Characterization of the Pyrroloquinoline Quinone Producing Rhodopseudomonas palustris as a Plant Growth-Promoting Bacterium under Photoautotrophic and Photoheterotrophic Culture Conditions.

Rhodopseudomonas palustris is a purple non-sulfide bacterium (PNSB), and some strains have been proven to promote plant growth. However, the mechanism underlying the effect of these PNSBs remains limited. Based on genetic information, R. palustris possesses the ability to produce pyrroloquinoline quinone (PQQ). PQQ is known to play a crucial role in stimulating plant growth, facilitating phosphorous solubilization, and acting as a reactive oxygen species scavenger. However, it is still uncertain whether growth conditions influence R. palustris's production of PQQ and other characteristics. In the present study, it was found that R. palustris exhibited a higher expression of genes related to PQQ synthesis under autotrophic culture conditions as compared to acetate culture conditions. Moreover, similar patterns were observed for phosphorous solubilization and siderophore activity, both of which are recognized to contribute to plant-growth benefits. However, these PNSB culture conditions did not show differences in Arabidopsis growth experiments, indicating that there may be other factors influencing plant growth in addition to PQQ content. Furthermore, the endophytic bacterial strains isolated from Arabidopsis exhibited differences according to the PNSB culture conditions. These findings imply that, depending on the PNSB's growing conditions, it may interact with various soil bacteria and facilitate their infiltration into plants.

Conversion of Escherichia coli into Mixotrophic CO2 Assimilation with Malate and Hydrogen Based on Recombinant Expression of 2-Oxoglutarate:Ferredoxin Oxidoreductase Using Adaptive Laboratory Evolution.

We report the mixotrophic growth of Escherichia coli based on recombinant 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO2 using malate as an auxiliary carbon source and hydrogen as an energy source. We employ a long-term (~184 days) two-stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli. In the first stage of evolution with serine, diauxic growth emerges as a prominent feature. At the end of the second stage of evolution with malate, the strain exhibits mixotrophy with CO2 as an essential substrate for growth. We expect this work will open new possibilities in the utilization of OGOR for microbial CO2 assimilation and future hydrogen-based electro-microbial conversion.

Utilization of Monosaccharides by Hungateiclostridium thermocellum ATCC 27405 through Adaptive Evolution.

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden-Mayerhof-Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture.

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC-MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

Biodegradation of dioxins by Burkholderia cenocepacia strain 869T2: Role of 2-haloacid dehalogenase.

Dioxin compounds are persistent carcinogenic byproducts of anthropogenic activities such as waste combustion and other industrial activities. The ubiquitous distribution of dioxins is global concerns these days. Among of recent techniques, bioremediation, an eco-friendly and cost-effective technology, uses bacteria or fungi to detoxify in dioxins; however, not many bacteria can degrade the most toxic dioxin congener 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD). In this study, the endophytic bacterium Burkholderia cenocapacia 869T2 was capable of TCDD degradation by nearly 95 % after one-week of an aerobic incubation. Through transcriptomic analysis of the strain 869T2 at 6 -h and 12 -h TCDD exposure, a number of catabolic genes involved in dioxin metabolism were detected with high gene expressions in the presence of TCDD. The transcriptome data also indicated that B. cenocepacia strain 869T2 metabolized the dioxin compounds from an early phase (at 6 h) of the incubation, and the initial outline for a general dioxin degradation pathway were proposed. One of the catabolic genes, l-2-haloacid dehalogenase (2-HAD) was cloned to investigate its contribution in dioxin dehalogenation. By detecting the increasing concentration of chloride ions released from TCDD, our results indicated that the dehalogenase played a crucial role in dehalogenation of dioxin in the aerobic condition.

Potential novel proteomic biomarkers for diagnosis of vertebral osteomyelitis identified using an immunomics protein array technique: Two cases reports.

