Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Characterization of mating-type idiomorphs suggests that <Emphasis Type="Italic">Morchella importuna,Mel-20</Emphasis> and <Emphasis Type="Italic">M. sextelata</Emphasis> are heterothallic

Morels (Morchella spp.) are highly prized for their culinary qualities and intensively collected worldwide by mycophiles. Morels are divided into three clades by phylogenetic analyses: black morels, yellow morels and the rufobrunnea clade. Morchella importuna, Mel-20 and M. sextelata are included in the black morel clade and are widely distributed in Yunnan province, China. M. importuna and M. sextelata have been artificially cultured in recent years, but their life cycles and reproductive systems are still poorly understood, which delays the progress of morel cultivation. In this study, the genomes of two ascospore isolates of M. importuna with opposite mating-type were sequenced and two idiomorphs, MAT1–1 and MAT1–2, were identified. The MAT1–2 idiomorph was 6.7 kb in length containing a single MAT1–2-1 gene, and the MAT1–1 idiomorph was 10.5 kb containing a MAT1–1-1 gene and two other open reading frames (ORFs), GME3123 and GME3124. These ORFs differed greatly from the homologues of previously published mating-type genes; therefore, we speculate that they are novel mating genes found only in morels. Single-ascospore populations of M. importuna, M. sextelata and Mel-20 were analysed, and the result indicated that the ratios of MAT1–1- and MAT1–2-harbouring idiomorphs were not significantly different from a 1:1 ratio. The results suggest that these three black morels are heterothallic.     

Development of SCoT-Based SCAR Marker for Rapid Authentication of <Emphasis Type="Italic">Taxus Media</Emphasis>

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530 bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.     

Morphological and physiological differences among cultivation lines of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> in a common garden experiment using a tank culture system

Undaria pinnatifida is grown for food and industrial materials worldwide; therefore, advanced breeding is needed to meet quality and productivity requirements. In this study, we examined regional lines of U. pinnatifida from five cultivation sites in Japan with different environmental conditions: Oga (OGA, the northern Sea of Japan coast), Hirota Bay (HRT, the northeastern Pacific coast), Matsushima Bay (MAT, the northeastern Pacific coast), Naruto (the Seto Inland Sea coast) and Shimonoseki (SIM, the southern Sea of Japan coast). The sporophytes of these lines were cultured in a tank culture system under controlled environmental conditions, and their morphological characteristics, nutrient uptake kinetics (V _max, K _s and V _max/K _s ), and carbon, nitrogen and phosphorus contents were determined. Sporophytes from MAT grew faster, whereas those from SIM were smaller than those from the other sites. Although the blade thickness of sporophytes cultivated in the sea significantly differs among cultivation sites in the previous study, there was no significant difference in blade thickness among the regional lines cultivated in the tank. Sporophytes from OGA had the greatest V _max/K _s values and significantly greater nitrogen contents than the other lines. Therefore, the morphological characteristics of MAT and SIM sporophytes, and the nutrient uptake kinetics of OGA sporophytes may have a genetic origin. This indicates that these lines may represent useful resources for selective breeding, with MAT sporophytes providing faster growth and OGA sporophytes being well-adapted to low-nutrient conditions.     

Metabolism of the thermophilic bacterium <Emphasis Type="Italic">Oceanithermus profundus</Emphasis>

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurred via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.     

Heat and hypoxia give a global invader, <Emphasis Type="Italic">Gambusia holbrooki,</Emphasis> the edge over a threatened endemic fish on Australian floodplains

Deciphering the mechanisms by which climate change interacts with invasive species to affect biodiversity is a major challenge of global change biology. We conducted experiments to determine whether the global invader, Gambusia holbrooki, was more resistant to high water temperature (heat) and low dissolved oxygen (hypoxia) than a threatened native fish, Nannoperca australis. Metabolic experiments conducted at 25 and 29 °C showed that G. holbrooki had at least four times the capacity for metabolic depression during hypoxia than N. australis. An increase in environmental temperature from 25 to 29 °C had no significant impact on the critical oxygen tension, P _crit, of G. holbrooki, but significantly and strongly increased P _crit of N. australis. Gambusia holbrooki also had a lower Q ₁₀ of standard metabolic rate than N. australis. Our results indicate that G. holbrooki have physiological traits conferring greater resistance to hypoxia than N. australis, and as temperature increases, the resistance of N. australis to hypoxia was more eroded than that of G. holbrooki. Intensive monitoring of the temperature and dissolved oxygen dynamics of wetlands showed that contemporary heat waves are already causing conditions that might give G. holbrooki the edge over N. australis on Australian floodplains. Our study adds weight to recent anecdotal reports of drought and heat waves causing localised extinction of N. australis, but the proliferation of G. holbrooki.     

