Fermented botanical fertilizer controls bacterial wilt of tomatoes caused by Ralstonia pseudosolanacearum.

This study demonstrates the effect of fermented botanical product (FBP) on Ralstonia pseudosolanacearum-induced bacterial wilt disease and unravels its action mechanism. Soaking with diluted FBP solutions (0.1%-0.5%) significantly suppressed bacterial wilt in tomato plants, and FBP-treated tomato plants grew well against R. pseudosolanacearum infection. Growth assays showed that FBP had no antibacterial effect but promoted R. pseudosolanacearum growth. In contrast, few or no R. pseudosolanacearum cells were detected in aerial parts of tomato plants grown in FBP-soaked soil. Subsequent infection assays using the chemotaxis-deficient mutant (ΔcheA) or the root-dip inoculation method revealed that FBP does not affect pathogen migration to plant roots during infection. Moreover, FBP-pretreated tomato plants exhibited reduced bacterial wilt in the absence of FBP. These findings suggest that the plant, but not the pathogen, could be affected by FBP, resulting in an induced resistance against R. pseudosolanacearum, leading to a suppressive effect on bacterial wilt.

A novel salt- and organic solvent-tolerant phosphite dehydrogenase from Cyanothece sp. ATCC 51142.

Phosphite dehydrogenase (PtxD) is a promising enzyme for NAD(P)H regeneration. To expand the usability of PtxD, we cloned, expressed, and analyzed PtxD from the marine cyanobacterium Cyanothece sp. ATCC 51142 (Ct-PtxD). Ct-PtxD exhibited maximum activity at pH 9.0°C and 50°C and high stability over a wide pH range of 6.0-10.0. Compared to previously reported PtxDs, Ct-PtxD showed increased resistance to salt ions such as Na+, K+, and NH4 +. It also exhibited high tolerance to organic solvents such as ethanol, dimethylformamide, and methanol when bound to its preferred cofactor, NAD+. Remarkably, these organic solvents enhanced the Ct-PtxD activity while inhibiting the PtxD activity of Ralstonia sp. 4506 (Rs-PtxD) at concentrations ranging from 10% to 30%. Molecular electrostatic potential analysis showed that the NAD+-binding site of Ct-PtxD was rich in positively charged residues, which may attract the negatively charged pyrophosphate group of NAD+ under high-salt conditions. Amino acid composition analysis revealed that Ct-PtxD contained fewer hydrophobic amino acids than other PtxD enzymes, which reduced the hydrophobicity and increased the hydration of protein surface under low water activity. We also demonstrated that the NADH regeneration system using Ct-PtxD is useful for the coupled chiral conversion of trimethylpyruvic acid into L-tert-leucine using leucine dehydrogenase under high ammonium conditions, which is less supported by the Rs-PtxD enzyme. These results imply that Ct-PtxD might be a potential candidate for NAD(P)H regeneration in industrial applications under the reaction conditions containing salt and organic solvent.

Bioaugmentation with marine sediment-derived microbial consortia in mesophilic anaerobic digestion for enhancing methane production under ammonium or salinity stress.

Ammonium (NH4+) and salinity (NaCl) inhibit CH4 production in anaerobic digestion. However, whether bioaugmentation using marine sediment-derived microbial consortia can relieve the inhibitory effects of NH4+ and NaCl stresses on CH4 production remains unclear. Thus, this study evaluated the effectiveness of bioaugmentation using marine sediment-derived microbial consortia in alleviating the inhibition of CH4 production under NH4+ or NaCl stress and elucidated the underlying mechanisms. Batch anaerobic digestion experiments under 5 gNH4-N/L or 30 g/L NaCl were performed with or without augmentation using two marine sediment-derived microbial consortia pre-acclimated to high NH4+ and NaCl. Compared with non-bioaugmentation, bioaugmentation reinforced CH4 production. Network analysis revealed the joint effects of microbial connections by Methanoculleus, which promoted the efficient consumption of propionate accumulated under NH4+ and NaCl stresses. In conclusion, bioaugmentation with pre-acclimated marine sediment-derived microbial consortia can mitigate the inhibition under NH4+ or NaCl stress and enhance CH4 production in anaerobic digestion.

Unexpectedly high thermostability of an NADP-dependent malic enzyme from a psychrophilic bacterium, Shewanella livingstonensis Ac10.