RATIONALE: Although vertebral osteomyelitis (VO) is commonly associated with high morbidity and high recurrence rate, effective diagnostic and prognostic biomarkers of VO are still lacking. PATIENTS CONCERNS: Case 1: a 60-year-old male had had upper back pain for 3 days. Case 2: a 71-year-old female presented upper back pain for 2 days. DIAGNOSES: Based on physical examination and findings of magnetic resonance imaging and findings by matrix-assisted laser desorption ionization-time of flight mass spectrometry, they were diagnosed with Staphylococcus aureus VO. INTERVENTIONS: Using Sengenics Immunome Protein Array by analyzing autoantibodies in both VO patients, potential biomarkers of VO were explored. OUTCOMES: Four subjects with more than 1600 antigens screened while the results showed that 14-3-3 protein gamma, pterin-4-alpha-carbinolamine dehydratase, fructose-bisphosphate aldolase A, and keratin type II cytoskeletal 8 were highly differentially expressed among VO and controls. Relevant auto-antibody profiles were discovered after intra-group and inter-group comparison, and based on functional rationality, an adapter protein 14-3-3 protein gamma, and pterin-4-alpha-carbinolamine dehydratase that involved in tetrahydrobiopterin biosynthesis, might serve as valuable diagnostic biomarkers. LESSONS: This pilot study on 4 subjects with more than 1600 antigens screened on the Sengenics Immunome protein array provided a general outlook on autoantibody biomarker profiles of VO subjects. Future large-scale trials with longer follow-up times are warranted.

Exploring Potential Proteomic Biomarkers for Prognosis of Infective Endocarditis through Profiled Autoantibodies by an Immunomics Protein Array Technique.

Though infective endocarditis (IE) is a life-threatening cardiac infection with a high mortality rate, the effective diagnostic and prognostic biomarkers for IE are still lacking. The aim of this study was to explore the potential applicable proteomic biomarkers for IE through the Immunome™ Protein Array system. The system was employed to profile those autoantibodies in IE patients and control subjects. Our results showed that interleukin-1 alpha (IL1A), nucleolar protein 4 (NOL4), tudor and KH domain-containing protein (TDRKH), G antigen 2B/2C (GAGE2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and X antigen family member 2 (XAGE2) are highly differentially-expressed among IE and non-IE control. Furthermore, bactericidal permeability-increasing protein (BPI), drebrin-like protein (DBNL), signal transducing adapter molecule 2 (STAM2), cyclin-dependent kinase 16 (CDK16), BAG family molecular chaperone regulator 4 (BAG4), and nuclear receptor-interacting protein 3 (NRIP3) are differentially-expressed among IE and healthy controls. On the other hand, those previously identified biomarkers for IE, including erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, procalcitonin, and N-terminal-pro-B-type natriuretic peptide demonstrated only minor significance. With scientific rationalities for those highly differentially-expressed proteins, they could serve as potential candidates for diagnostic biomarkers of IE for further analysis.

Manipulating ATP supply improves in situ CO2 recycling by reductive TCA cycle in engineered Escherichia coli.

The reductive tricarboxylic acid (rTCA) cycle was reconstructed in Escherichia coli by introducing pGETS118KAFS, where kor (encodes α-ketoglutarate:ferredoxin oxidoreductase), acl (encodes ATP-dependent citrate lyase), frd (encodes fumarate reductase), and sdh (encodes succinate dehydrogenase) were tandemly conjugated by the ordered gene assembly in Bacillus subtilis (OGAB). E. coli MZLF (E. coli BL21(DE3) Δzwf, Δldh, Δfrd) was employed so that the C-2/C-1 [(ethanol + acetate)/(formate + CO2)] ratio can be used to investigate the effectiveness of the recombinant rTCA for in situ CO2 recycling. It has been shown that supplying ATP through the energy pump (the EP), where formate donates electron to nitrate to form ATP, elevates the C-2/C-1 ratio from 1.03 ± 0.00 to 1.49 ± 0.02. Similarly, when ATP production is increased by the introduction of the heterologous ethanol production pathway (pLOI295), the C-2/C-1 ratio further increased to 1.79 ± 0.02. In summary, the ATP supply is a rate-limiting step for in situ CO2 recycling by the recombinant rTCA cycle. The decrease in C-1 is significant, but the destination of those recycled C-1 is yet to be determined.

