higher under aerobic conditions than that under anaerobic conditions with fumarate as an electron acceptor, whereas expression from PomcA exhibited the opposite trend. Deletion of the region upstream of the CRP-binding site of PomcA resulted in a significant increase in promoter activity under aerobic conditions, demonstrating that the deleted region is involved in the negative regulation of PomcA. Conclusions: Taken together, the present results indicate that transcription of the mtr genes is regulated by multiple promoters and regulatory systems, including the CRP/cAMP-dependent regulatory system and yet-unidentified negative regulators. [ABSTRACT FROM AUTHOR]

In this article, the authors focus on the effect of OsJARl rice genome on the biosynthesis of the jasmonoyl-isoleucine (JA-Ile) related to in defense responses in rice. They inform that osjarl mutant has been impaired in JA-induced expression of JA-Ile responsive genes. They mention that the mutant is more susceptible to blast fungus and JA-Ile is indispensable in the production of a flavonoid phytoalexin.

Background: Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDOO, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex. Results: In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding. Conclusions: The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O–O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cisdihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism. [ABSTRACT FROM AUTHOR]

Abstract  Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants. [ABSTRACT FROM AUTHOR]

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazoledegrading capacity of host bacterial cells. [ABSTRACT FROM AUTHOR]

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C5 units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants. [ABSTRACT FROM AUTHOR]

Cumene dioxygenase (CumDO) is an initial enzyme in the cumene degradation pathway of Pseudomonas fluorescens IP01 and is a Rieske non-heme iron oxygenase (RO) that comprises two electron transfer components (reductase [CumDO-R] and Rieske-type ferredoxin [CumDO-F]) and one catalytic component (α3β3-type oxygenase [CumDO-O]). Catalysis is triggered by electrons that are transferred from NAD(P)H to CumDO-O by CumDO-R and CumDO-F. To investigate the binding mode between CumDO-F and CumDO-O and to identify the key CumDO-O amino acid residues for binding, we simulated docking between the CumDO-O crystal structure and predicted model of CumDO-F and identified two potential binding sites: one is at the side-wise site and the other is at the top-wise site in mushroomlike CumDO-O. Then, we performed alanine mutagenesis of 16 surface amino acid residues at two potential binding sites. The results of reduction efficiency analyses using the purified components indicated that CumDO-F bound at the side-wise site of CumDO-O, and K117 of the α-subunit and R65 of the b-subunit were critical for the interaction. Moreover, these two positively charged residues are well conserved in α3β3-type oxygenase components of ROs whose electron donors are Rieske-type ferredoxins. Given that these residues were not conserved if the electron donors were different types of ferredoxins or reductases, the side-wise site of the mushroom-like structure is thought to be the common binding site between Rieske-type ferredoxin and α3β3-type oxygenase components in ROs. [ABSTRACT FROM AUTHOR]

MvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT-homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB, are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage. [ABSTRACT FROM AUTHOR]

Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype. [ABSTRACT FROM AUTHOR]

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy encoding gene from Janthinobacterium sp. strain J3 generated the 1262 V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates.The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Glyl78 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15,25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Glyl78 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C- 9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Glyl78. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate binding pocket in ROs causes a slight shift in the substrate binding position that, in turn, favors specific oxygenation reactions toward various substrates. [ABSTRACT FROM AUTHOR]

The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCARl-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a "transient" host of pCAR1. [ABSTRACT FROM AUTHOR]

The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed. [ABSTRACT FROM AUTHOR]

Pseudomonas putida KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of pplR1 to pplR3 showed that a triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5'-T(G/A)TACAN12TGTA(C/T)A-3'. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV (where LOV indicates "light, oxygen, or voltage") and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria. [ABSTRACT FROM AUTHOR]