Psychrophilic enzymes are generally active at low temperatures, and their functions have attracted much interest in food processing, biochemical research, and chemical industry. However, their activities are usually lost above their growth temperature because of their flexible and unstable structure. Here, we unexpectedly found that a homodimeric NADP-dependent malic enzyme from a psychrophilic bacterium, Shewanella livingstonensis Ac10 (SL-ME) showed sufficient activity with 60°C treatment, similar to its counterpart from mesophilic Escherichia coli (MaeB). Consistently, SL-ME and MaeB irreversibly denatured at 71.9°C and 64.5°C, respectively. Therefore, SL-ME shows robust catalytic activity, which appears to be advantageous for its application in the bioconversion of NADP to NADPH, an essential ingredient for membrane phospholipid synthesis.

Chemotactic disruption as a method to control bacterial wilt caused by Ralstonia pseudosolanacearum.

We have demonstrated that chemotaxis to l-malate facilitated motility of Ralstonia pseudosolanacearum MAFF 106611, a causative agent of bacterial wilt, to plant roots. Here, we evaluated the assumption that the disruption of chemotaxis to l-malate leads to inhibition of plant infection by R. pseudosolanacearum MAFF 106611. Chemotactic assays revealed that chemotaxis to l-malate was completely or partially inhibited in the presence of l-, d-, and dl-malate, respectively. Moreover, l-malate served as a carbon and energy source for R. pseudosolanacearum MAFF 106611, while d-malate inhibited the growth of this bacterium. In the sand-soak inoculation virulence assay for tomato plants, the addition of l-, d-, and dl-malate to sand suppressed the plant infection. We concluded that supplementation of l- and dl-malate suppresses tomato plant infection with R. pseudosolanacearum MAFF 106611 by disrupting its chemotaxis to l-malate, while d-malate suppresses it by both the disruption of l-malate chemotaxis and inhibition of growth.

Psychrophile-based simple biocatalysts for effective coproduction of 3-hydroxypropionic acid and 1,3-propanediol.

3-Hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) have tremendous potential markets in many industries. This study evaluated the simultaneous biosynthesis of the 2 compounds using the new psychrophile-based simple biocatalyst (PSCat) reaction system. The PSCat method is based on the expression of glycerol dehydratase, 1,3-propanediol dehydrogenase, and aldehyde dehydrogenase from Klebsiella pneumoniae in Shewanella livingstonensis Ac10 and Shewanella frigidimarina DSM 12253, individually. Heat treatment at 45 °C for 15 min deactivated the intracellular metabolic flux, and the production process was started after adding substrate, cofactor, and coenzyme. In the solo production process after 1 h, the maximum production of 3-HP was 62.0 m m. For 1,3-PDO, the maximum production was 25.0 m m. In the simultaneous production process, productivity was boosted, and the production of 3-HP and 1,3-PDO increased by 13.5 and 4.9 m m, respectively. Hence, the feasibility of the individual production and the simultaneous biosynthesis system were verified in the new PSCat approach.

Efficient production of 1,3-propanediol by psychrophile-based simple biocatalysts in Shewanella livingstonensis Ac10 and Shewanella frigidimarina DSM 12253.

1,3-Propanediol (1,3-PDO) is a valuable compound with a large potential market in many industries. This study aimed to evaluate the abilities of the Psychrophile-based Simple bioCatalyst (PSCat) reaction system to biosynthesize 1,3-PDO. This biocatalyst has a potential platform that replaces the chemical-based production counterparts. The two genes involved in the metabolic pathway were expressed both individually and together in the psychrophilic host bacterium. The intracellular metabolic flux was deactivated using heat treatment, at 45 °C for 15 min. After individual gene expression (25.0 mM), 1,3-PDO productivity of the cells increased by approximately 2.5 times, in comparison to when genes were expressed together (10.2 mM). Productivity was boosted (31.1 mM) when the cofactor regeneration system was activated in the biocatalyst. Hence, both the ability of individual gene expression and the cofactor regeneration system were verified in the PSCat approach. Nonetheless, further research is necessary to develop and optimize this process for industrial production.

Characterization of methyl-accepting chemotaxis proteins (MCPs) for amino acids in plant-growth-promoting rhizobacterium Pseudomonas protegens CHA0 and enhancement of amino acid chemotaxis by MCP genes overexpression.

