Membrane Potential-requiring Succinate Dehydrogenase Constitutes the Key to Propionate Oxidation and Is Unique to Syntrophic Propionate-oxidizing Bacteria.

Propionate oxidation in Pelotomaculum thermopropionicum is performed under a thermodynamic limit. The most energetically unfavorable reaction in the propionate oxidation pathway is succinate oxidation. Based on previous genomic and transcriptomic ana-lyses, succinate oxidation in P. thermopropionicum under propionate-oxidizing conditions is conducted by the membrane-bound forms of two succinate dehydrogenases (SDHs). We herein examined the activity of SDH, the mechanisms underlying the succinate oxidation reaction in P. thermopropionicum, and the importance of the protein sequences of related genes. SDH activity was highly localized to the membrane fraction. An ana-lysis of the soluble fraction revealed that fumarate reductase received electrons from NADH, suggesting the involvement of membrane-bound SDH in propionate oxidation. We utilized an uncoupler and inhibitors of adenosine triphosphate (ATP) synthase and membrane-bound SDH to investigate whether the membrane potential of P. thermopropionicum supports propionate oxidation alongside hydrogen production. These chemicals inhibited hydrogen production, indicating that membrane-bound SDH requires a membrane potential for succinate oxidation, and this membrane potential is maintained by ATP synthase. In addition, the phylogenetic distribution of the flavin adenine dinucleotide-binding subunit and conserved amino acid sequences of the cytochrome b subunit of SDHs in propionate-oxidizing bacteria suggests that membrane-bound SDHs possess specific conserved amino acid residues that are strongly associated with efficient succinate oxidation in syntrophic propionate-oxidizing bacteria.

Molecular Analysis of MgO Nanoparticle-Induced Immunity against Fusarium Wilt in Tomato.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a devastating soilborne disease in tomatoes. Magnesium oxide nanoparticles (MgO NPs) induce strong immunity against Fusarium wilt in tomatoes. However, the mechanisms underlying this immunity remain poorly understood. Comparative transcriptome analysis and microscopy of tomato roots were performed to determine the mechanism of MgO NP-induced immunity against FOL. Eight transcriptomes were prepared from tomato roots treated under eight different conditions. Differentially expressed genes were compared among the transcriptomes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that in tomato roots pretreated with MgO NPs, Rcr3 encoding apoplastic protease and RbohD encoding NADPH oxidase were upregulated when challenge-inoculated with FOL. The gene encoding glycine-rich protein 4 (SlGRP4) was chosen for further analysis. SlGRP4 was rapidly transcribed in roots pretreated with MgO NPs and inoculated with FOL. Immunomicroscopy analysis showed that SlGRP4 accumulated in the cell walls of epidermal and vascular vessel cells of roots pretreated with MgO NPs, but upon FOL inoculation, SlGRP4 further accumulated in the cell walls of cortical tissues within 48 h. The results provide new insights into the probable mechanisms of MgO NP-induced tomato immunity against Fusarium wilt.

Gene Deletion Algorithms for Minimum Reaction Network Design by Mixed-Integer Linear Programming for Metabolite Production in Constraint-Based Models: gDel_minRN.

Genome-scale constraint-based metabolic networks play an important role in the simulation of growth-coupled production, which means that cell growth and target metabolite production are simultaneously achieved. For growth-coupled production, a minimal reaction-network-based design is known to be effective. However, the obtained reaction networks often fail to be realized by gene deletions due to conflicts with gene-protein-reaction (GPR) relations. Here, we developed gDel_minRN that determines gene deletion strategies using mixed-integer linear programming to achieve growth-coupled production by repressing the maximum number of reactions via GPR relations. The results of computational experiments showed that gDel_minRN could determine the core parts, which include only 30% to 55% of whole genes, for stoichiometrically feasible growth-coupled production for many target metabolites, which include useful vitamins such as biotin (vitamin B7), riboflavin (vitamin B2), and pantothenate (vitamin B5). Since gDel_minRN calculates a constraint-based model of the minimum number of gene-associated reactions without conflict with GPR relations, it helps biological analysis of the core parts essential for growth-coupled production for each target metabolite. The source codes, implemented in MATLAB using CPLEX and COBRA Toolbox, are available on https://github.com/MetNetComp/gDel-minRN.

Biosynthesis of dillapiole/apiole in dill (Anethum graveolens): characterization of regioselective phenylpropene O-methyltransferase.

