Early Logarithmic Phase Research Articles

Inhibitor Tolerance Capacity of Pichia kudriavzevii NBRC1279 and NBRC1664

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD+-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose.

Taxonomic and physiological studies of Parachlorella kimitsuensis sp. nov. (Trebouxiophyceae), which shows high ammonium tolerance

Biological treatments using microalgae represent an environmental-friendly approach to the decontamination of coking wastewater, which contains toxic levels of ammonium; however, few algal species can tolerate such high ammonium levels. In this study, we investigated the morphology, ultrastructure, molecular phylogeny, and physiological characteristics of Parachlorella kimitsuensis sp. nov., a new algal species with high ammonium tolerance. This species was isolated from coking wastewater obtained near a steelworks factory, where the ratio of nitrogen to phosphorus (N/P) exceeded 40, approximately 100-fold that of general culture medium. The growth inhibition test estimated that the effective concentration at 10 % inhibition (EC10) was 3.2 ppm NH4+-N, and the half maximal effective concentration (EC50) was 98.0 ppm NH4+-N, indicating tolerance to fairly high concentrations of ammonium in this species. Parachlorella kimitsuensis has spherical, green cells resembling those of Chlorella species. The major diameter of the cells was 3.7 ± 0.39 μm; cells containing eight autospores appearing in the early logarithmic growth phase were 6.9 ± 0.78 μm in diameter. Molecular phylogenetic analysis showed that the alga was a member of the genus Parachlorella, with 100 % bootstrap support. However, the results of vegetative cell morphology and phylogenetic analyses using 18S rRNA genes were inconsistent with those of other described Parachlorella species. Therefore, we identified the isolate as a new species in the genus Parachlorella. We anticipate that this species will be suitable for biological treatment of wastewater containing high ammonium levels, such as coking wastewater.

Multiobjective response and chemometric approaches to enhance the phytochemicals and biological activities of beetroot leaves: an unexploited organic waste.

Research on medicinal plants is developing each day due to inborn phytochemicals, which can encourage the progress of novel drugs. Most plant-based phytochemicals have valuable effects on well-being. Among them, beetroot leaves (BL) are known for their therapeutic properties. Here, three solvents, namely, acetonitrile, ethanol, and water, and their combinations were developed for BL extraction and simultaneous assessment of phytochemical compounds and antioxidant and antifoodborne pathogen bacteria activities. By using the augmented simplex-centroid mixture design, 40.40% acetonitrile diluted in water at 38.74% and ethanol at 20.86% favored the recovery of 49.28mg GAE/mL (total phenolic content (TPC)) and 0.314mg QE/mL (total flavonoid content (TFC)), respectively. Acetonitrile diluted in water at 50% guarantees the best antioxidant activity, whereas the optimal predicted mixture for the highest antibacterial activity matches 24.58, 50.17, and 25.25% of acetonitrile, ethanol, and water, respectively. These extraction conditions ensured inhibition of Staphylococcus aureus, Salmonella enterica, and Escherichia coli, respectively, at 0.402, 0.497, and 0.207mg/mL. Under optimized conditions, at three concentrations of BL, minimal inhibitory concentration (MIC), 2 × MIC, and 4 × MIC, a linear model was employed to investigate the inhibition behavior against the three tested bacteria. The early logarithmic growth phase of these bacteria illustrated the bactericidal effect of optimized extracted BL with a logarithmic growth phase inferior to 6h. Therefore, BL extract at 4 × MIC, which corresponds to 1.608, 1.988, and 0.828mg/mL, was more efficient against S. aureus, S. enterica, and E. coli.

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

BackgroundOur recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction.ResultsBased on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction.ConclusionThis study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

Synthesis and antibacterial activity evaluation of N (7) position-modified balofloxacins.