Detailed profiling of carbon fixation of in silico synthetic autotrophy with reductive tricarboxylic acid cycle and Calvin-Benson-Bassham cycle in Esherichia coli using hydrogen as an energy source.

Carbon fixation is the main route of inorganic carbon in the form of CO2 into the biosphere. In nature, RuBisCO is the most abundant protein that photosynthetic organisms use to fix CO2 from the atmosphere through the Calvin-Benson-Bassham (CBB) cycle. However, the CBB cycle is limited by its low catalytic rate and low energy efficiency. In this work, we attempt to integrate the reductive tricarboxylic acid and CBB cycles in silico to further improve carbon fixation capacity. Key heterologous enzymes, mostly carboxylating enzymes, are inserted into the Esherichia coli core metabolic network to assimilate CO2 into biomass using hydrogen as energy source. Overall, such a strain shows enhanced growth yield with simultaneous running of dual carbon fixation cycles. Our key results include the following. (i) We identified two main growth states: carbon-limited and hydrogen-limited; (ii) we identified a hierarchy of carbon fixation usage when hydrogen supply is limited; and (iii) we identified the alternative sub-optimal growth mode while performing genetic perturbation. The results and modeling approach can guide bioengineering projects toward optimal production using such a strain as a microbial cell factory.

Characterizing an engineered carotenoid-producing yeast as an anti-stress chassis for building cell factories.

BACKGROUND: A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. RESULTS: Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. CONCLUSIONS: The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.

Genomic sequence analysis of a plant-associated Photobacterium halotolerans MELD1: from marine to terrestrial environment?

Mercury impacts the function and development of the central nervous system in both humans and wildlife by being a potent neurotoxin. Microbial bioremediation is an important means of remediation of mercury-contaminated soil. The rhizospheric Photobacterium halotolerans strain MELD1 was isolated from mercury and dioxin contaminated site from Tainan, Taiwan. It has been shown to reduce Hg(2+) to Hg(0). The 4,758,027 bp genome of P. halotolerans MELD1 has a G + C content of 50.88 % and contains 4198 protein-coding and 106 RNA genes. Genomic analysis revealed the presence of a number of interesting gene cluster that maybe involved in heavy metal resistance, rhizosphere competence and colonization of the host plant.

Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA.

The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.

Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP's RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.

Recent insights into the development of therapeutics against coronavirus diseases by targeting N protein.

The advent of severe acute respiratory syndrome (SARS) in the 21st century and the recent outbreak of Middle-East respiratory syndrome (MERS) highlight the importance of coronaviruses (CoVs) as human pathogens, emphasizing the need for development of novel antiviral strategies to combat acute respiratory infections caused by CoVs. Recent studies suggest that nucleocapsid (N) proteins from coronaviruses and other viruses can be useful antiviral drug targets against viral infections. This review aims to provide readers with a concise survey of the structural features of coronavirus N proteins and how these features provide insights into structure-based development of therapeutics against coronaviruses. We will also present our latest results on MERS-CoV N protein and its potential as an antiviral drug target.

Synergistic collaboration of gut symbionts in Odontotermes formosanus for lignocellulosic degradation and bio-hydrogen production.

In this work, gut microbes from the macrotermitine termite Odontotermes formosanus the cellulolytic Bacillus and fermentative Clostridium were studied in batch experiments using different carbon substrates to bio-mimic the termite gut for hydrogen production. Their fungus comb aging and the in vitro lignocellulosic degradation of the mango tree substrates by the synergistic interaction of Bacillus, Clostridium and Termitomyces were detected by Solid-state NMR. From the results, Bacillus species acted as a mutualist, by initiating an anaerobic environment for the growth of Clostridium, for bio-hydrogen production and the presence of Termitomyces enhanced the lignocellulosic degradation of substrates in vitro and in vivo. Thus, the synergistic collaboration of these three microbes can be used for termite-derived bio-fuel processing technology.

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase.

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 °C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 °C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.