Frequency of <Emphasis Type="Italic">Aedes sp</Emphasis>. Linnaeus (Diptera: Culicidae) and Associated Entomofauna in Bromeliads from a Forest Patch within a densely Urbanized Area

Little is known about the relationship between the presence of Aedes, abiotic factors and the entomofauna existing in phytotelmatas. The aim of this study was to identify biotic and abiotic factors associated with the presence of Aedes in bromeliads sites located in a forest fragment in Rio de Janeiro, Brazil. In the study area, eight bromeliads from the Aechmea genus were chosen and kept in landscape form. Physical and chemical variables were measured. Collected aquatic insects were identified according to the literature. A total of 3102 immature insects were collected. The presence of Aedes aegypti (Linnaeus) (2.29%) was rare. Few specimens were found concentrated in urban-adjacent areas during summer. On the other hand, Aedes albopictus (Skuse) (17.57%) was found throughout the year in 0%–80% of the sites, averaging 1.0 mosq/bromelia. Aedes albopictus was found predominantly in central sites of the forest fragment. The Canonical Correspondence Analysis indicates that most taxa had a moderate association with temperature, dissolved oxygen, and pH. The abiotic variables, such as temperature and dissolved oxygen, affect the distribution of the genus Aedes vectors, while most of the other variables did not.     

Microaerophilic alkane degradation in <Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis>: a transcriptomic and physiological approach

Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13–C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments.     

Different responses in the expression of alginases,alginate polymerase and acetylation genes during alginate production by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> under oxygen-controlled conditions

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate \((q_{{{\text{O}}_{ 2} }} )\) from 10.9 to 45.3 mmol g^?1 h^?1 by changes in the dilution rate (D) from 0.06 to 0.10 h^?1, whereas under 1% DOT the \(q_{{{\text{O}}_{ 2} }}\) was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.     

ClpXP affects the cell metabolism of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> partially in an RpoS-dependent manner

Introduction

ClpXP protease is an important proteolytic system in Salmonella enterica serovar typhimurium (S. typhimurium). Inactivation of ClpXP by deletion of clpP resulted in overproduction of RpoS and a growth defect phenotype. Only one report has indicated that deleting rpoS can restore the growth of a S. typhimurium clpP mutant to the wild-type level. Whether overproduction of RpoS is responsible for the growth deficiency resulting from clpP disruption and how ClpXP affects the cell metabolism of S. typhimurium remain to be elucidated.

Objectives

The aim of this study is to investigate the effect of ClpXP on cell metabolism of S. typhimurium and explore the possible co-effect of RpoS associated with ClpXP in cell metabolism.

Method

We constructed a clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) using a two-step phage transduction technique. We then compared the metabolite fingerprints of Salmonella rpoS deletion mutant TT-14 (ΔrpoS TT-1), clpP deletion mutant TT-16 (ΔclpP TT-1), and clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) with those of the wild-type strain TT-1 by using gas chromatography coupled with mass spectrometry (GC–MS).

Results

Deletion of rpoS recovered only a part of the growth of Salmonella clpP mutant. Further metabolome analysis indicated that clpP disruption changed the levels of 16 extra- and 19 intracellular substances, while the extracellular concentrations of 4 compounds (serine, l-5-oxoproline, l-glutamic acid, and l-tryptophan) and intracellular concentrations of 10 compounds (l-isoleucine, glycine, serine, l-methionine, l-phenylalanine, malic acid, citric acid, urea, putrescine, and 6-hydroxypurine) returned to their wild-type levels when rpoS was also deleted.

Conclusion

ClpXP affects the cell metabolism of S. typhimurium partially in an RpoS-dependent manner.

     

Oxygen transfer as a tool for fine-tuning recombinant protein production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> under glyceraldehyde-3-phosphate dehydrogenase promoter

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q _O/V = 3 and 10 vvm, and agitation rate was N = 900 min^?1; while the other one was based on constant dissolved oxygen concentrations, C _DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ _o = 0.15 h^?1. The highest cell concentration was obtained as 44 g L^?1 at t = 9 h of the glucose fed-batch phase at C _DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L^?1 and 126 U g^?1 cell, respectively at C _DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C _DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K _L a = 0.045 s^?1 and OUR = 8.91 mmol m^?3 s^?1, respectively.     

The eurythermal adaptivity and temperature tolerance of a newly isolated psychrotolerant Arctic <Emphasis Type="Italic">Chlorella</Emphasis> sp.

A new strain of Chlorella sp. (Chlorella-Arc), isolated from Arctic glacier melt water, was found to have high specific growth rates (μ) between 3 and 27 °C, with a maximum specific growth rate of 0.85 day^?1 at 15 °C, indicating that this strain was a eurythermal strain with a broad temperature tolerance range. To understand its acclimation strategies to low and high temperatures, the physiological and biochemical responses of the Chlorella-Arc to temperature were studied and compared with those of a temperate Chlorella pyrenoidosa strain (Chlorella-Temp). As indicated by declining F _v/F _m, photoinhibition occurred in Chlorella-Arc at low temperature. However, Chlorella-Arc reduced the size of the light-harvesting complex (LHC) to alleviate photoinhibition, as indicated by an increasing Chl a/b ratio with decreasing temperatures. Interestingly, Chlorella-Arc tended to secrete soluble sugar into the culture medium with increasing temperature, while its intracellular soluble sugar content did not vary with temperature changes, indicating that the algal cells might suffer from osmotic stress at high temperature, which could be adjusted by excretion of soluble sugar. Chlorella-Arc accumulated protein and lipids under lower temperatures (<15 °C), and its metabolism switched to synthesis of soluble sugar as temperatures rose. This reflects a flexible ability of Chlorella-Arc to regulate carbon and energy distribution when exposed to wide temperature shifts. More saturated fatty acids (SFA) in Chlorella-Arc than Chlorella-Temp also might serve as the energy source for growth in the cold and contribute to its cold tolerance.