A series of small-molecule fluoroquinolones were synthesized, characterized by HRMS and NMR spectroscopy, and screened for their antibacterial activity against MRSA, P. aeruginosa, and E. coli as model G+/G− pathogens. Compounds 2-e, 3-e, and 4-e were more potent than the reference drug balofloxacin against MRSA and P. aeruginosa (MIC values of 0.0195 and 0.039 μg/ml for 2-e, 0.039 and 0.078 μg/ml for each of 3-e and 4-e, respectively). Analysis of the time-dependent antibacterial effect of compound 2-e toward MRSA showed that in the early logarithmic growth phase, bactericidal effects occurred, while in the late logarithmic growth phase, bacterial inhibition occurred because of concentration effects and possibly the development of drug resistance. Compound 2-e exhibited low toxicity toward normal mammalian cell lines 3T3 and L-02 and tumor cell lines A549, H520, BEL-7402, and MCF-7. The compound was not hemolytic. Atomic force microscopy (AFM) revealed that compound 2-e could effectively destroy the membrane and wall of MRSA cells, resulting in the outflow of the cellular contents. Docking studies indicated the good binding profile of these compounds toward DNA gyrase and topoisomerase IV. ADMET’s prediction showed that most of the synthesized compounds followed Lipinski’s “rule of five” and possessed good drug-like properties. Our data suggested that compound 2-e exhibited potent anti-MRSA activity and is worthy of further investigation.

Homeophasic Adaptation in Response to UVA Radiation in Pseudomonas aeruginosa: Changes of Membrane Fatty Acid Composition and Induction of desA and desB Expression.

In bacteria, exposure to changes in environmental conditions can alter membrane fluidity, thereby affecting its essential functions in cell physiology. To adapt to these changes, bacteria maintain appropriate fluidity by varying the composition of the fatty acids of membrane phospholipids, a phenomenon known as homeophasic adaptation. In Pseudomonas aeruginosa, this response is achieved mainly by two mechanisms of fatty acid desaturation: the FabA-FabB and DesA-DesB systems. This study analyzed the effect of ultraviolet-A (UVA) radiation-the major fraction of solar UV radiation reaching the Earth's surface-on the homeophasic process. The prototypical strain PAO1 was grown under sublethal UVA doses or in the dark, and the profiles of membrane fatty acids were compared at early logarithmic, logarithmic and stationary growth phases. In the logarithmic growth phase, it was observed that growth under sublethal UVA doses induced the expression of the desaturase-encoding genes desA and desB and increased the proportion of unsaturated fatty acids; in addition, membrane fluidity could also increase, as suggested by the indices used as indicators of this parameter. The opposite effect was observed in the stationary growth phase. These results demonstrate the relevant role of UVA on the homeophasic response at transcriptional level.

Adhesion mechanisms of Bifidobacterium animalis subsp. lactis JCM 10602 to dietary fiber.

Adherence of probiotics to dietary fibers present in the intestinal tract may affect adhesion to intestinal epithelial cells. The properties of the adhesion of bifidobacteria to mucin or epithelial cells have been well studied; however, adhesion of bifidobacteria to dietary fiber has not been investigated. The adhesion ratio of six Bifidobacterium strains to cellulose and chitin was examined; among the strains, Bifidobacterium animalis subsp. lactis JCM 10602 showed high adherence to both cellulose and chitin, and two strains showed high adherence to only chitin. The ratios of adhesion of B. animalis to cellulose and chitin were positively and negatively correlated with ionic strength, respectively. These data suggest that hydrophobic and electrostatic interactions are involved in the adhesion to cellulose and chitin, respectively. The adhesion ratios of the cells in the late logarithmic phase to cellulose and chitin decreased by approximately 40% and 70% of the cells in the early logarithmic phase, respectively. Furthermore, the adhesion ratio to cellulose decreased with increasing bile concentration regardless of the culture phase of the cells. On the other hand, the adhesion ratio to chitin of cells in the early logarithmic phase decreased with increasing bile concentration; however, that of cells in the late logarithmic phase increased slightly, suggesting that adhesins differ depending on the culture phase. Our results indicated the importance of considering adhesion to both dietary fibers and the intestinal mucosa when using bifidobacteria as probiotics.

Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model.