Pseudomonas protegens CHA0, known as plant-growth-promoting rhizobacterium, showed positive chemotactic responses toward proteinaceous L-amino acids. Genomic analysis revealed that P. protegens CHA0 possesses four putative chemoreceptors for amino acids (designated CtaA, CtaB, CtaC, and CtaD, respectively). Pseudomonas aeruginosa PCT2, a mutant defective in chemotaxis to amino acids, harboring a plasmid containing each of ctaA, ctaB, ctaC, and ctaD showed chemotactic responses to 20, 4, 4, and 11 types of amino acids, respectively. To enhance chemotaxis toward amino acids, we introduced the plasmids containing ctaA, ctaB, ctaC, or ctaD into P. protegens CHA0. By overexpression of the genes, we succeeded in enhancing chemotaxis toward more than half of the tested ligands. However, unexpectedly, the P. protegens CHA0 transformants showed unchanged or decreased responses to some amino acids when compared to wild-type CHA0. We speculate that alternation of expression of a chemoreceptor may affect the abundance of other chemoreceptors. ABBREVIATIONS: cDNA: complementary DNA; LBD: ligand-binding domain; MCP: methyl-accepting chemotaxis protein; PDC: PhoQ/DcuS/CitA; PGPR: plant-growth-promoting rhizobacteria; qRT-PCR: quantitative reverse transcription PCR.

Accelerating itaconic acid production by increasing membrane permeability of whole-cell biocatalyst based on a psychrophilic bacterium Shewanella livingstonensis Ac10.

Whole-cell biocatalysts have numerous advantages including ease of preparation and coenzyme recovery over purified industrially used enzymes. However, the cell membrane can occasionally hinder cytoplasmic diffusion of the substrate, resulting in reduced biotransformation efficiency. Psychrophiles can grow and reproduce at low temperatures; their cell membranes are highly flexible, and their permeability can be improved via heat treatment at a moderate temperature. The aim of this study was to generate a psychrophile-based simple biocatalyst (PSCats) using Shewanella livingstonensis Ac10. This biocatalyst contained two enzymes that were heterologously expressed and converted citric acid to itaconic acid, thereby serving as a potential platform replacing the petroleum-based counterparts. The efficiency of the biocatalyst was increased via heat treatment at 45 °C for 15 min, and itaconic acid productivity of the cells after heat treatment (1.41 g/L/h) was increased around 6-fold in comparison with those without heat treatment (0.22 g/L/h). A large part of the productivity remained (67.3 %) when the cells were reused for 5 times (10 h for each reaction). Therefore, the potential of this heat-permeabilized psychrophile host to increase the productivity of whole-cell biocatalyst was proved; however, further research is necessary to understand the underlying mechanism.

Ethanol yield and sugar usability in thermophilic ethanol production from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

Bioconversion from inexpensive renewable resource, such as biomass, to liquid fuel is one of the promising technologies to reduce the use of petroleum. We previously reported the genetically engineered Moorella thermoacetica could produce ethanol from the lignocellulosic feedstock. However, it was still unclear which carbon source in the substrate was preferentially consumed to produce ethanol. To identify the hierarchy of the sugar utilization during ethanol fermentation of this strain, we analyzed the sugar composition of lignocellulosic feedstock, and consumption rate of sugars during the fermentation process. The hydrolysates after acid pretreatment and enzymatic saccharification contained glucose, xylose, galactose, arabinose, and mannose. Time course data suggested that xylose was the most preferred carbon source among those sugars during ethanol fermentation. Ethanol yield was 0.40 ± 0.06 and 0.40 ± 0.12 g/g-total sugar, from lignocellulosic hydrolysates of Japanese cedar (Cryptomeria japonica) and rice straw (Oryza sativa), respectively. The results demonstrated that the genetically engineered M. thermoacetica is a promising candidate for thermophilic ethanol fermentation of lignocellulosic feedstocks, especially hemicellulosic sugars.

Two citrate chemoreceptors involved in chemotaxis to citrate and/or citrate-metal complexes in Ralstonia pseudosolanacearum.

The bacterial wilt pathogen Ralstonia pseudosolanacearum Ps29 exhibited chemotactic responses to citrate. This pathogen expresses 22 putative chemoreceptors. In screening a complete collection of mcp single-gene deletion mutants of Ps29, none showed a significant decrease in response to citrate compared with the wild-type strain. Analysis of a collection of stepwise- and multiple-deletion mutants of Ps29 revealed that the RS_RS07350 homolog (designated McpC) and McpP (chemoreceptor mediating both positive chemotaxis to phosphate and negative chemotaxis to maleate) are chemoreceptors for citrate. Double deletion of mcpC and mcpP markedly reduced the response to citrate, indicating that McpC and McpP are major chemoreceptors for citrate. Wild-type Ps29 was attracted to both free citrate and citrate complexed with divalent metal cations such as magnesium and calcium. The mcpC mcpP double-deletion mutant also showed significant reduction in chemotaxis to Mg2+- and Ca2+-citrate complexes. Introduction of a plasmid harboring the mcpC gene (but not the mcpP gene) restored the ability to respond to these citrate-metal complexes, demonstrating that McpC can sense complexes of citrate and metal ions such as Mg2+ and Ca2+ as well as free citrate. Thus, R. pseudosolanacearum Ps29 expresses two chemoreceptors for citrate. In plant infection assays using tomato seedlings, the mcpC and mcpP single- and double-deletion mutants of the highly virulent R. pseudosolanacearum MAFF106611 strain were as infectious as the wild-type strain, suggesting that citrate chemotaxis does not play an important role in infection of tomato plants in this assay system.