The phenylpropene volatiles dillapiole and apiole impart one of the characteristic aromas of dill (Anethum graveolens) weeds. However, very few studies have been conducted to investigate the chemical composition of volatile compounds from different developmental stages and plant parts of A. graveolens. In this study, we examined the distribution of volatile phenylpropenes, including dillapiole, in dill plants at various developmental stages. We observed that young dill seedlings accumulate high levels of dillapiole and apiole, whereas a negligible proportion was found in the flowering plants and dry seeds. Based on transcriptomics and co-expression approaches with phenylpropene biosynthesis genes, we identified dill cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase 1 (AgOMT1), an enzyme that can convert 6- and 2-hydroxymyristicin to dillapiole and apiole, respectively, via the methylation of the ortho-hydroxy group. The AgOMT1 protein shows an apparent Km value of 3.5 µm for 6-hydroxymyristicin and is 75% identical to the anise (Pimpinella anisum) O-methyltransferase (PaAIMT1) that can convert isoeugenol to methylisoeugenol via methylation of the hydroxy group at the para-position of the benzene ring. AgOMT1 showed a preference for 6-hydroxymyristicin, whereas PaAIMT1 displayed a large preference for isoeugenol. In vitro mutagenesis experiments demonstrated that substituting only a few residues can substantially affect the substrate specificity of these enzymes. Other plants belonging to the Apiaceae family contained homologous O-methyltransferase (OMT) proteins highly similar to AgOMT1, converting 6-hydroxymyristicin to dillapiole. Our results indicate that apiaceous phenylpropene OMTs with ortho-methylating activity evolved independently of phenylpropene OMTs of other plants and the enzymatic function of AgOMT1 and PaAIMT1 diverged recently.

Highly efficient production of 2,3-butanediol from xylose and glucose by newly isolated thermotolerant Cronobacter sakazakii.

BACKGROUND: 2,3-Butanediol (2,3-BD), a valuable compound used for chemicals, cosmetics, pesticides and pharmaceuticals, has been produced by various microbes. However, no high-temperature fermentation of the compound at high productivity has been reported. METHODS: Thermotolerant xylose-utilizing microbes were isolated from 6 different districts in Laos and screened for a low accumulation of xylitol in a xylose medium at 37 ˚C. One isolate was found to produce 2,3-BD and identified by 16S rDNA sequencing. The 2,3-BD fermentation capacity was investigated at different temperatures using xylose and glucose as carbon sources, and the fermentation parameters were determined by a high-performance liquid chromatography system. RESULTS: By screening for a low accumulation of xylitol in a xylose medium, one isolate that accumulated almost no xylitol was obtained. Further analyses revealed that the isolate is Cronobacter sakazakii and that it has the ability to produce 2,3-BD at high temperatures. When xylose and glucose were used, this strain, named C. sakazakii OX-25, accumulated 2,3-BD in a short period before the complete consumption of these sugars and then appeared to convert 2,3-BD to acetoin. The optimum temperature of the 2,3-BD fermentation was 42 ˚C to 45 ˚C, and the maximum yield of 2,3-BD was 0.3 g/g at 12 h in 20 g/l xylose medium and 0.4 g/g at 6 h in 20 g/l glucose medium at 42 ˚C. The 2,3-BD productivity of the strain was higher than the 2,3-BD productivities of other non-genetically engineered microorganisms reported previously, and the highest productivity was 0.6 g/l·h and 1.2 g/l·h for xylose and glucose, respectively. CONCLUSIONS: Among thermotolerant microbes isolated in Laos, we discovered a strain, C. sakazakii OX-25, that can convert xylose and glucose to 2,3-BD with high efficiency and high productivity at high temperatures, suggesting that C. sakazakii OX-25 has the potential for industrial application to produce 2,3-BD as an important platform chemical.

Evolutionary Adaptation by Repetitive Long-Term Cultivation with Gradual Increase in Temperature for Acquiring Multi-Stress Tolerance and High Ethanol Productivity in Kluyveromyces marxianus DMKU 3-1042.