The increasing challenge of antibiotic resistance requires not only the discovery of new antibiotics, but also the development of new alternative approaches. Therefore, in the present study, we investigated for the first time the antibacterial potential of phytic acid (myo-inositol hexakisphosphate, IP6), a natural molecule that is 'generally recognized as safe' (FDA classification), against the proliferation of common foodborne bacterial pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella Typhimurium. Interestingly, compared to citric acid, IP6 was found to exhibit significantly greater inhibitory activity (P<0.05) against these pathogenic bacteria. The minimum inhibitory concentration of IP6 varied from 0.488 to 0.97 mg/ml for the Gram-positive bacteria that were tested, and was 0.244 mg/ml for the Gram-negative bacteria. Linear and general models were used to further explore the antibacterial effects of IP6. The developed models were validated using experimental growth data for L. monocytogenes, S. aureus and S. Typhimurium. Overall, the models were able to accurately predict the growth of L. monocytogenes, S. aureus, and S. Typhimuriumin Polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol (PALCAM), Chapman broth, and xylose lysine xeoxycholate (XLD) broth, respectively. Remarkably, the early logarithmic growth phase of S. Typhimurium showed a rapid and severe decrease in a period of less than one hour, illustrating the bactericidal effect of IP6. These results suggest that IP6 is an efficient antibacterial agent and can be used to control the proliferation of foodborne pathogens. It has promising potential for environmentally friendly applications in the food industry, such as for food preservation, food safety, and for prolonging shelf life.

Determination of the Effects of Nitrogen and Sulfur Starvationon Chlorophyll Content, Cell Density and Lipid Content in Chlamydomonas reinhardtii

The severe environmental impacts of the use of fossil fuels has led to the development of biofuels as an alternative source of renewable green energy. Currently, algae is being studied as a source for biofuel because it does not compete for arable land and has a fast generation time. The microalgae, Chlamydomonas reinhardtii has potential for biofuel production due to its high lipid body synthesis when subjected to nutrient deprivation. The lipid bodies, predominantly triacylglycerides (TAGs) can be converted into biodiesel which could then be used as a form of biofuel. There is still a lack of research comparing the effects of nutrient starvation in strains of C. reinhardtii. The objectives of this study are: to determine the optimum time at which C. reinhardtii produces the highest amount of lipids under nutrient starvation and to compare the effects of nitrogen, sulfur and both nitrogen sulfur starvation on cell density, chlorophyll content and lipid content of strains cc125. We hypothesize that the nitrogen starvation treatment of C. reinhardtii strain cc125 will produce higher lipid synthesis and the sulfur starvation treatment will produce the highest biomass synthesis.C. reinhardtii strains cc125 were grown to early logarithmic phase in TAP medium liquid culture under constant light on a rotary shaker. Once logarithmic phase was reached, cells were subjected to nitrogen (TAP‐N), sulfur (TAP‐S) and combined nitrogen and sulfur (TAP‐NS) deprivation over a 6 day period to promote lipid accumulation. Samples for cell density, chlorophyll content, and confocal imaging were collected for each strain and nutrient treatment every 2 days. The change in chlorophyll a, chlorophyll b, total chlorophyll and cell density at day 2, 4 and 6 from day 0 were determined. Results showed that changes in chlorophyll a, chlorophyll b and total chlorophyll among all treatments were statistically not significantly different (p&gt;0.05). Chlorophyll a (3.02 ± 2.31), chlorophyll b (1.68 ± 0.85) and total chlorophyll (4.66 ± 2.98) were all highest in treatment TAP‐N and was lowest in TAP‐NS, (chlorophyll a, 0.34 ± 2.30; chlorophyll b, 1.12 ± 0.86; total chlorophyll, 0.6 ± 2.99). There was no statistical difference in cell density among treatments (p&gt;0.05). There was highest increase in cell density in TAP‐S treatment while highest decrease in cell density was observed in TAP‐NS treatment. In addition, there was no statistical difference among days for all treatments in chlorophyll a, chlorophyll b, total chlorophyll and cell density. Results for biomass determination, lipid extractions and quantification; and the qualitative detection of lipid bodies by confocal microscopy will be reported. C. reinhardtii strain sta6 will also be studied. Determining which strain of C. reinhardtii and at which condition will produce high TAGs is essential for engineering and mass producing algae as an established source of biofuel production. This can lead to the adaptation of a viable fuel production system that would resolve the ever growing demand for environmentally sustainable energy resources.Support or Funding InformationACT On IDEASCollege of Arts and Sciences

The ROK like protein of Myxococcus xanthus DK1622 acts as a pleiotropic transcriptional regulator for secondary metabolism

Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias.This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.