Isolation of High Carotenoid-producing Aurantiochytrium sp. Mutants and Improvement of Astaxanthin Productivity Using Metabolic Information.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe2+), as an oxidative stress compound, to the culture medium significantly enhanced the production of astaxanthin by the mutants. These approaches improved the productivity of astaxanthin up to 9.5 mg/L/day at the flask scale using not only glucose but also fructose which is the main carbon source in fermentation systems with syrup and brown algae as the raw materials.

Efficient conversion of mannitol derived from brown seaweed to fructose for fermentation with a thraustochytrid.

Macroalgae are a promising biomass feedstock for energy and valuable chemicals. Mannitol and alginate are the major carbohydrates found in the microalga Laminaria japonica (Konbu). To convert mannitol to fructose for its utilization as a carbon source in mannitol non-assimilating bacteria, a psychrophile-based simple biocatalyst (PSCat) was constructed using a psychrophile as a host by expressing mesophilic enzymes, including mannitol 2-dehydrogenase for mannitol oxidation, and NADH oxidase and alkyl hydroxyperoxide reductase for NAD+ regeneration. PSCat was treated at 40 °C to inactivate the psychrophilic enzymes responsible for byproduct formation and to increase the membrane permeability of the substrate. PSCat efficiently converted mannitol to fructose with high conversion yield without additional input of NAD+. Konbu extract containing mannitol was converted to fructose with hydroperoxide scavenging, inhibiting the mannitol dehydrogenase activity. Auranthiochytrium sp. could grow well in the presence of fructose converted by PSCat. Thus, PSCat is a potential carbohydrate converter for mannitol non-assimilating microorganism.

Involvement of many chemotaxis sensors in negative chemotaxis to ethanol in Ralstonia pseudosolanacearum Ps29.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

Improved methanization and microbial diversity during batch mode cultivation with repetition of substrate addition using defined organic matter and marine sediment inoculum at seawater salinity.

The activation of microbes, which are needed to initiate continuous methane production, can be accomplished by fed-batch methanization. In the present study, marine sediment inoculum was activated by batch mode methanization with repetition of substrate addition using defined organic matter from sugar, protein, or fat at seawater salinity to investigate the potential for application of the activation method to various types of saline waste and microbial community compositions. All substrates had methane potentials close to the theoretical value except for bovine serum albumin (BSA) whose methane potential was lower, but the maximum methane potential reached the value during repeated methanization. Beta diversity analysis revealed that substrate (especially BSA)-fed and non-fed cultures had distinct microbial community compositions. Bacterial members depended on substrate. Thus, marine sediment inocula activated via the methanization method can be used to effectively treat various types of saline waste.

Identification of boric acid as a novel chemoattractant and elucidation of its chemoreceptor in Ralstonia pseudosolanacearum Ps29.

Chemotaxis enables bacteria to move toward more favorable environmental conditions. We observed chemotaxis toward boric acid by Ralstonia pseudosolanacearum Ps29. At higher concentrations, the chemotactic response of R. pseudosolanacearum toward boric acid was comparable to or higher than that toward L-malate, indicating that boric acid is a strong attractant for R. pseudosolanacearum. Chemotaxis assays under different pH conditions suggested that R. pseudosolanacearum recognizes B(OH)3 (or B(OH3) + B(OH)4-) but not B(OH)4- alone. Our previous study revealed that R. pseudosolanacearum Ps29 harbors homologs of all 22R. pseudosolanacearum GMI1000 mcp genes. Screening of 22 mcp single-deletion mutants identified the RS_RS17100 homolog as the boric acid chemoreceptor, which was designated McpB. The McpB ligand-binding domain (LBD) was purified in order to characterize its binding to boric acid. Using isothermal titration calorimetry, we demonstrated that boric acid binds directly to the McpB LBD with a K D (dissociation constant) of 5.4 µM. Analytical ultracentrifugation studies revealed that the McpB LBD is present as a dimer that recognizes one boric acid molecule.

Negative chemotaxis of Ralstonia pseudosolanacearum to maleate and identification of the maleate chemosensory protein.

Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R. pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R. pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R. pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant.

Thermophilic ethanol fermentation from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

A transformant of Moorella thermoacetica was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, pdul1 and pdul2, and introducing the native aldehyde dehydrogenase gene (aldh) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production.

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073.

For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.