During ethanol fermentation, yeast cells are exposed to various stresses that have negative effects on cell growth, cell survival, and fermentation ability. This study, therefore, aims to develop Kluyveromyces marxianus-adapted strains that are multi-stress tolerant and to increase ethanol production at high temperatures through a novel evolutionary adaptation procedure. K. marxianus DMKU 3-1042 was subjected to repetitive long-term cultivation with gradual increases in temperature (RLCGT), which exposed cells to various stresses, including high temperatures. In each cultivation step, 1% of the previous culture was inoculated into a medium containing 1% yeast extract, 2% peptone, and 2% glucose, and cultivation was performed under a shaking condition. Four adapted strains showed increased tolerance to ethanol, furfural, hydroxymethylfurfural, and vanillin, and they also showed higher production of ethanol in a medium containing 16% glucose at high temperatures. One showed stronger ethanol tolerance. Others had similar phenotypes, including acetic acid tolerance, though genome analysis revealed that they had different mutations. Based on genome and transcriptome analyses, we discuss possible mechanisms of stress tolerance in adapted strains. All adapted strains gained a useful capacity for ethanol fermentation at high temperatures and improved tolerance to multi-stress. This suggests that RLCGT is a simple and efficient procedure for the development of robust strains.

Distinct Metabolic Flow in Response to Temperature in Thermotolerant Kluyveromyces marxianus.

The intrinsic mechanism of the thermotolerance of Kluyveromyces marxianus was investigated by comparison of its physiological and metabolic properties at high and low temperatures. After glucose consumption, the conversion of ethanol to acetic acid became gradually prominent only at a high temperature (45°C) and eventually caused a decline in viability, which was prevented by exogenous glutathione. Distinct levels of reactive oxygen species (ROS), glutathione, and NADPH suggest a greater accumulation of ROS and enhanced ROS-scavenging activity at a high temperature. Fusion and fission forms of mitochondria were dominantly observed at 30°C and 45°C, respectively. Consistent results were obtained by temperature upshift experiments, including transcriptomic and enzymatic analyses, suggesting a change of metabolic flow from glycolysis to the pentose phosphate pathway. The results of this study suggest that K. marxianus survives at a high temperature by scavenging ROS via metabolic change for a period until a critical concentration of acetate is reached. IMPORTANCE Kluyveromyces marxianus, a thermotolerant yeast, can grow well at temperatures over 45°C, unlike Kluyveromyces lactis, which belongs to the same genus, or Saccharomyces cerevisiae, which is a closely related yeast. K. marxianus may thus bear an intrinsic mechanism to survive at high temperatures. This study revealed the thermotolerant mechanism of the yeast, including ROS scavenging with NADPH, which is generated by changes in metabolic flow.

Cell Lysis Directed by SulA in Response to DNA Damage in Escherichia coli.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

Enhancement of Thermal Resistance by Metal Ions in Thermotolerant Zymomonas mobilis TISTR 548.

The thermal resistance of fermenting microbes is a key characteristic of stable fermentation at high temperatures. Therefore, the effects of various metal ions on the growth of Zymomonas mobilis TISTR 548, a thermotolerant ethanologenic bacterium, at a critical high temperature (CHT) were examined. Addition of Mg2+ and K+ increased CHT by 1°C, but the effects of the addition of Mn2+, Ni2+, Co2+, Al3+, Fe3+, and Zn2+ on CHT were negligible. To understand the physiological functions associated with the addition of Mg2+ or K+, cell morphology, intracellular reactive oxygen species (ROS) level, and ethanol productivity were investigated at 39°C (i.e., above CHT). Cell elongation was repressed by Mg2+, but not by K+. Addition of both metals reduced intracellular ROS level, with only K+ showing the highest reduction strength, followed by both metals and only Mg2+. Additionally, ethanol productivity was recovered with the addition of both metals. Moreover, the addition of Mg2+ or K+ at a non-permissive temperature in 26 thermosensitive, single gene-disrupted mutants of Z. mobilis TISTR 548 revealed that several mutants showed metal ion-specific growth improvement. Remarkably, K+ repressed growth of two mutants. These results suggest that K+ and Mg2+ enhance cell growth at CHT via different mechanisms, which involve the maintenance of low intracellular ROS levels.

Author Correction: MIG1 as a positive regulator for the histidine biosynthesis pathway and as a global regulator in thermotolerant yeast Kluyveromyces marxianus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characteristics of physiology of and genomic mutations in aggregation-enhanced mutants of Methanothermobacter sp. CaT2.

The thermophilic hydrogenotrophic methanogen Methanothermobacter sp. CaT2 aggregates by itself. CaT2 is known to have a surface sugar layer and extracellular proteins that may be related to its aggregation. Aggregation-enhanced mutants, CHA001 and CHA002, were isolated after repeated cultivation for more than two years. When treated with proteinase K, CHA001 and CaT2 similarly exhibited a very low degree of aggregation and CHA002 exhibited less aggregation but still retained aggregation, suggesting protein-based aggregation via extracellular proteins in both CHA001 and CHA002, presumably via a putative membrane-bound and extracellularly protruding protein, MTCT_1020, identified previously. Genomic analysis revealed that CHA001 and CHA002 shared a missense mutation of MTCT_1348 and had distinct mutations. These results suggested that the MTCT_1348 mutation provides subsidiary support to the adhesive function of extracellular proteins and that there is an additional mutation(s) in CHA002 for the non-proteinous aggregation capability.