Comparative evaluation of eight in vitro pharmacodynamic models of infection: Activity of moxifloxacin against Escherichia coli and Streptococcus pneumoniae as an exemplary example

Use of pharmacodynamic in vitro models provides more clinically relevant information about the activities of antibiotics than static endpoints. Several models are used to simulate pharmacokinetics by dilution of the medium. It is discussed whether this procedure would result in a washout of bacteria, particularly if profiles with a short half-life are simulated. Methods have been developed to minimise the washout of bacteria. Bacteria are retained in the system either by centrifugation and resuspension, use of filters, a capillary unit, dialysis tubing or mathematical correction, versus systems with an unprotected outflow allowing a continuous washout of bacteria. None of these eight models has been directly compared with another. Therefore, an interlaboratory study was performed to address the question of whether or not washout matters. All laboratories used identical batches of media, bacteria, antibiotics and simulated pharmacokinetic profiles with a short or long half-life. Values of area under the bacterial kill-time curve (AUBKC), single-point kill rate and time to 3-log10 reduction of inoculum were calculated. These parameters did not differ significantly between the models. Differences were noted if the inoculum was prepared from the early logarithmic growth phase compared with the late logarithmic or stationary growth phase, resulting either in a pronounced or reduced antibacterial activity. Thus, preparation of inocula affects the results generated, whereas washout of bacteria has apparently a negligible impact on antibacterial activities.

Characterization of Hydrogen Production by Escherichia coli Wild-type and Mutants of Hydrogenases Utilizing Xylose as Fermentation Substrate

Xylose, one of the most common sugars derived from lignocellulosic material, can be fermented by bacteria. Escherichia coli uses natural sugars to produce biomass and H2. The latter can be formed from the formate via formate hydrolysis (FHL) during the fermentation of xylose or glucose; [Ni-Fe]-hydrogenase (Hyd) is involved in H2 formation. The growth, H2 production characteristics of E. coli BW25113 (wild-type), and ΔhyaB, ΔhybC, ΔhycE, or ΔhyfG mutant strains with deletions of catalytic subunits of Hyd-1 to Hyd-4, respectively, were monitored with addition of 0, 0.5, to 1% xylose on peptone and minimal salt-based mediums, pH 5.5 and pH 7.5. At pH 5.5, with the growth of bacteria on both media, H2 produced in the early logarithmic phase (1.40–0.02 mmol H2 L−1), whereas at pH 7.5 it is noticeably delayed. Results showed that during the fermentation of xylose, Hyd-3 and Hyd-4 are important for both bacterial growth and production of H2, mainly at pH 5.5. In addition, high xylose concentrations can stimulate the activity of the Hyd-1 enzyme during bacterial growth at pH 7.5. These results are new and important for developing advanced H2 production technologies using xylose as a feedstock.

A novel fed-batch strategy to boost cyst cells production based on the understanding of intracellular carbon and nitrogen metabolism in Haematococcus pluvialis

Haematococcus pluvialis is a prominent feedstock of astaxanthin. The ratio of carbon to nitrogen (C/N) strongly influences the metabolic pathways of mixotrophic-grown microalgae, however, its role involved in astaxanthin biosynthesis is still not fully understood. In this study, integrative metabolic and physiologic profiles were analyzed in elucidating how C/N affected carbon and nitrogen assimilation and thereby exerted influence on astaxanthin biosynthesis. It was demonstrated that high C/N up-regulated the activities of acetate kinase by increase of 5.76 folds in early logarithmic phase, leading a significant increase of acetyl-CoA. The increased carbon skeletons were then funneled into astaxanthin biosynthesis. Additionally, high C/N increased the proportion of carotenoid-intermediates in cytoplasm from chloroplast. Finally, a fed-batch cultivation strategy based on C/N gradient was developed. Biomass attained 9.18 g L−1 in 100% type of immotile cyst cells, which presented astaxanthin productivity at 15.45 mg L−1 d−1 afterward, exhibiting a promising paradigm in commercial production.

Time-resolved decoding of metabolic signatures of in vitro growth of the hemibiotrophic pathogen Colletotrichum sublineolum

Metabolomics has emerged as a powerful approach to comprehensively interrogate cellular biochemistry. As such, we applied an untargeted liquid chromatography-mass spectrometry metabolomic strategy to elucidate metabolome changes in the anthracnose-causing hemibiotrophic sorghum pathogen, Colletotrichum sublineolum. An in vitro batch culture study model with different carbon sources, glucose, arabinose and rhamnose, were used to support fungal growth over a period of twelve days. Metabolites representing the intracellular and extracellular (secreted) metabolomes were extracted with methanol and subjected to LC-MS analyses. Chemometric modelling revealed a metabolic variation trajectory, comprising three distinct stages that metabolically describe the adaptation of the fungus to diminishing nutrients. Selected marker gene expression indicated stage one (0–3 d.p.i) as corresponding to the early logarithmic phase. Stage two can be interpreted as an intermediate transitionary stage with stage three corresponding to the stationary phase (9–12 d.p.i). Stage one was characterised by up-regulation of endo-metabolites such as ferricrocin, fatty acids and flavone-conjugates, while stage three was characterised by the secretion of phytotoxins, including colletotrichin and colletotric acid. Ultimately, results from our in vitro model reveal previously unknown insights into the dynamic aspects of metabolome reprogramming in the growth phases of Colletotrichum spp as determined by nutrients obtainable from plant cell walls.

Development of a promising microbial platform for the production of dicarboxylic acids from biorenewable resources

BackgroundAs a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast Candida tropicalis has been used as a host for DCA production, additional strains are continually investigated to meet productivity thresholds and industrial needs. In this regard, the yeast Wickerhamiella sorbophila, a potential candidate strain, has been screened. However, the lack of genetic and physiological information for this uncommon strain is an obstacle that merits further research. To overcome this limitation, we attempted to develop a method to facilitate genetic recombination in this strain and produce high amounts of DCAs from methyl laurate using engineered W. sorbophila.ResultsIn the current study, we first developed efficient genetic engineering tools for the industrial application of W. sorbophila. To increase homologous recombination (HR) efficiency during transformation, the cell cycle of the yeast was synchronized to the S/G2 phase using hydroxyurea. The HR efficiency at POX1 and POX2 loci increased from 56.3% and 41.7%, respectively, to 97.9% in both cases. The original HR efficiency at URA3 and ADE2 loci was nearly 0% during the early stationary and logarithmic phases of growth, and increased to 4.8% and 25.6%, respectively. We used the developed tools to construct W. sorbophila UHP4, in which β-oxidation was completely blocked. The strain produced 92.5 g/l of dodecanedioic acid (DDDA) from methyl laurate over 126 h in 5-l fed-batch fermentation, with a productivity of 0.83 g/l/h.ConclusionsWickerhamiella sorbophila UHP4 produced more DDDA methyl laurate than C. tropicalis. Hence, we demonstrated that W. sorbophila is a powerful microbial platform for vegetable oil-based DCA production. In addition, by using the developed genetic engineering tools, this emerging yeast could be used for the production of a variety of fatty acid derivatives, such as fatty alcohols, fatty aldehydes, and ω-hydroxy fatty acids.

Cellular accumulation of cesium in the unicellular red alga Galdieria sulphuraria under mixotrophic conditions

As a consequence of the Fukushima Daiichi Nuclear Power Plant accident which occurred in Japan in 2011, huge amounts of radioactive cesium (Cs) spread across a large area at low concentrations. Bioremediation is known as an effective method for the recovery and concentration of metals at low concentrations. In the present study we showed that G. sulphuraria cells recovered 52 ± 15% of the Cs present in a potassium-deficient medium containing 30 μg L−1 Cs in 10 days. Because of the advantages in biotechnological use, G. sulphuraria is a good candidate for the decontamination of radioactive Cs. In G. sulphuraria cells, Cs was accumulated specifically under mixotrophic condition of the three different culture conditions used (autotorophic, mixotrophic, and heterotrophic condition). Cells in the early logarithmic phase of growth did not accumulate Cs, whereas those in the late logarithmic phase did. Cs accumulated in the cell exists mainly in a soluble form (e.g., salt, chelating complex).