Integration of comprehensive data and biotechnological tools for industrial applications of Kluyveromyces marxianus.

Among the so-called non-conventional yeasts, Kluyveromyces marxianus has extremely potent traits that are suitable for industrial applications. Indeed, it has been used for the production of various enzymes, chemicals, and macromolecules in addition to utilization of cell biomass as nutritional materials, feed and probiotics. The yeast is expected to be an efficient ethanol producer with advantages over Saccharomyces cerevisiae in terms of high growth rate, thermotolerance and a wide sugar assimilation spectrum. Results of comprehensive analyses of its genome and transcriptome may accelerate studies for applications of the yeast and may further increase its potential by combination with recent biotechnological tools including the CRISPR/Cas9 system. We thus review published studies by merging with information obtained from comprehensive data including genomic and transcriptomic data, which would be useful for future applications of K. marxianus.

MIG1 as a positive regulator for the histidine biosynthesis pathway and as a global regulator in thermotolerant yeast Kluyveromyces marxianus.

Kmmig1 as a disrupted mutant of MIG1 encoding a regulator for glucose repression in Kluyveromyces marxianus exhibits a histidine-auxotrophic phenotype. Genome-wide expression analysis revealed that only HIS4 in seven HIS genes for histidine biosynthesis was down-regulated in Kmmig1. Consistently, introduction of HIS4 into Kmmig1 suppressed the requirement of histidine. Considering the fact that His4 catalyzes four of ten steps in histidine biosynthesis, K. marxianus has evolved a novel and effective regulation mechanism via Mig1 for the control of histidine biosynthesis. Moreover, RNA-Seq analysis revealed that there were more than 1,000 differentially expressed genes in Kmmig1, suggesting that Mig1 is directly or indirectly involved in the regulation of their expression as a global regulator.

Correction: Capacity for survival in global warming: Adaptation of mesophiles to the temperature upper limit.

[This corrects the article DOI: 10.1371/journal.pone.0215614.].

An Aggregation-defective Mutant of Methanothermobacter sp. CaT2 Reveals Unique Protein-dependent Aggregation.

The thermophilic hydrogenotrophic methanogen, Methanothermobacter sp. CaT2, which possesses an extracellular sugar layer, commonly aggregates by itself or with other microorganisms. To elucidate the molecular mechanisms responsible for this aggregation, the aggregation-defective mutant, CLA160, was isolated. Optical and electron microscopy observations revealed that the mutant exhibited a significant reduction in aggregation. Genomic sequencing showed that CLA160 has a single point mutation, causing a nonsense mutation in MTCT_1020, which encodes a hypothetical protein. Motif and domain analyses indicated that the hypothetical protein bears two membrane-spanning segments at the N- and C-terminal regions and a large middle repeat-containing region. The results of a bioinformatic analysis suggested that the first middle region (RII) of the protein or the whole structure is responsible for the function of the product of MTCT_1020 in the aggregation of CaT2. A treatment with proteinase K suppressed sedimentation in CaT2, indicating a reduction in aggregation, with almost no effect on sedimentation in CLA160. The addition of Ca2+ or Mg2+ ions enhanced sedimentation in CaT2, whereas a DNase treatment had no effect on sedimentation in either strain. These results suggest that the hypothetical protein encoded by MTCT_1020 plays a key role as a membrane-bound adhesion protein in the aggregation of CaT2, which is enhanced by the addition of Ca2+ or Mg2+ ions.

Capacity for survival in global warming: Adaptation of mesophiles to the temperature upper limit.

The Intergovernmental Panel on Climate Change recommends keeping the increase in temperature to less than a two-degree increase by the end of the century, but the direct impact of global warming on ecosystems including microbes has not been investigated. Here we performed thermal adaptation of two species and three strains of mesophilic microbes for improvement of the survival upper limit of temperature, and the improvement was evaluated by a newly developed method. To understand the limitation and variation of thermal adaptation, experiments with mutators and by multiple cultures were performed. The results of experiments including genome sequencing and analysis of the characteristics of mutants suggest that these microbes bear a genomic potential to endure a 2-3°C rise in temperature but possess a limited variation of strategies for thermal adaptation.

Flagellum-mediated motility in Pelotomaculum thermopropionicum SI.

The basic functions of a propionate-oxidizing bacterium Pelotomaculum thermopropionicum flagellum, such as motility and chemotaxis, have not been studied. To investigate its motility, we compared with that of Syntrophobacter fumaroxidans, an aflagellar propionate-oxidizing bacterium, in soft agar medium. P. thermopropionicum cells spread, while S. fumaroxidans cells moved downward slightly, indicating flagellum-dependent motility in P. thermopropionicum SI. The motility of P. thermopropionicum was inhibited by the addition of carbonyl cyanide m-chlorophenyl hydrazone, a proton uncoupler, which is consistent with the fact that stator protein, MotB of P. thermopropionicum, shared sequence homology with proton-type stators. In addition, 5-N-ethyl-N-isopropyl amiloride, an Na+ channel blocker, showed no inhibitory effect on the motility. Furthermore, motAB of P. thermopropionicum complemented the defective swimming ability of Escherichia coli ∆motAB. These results suggest that the motility of P. thermopropionicum SI depends on the proton-type flagellar motor.

Improvement of Thermotolerance of Zymomonas mobilis by Genes for Reactive Oxygen Species-Scavenging Enzymes and Heat Shock Proteins.

Thermotolerant genes, which are essential for survival at a high temperature, have been identified in three mesophilic microbes, including Zymomonas mobilis. Contrary to expectation, they include only a few genes for reactive oxygen species (ROS)-scavenging enzymes and heat shock proteins, which are assumed to play key roles at a critical high temperature (CHT) as an upper limit of survival. We thus examined the effects of increased expression of these genes on the cell growth of Z. mobilis strains at its CHT. When overexpressed, most of the genes increased the CHT by about one degree, and some of them enhanced tolerance against acetic acid. These findings suggest that ROS-damaged molecules or unfolded proteins that prevent cell growth are accumulated in cells at the CHT.

Functional analysis of Mig1 and Rag5 as expressional regulators in thermotolerant yeast Kluyveromyces marxianus.

To analyze the glucose repression mechanism in the thermotolerant yeast Kluyveromyces marxianus, disrupted mutants of genes for Mig1 and Rag5 as orthologs of Mig1 and Hxk2, respectively, in Saccharomyces cerevisiae were constructed, and their characteristics were compared with those of the corresponding mutants of S. cerevisiae. MIG1 mutants of both yeasts exhibited more resistance than the corresponding parental strains to 2-deoxyglucose (2-DOG). Histidine was found to be essential for the growth of Kmmig1, but not that of Kmrag5, suggesting that MIG1 is required for histidine biosynthesis in K. marxianus. Moreover, Kmrag5 and Schxk2 were more resistant than the corresponding MIG1 mutant to 2-DOG, and only the latter increased the utilization speed of sucrose in the presence of glucose. Kmrag5 exhibited very low activities for gluco-hexokinase and hexokinase and, unlike Schxk2, showed very slow growth and a low level of ethanol production in a glucose medium. Furthermore, Kmrag5, but not Kmmig1, exhibited high inulinase activity in a glucose medium and exhibited greatly delayed utilization of accumulated fructose in the medium containing both glucose and sucrose. Transcription analysis revealed that the expression levels of INU1 for inulinase and GLK1 for glucokinase in Kmrag5 were higher than those in the parental strain; the expression level of INU1 in Kmmig1 was higher, but the expression levels of RAG1 for a low-affinity glucose transporter in Kmmig1 and Kmrag5 were lower. These findings suggest that except for regulation of histidine biosynthesis, Mig1 and Rag5 of K. marxianus play similar roles in the regulation of gene expression and share some functions with Mig1 and Hxk2, respectively, in S. cerevisiae.

Update of thermotolerant genes essential for survival at a critical high temperature in Escherichia coli.

Previous screening of a single-gene knockout library consisting of 3,908 disrupted-mutant strains allowed us to identify 51 thermotolerant genes that are essential for survival at a critical high temperature (CHT) in Escherichia coli [Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, Kosaka T, Oshima T, Ogasawara N, Yamada M (2011) PLoS ONE 6: e20063]. In this study, we identified another 21 thermotolerant genes. E. coli thus has 72 thermotolerant genes in total. The genes are classified into 8 groups: genes for energy metabolism, outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control, cell division and transporters. This classification and physiological analysis indicate the existence of fundamental strategies for survival at a CHT, which seems to exclude most of the heat shock responses.