Isolation and preliminary characterisation of bacteriocin produced by Enterococcus faecium GHB21 isolated from Algerian paste of dates “ghars”

The bacteriocin-producing strain GHB21 isolated from the Algerian traditional paste of dates “ghars” was identified as Enterococcus faecium based on 16S rRNA sequence analysis. The bacteriocin produced by Enterococcus faecium GHB21 was sensitive to proteases, heat-stable, resistant to a wide range of pH and also to several chemicals. Recorded bacteriocin activity was higher when GHB21 was fermented in a bioreactor compared with static fermentation in a flask; it was also shown to be growth-associated and to display primary metabolite kinetics as its maximal production was detected in the early logarithmic growth phase. GHB21 presented a large activity spectrum towards different genera, and proved active against either Gram positive or Gram negative bacteria, including some pathogenic and/or spoilage bacteria. Partial purification of this bacteriocin by an adsorption-desorption method led to a fraction that lost its entire activity after 1 week of storage at 4 °C, or after 15 days when supplemented with bovine serum albumin; nevertheless the lyophilised fraction could remain stable with full activity even after 1 year. The partially purified bacteriocin exhibited a bactericidal effect on Listeria ivanovii subsp. ivanovii ATCC 19119 and could lead to its total inhibition after only 6 h but without concomitant cell lysis. Tricine-SDS-PAGE analysis of the partially purified bacteriocin indicated a low molecular weight of approximately 5 kDa.

The Neisseria gonorrhoeae Obg protein is an essential ribosome-associated GTPase and a potential drug target.

BackgroundNeisseria gonorrhoeae (GC) is a Gram-negative pathogen that most commonly infects mucosal surfaces, causing sexually transmitted urethritis in men and endocervicitis in women. Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility. The incidence of gonorrhea cases remains high globally while antibiotic treatment options, the sole counter measures against gonorrhea, are declining due to the remarkable ability of GC to acquire resistance. Evaluating of potential drug targets is essential to provide opportunities for developing antimicrobials with new mechanisms of action. We propose the GC Obg protein, belonging to the Obg/CgtA GTPase subfamily, as a potential target for the development of therapeutic interventions against gonorrhea, and in this study perform its initial functional and biochemical characterization.ResultsWe report that NGO1990 encodes Obg protein, which is an essential factor for GC viability, associates predominantly with the large 50S ribosomal subunit, and is stably expressed under conditions relevant to infection of the human host. The anti-Obg antisera cross-reacts with a panel of contemporary GC clinical isolates, demonstrating the ubiquitous nature of Obg. The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth. The in vitro binding and hydrolysis of the fluorescent guanine nucleotide analogs mant-GTP and mant-GDP by recombinant wild type and T192AT193A mutated variants of Obg are also assessed.ConclusionsCharacterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0453-1) contains supplementary material, which is available to authorized users.

Streptomyces subrutilus P5가 생산하는 철 함유 superoxide dismutase의 분비

We tried to analyze the growth time for secretion of the iron containing superoxide dismutase by comparing the intra-and extracellular enzyme activity from Streptomyces subrutilus P5 and analyze possible genetic information for this enzyme secretion. The mycelial dry weights and glucose concentrations in culture filtrates were determined during growth. Glucose was consumed rapidly during logarithmic growth phase and almost exhausted at 24 h of cultivation. While the intracellular activity of iron containing superoxide dismutase was first appeared at three hours, the extracellular activity of this enzyme appeared from 7.5 h of cultivation, early logarithmic growth phase. This early presence of the superoxide dismutase might not be the result of cell lysis but active secretion pathway. There was no information for signal peptide responsible for the enzyme secretion in sodF. However, we found a type three secretion box in the promoter region of sodF that has been known for the genes of type III secreted proteins in other bacteria. This is the first report on the possible existence of type III secretion in Streptomyces.

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane.

This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.