Overexpression Strain Research Articles

Effects of SNF1 on Maltose Metabolism and Leavening Ability of Baker's Yeast in Lean Dough.

Maltose metabolism of baker's yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression, thereby delaying the dough fermentation. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. The Snf1 protein kinase is well known to be essential for the response to glucose repression and required for transcription of glucose-repressed genes including the maltose-utilization genes (MAL). In this study, the SNF1 overexpression and deletion industrial baker's yeast strains were constructed and characterized in terms of maltose utilization, growth and fermentation characteristics, mRNA levels of MAL genes (MAL62 encoding the maltase and MAL61 encoding the maltose permease) and maltase and maltose permease activities. Our results suggest that overexpression of SNF1 was effective to glucose derepression for enhancing MAL expression levels and enzymes (maltase and maltose permease) activities. These enhancements could result in an 18% increase in maltose metabolism of industrial baker's yeast in LSMLD medium (the low sugar model liquid dough fermentation medium) containing glucose and maltose and a 15% increase in leavening ability in lean dough. These findings provide a valuable insight of breeding industrial baker's yeast for rapid fermentation.

Dme-miR-314-3p modulation in Cr(VI) exposed Drosophila affects DNA damage repair by targeting mus309

microRNAs (miRNAs) as one of the major epigenetic modulators negatively regulate mRNAs at post transcriptional level. It was therefore hypothesized that modulation of miRNAs by hexavalent Chromium [Cr(VI)], a priority environmental chemical, can affect DNA damage. In a genetically tractable model, Drosophila melanogaster, role of maximally up-regulated miRNA, dme-miR-314-3p, on DNA damage was examined by exposing the third instar larvae to 5.0–20.0μg/ml Cr(VI) for 24 and 48h. mus309, a Drosophila homologue of human Bloom’s syndrome and predicted as one of the potential targets of this miRNA, was confirmed as its target by 5′RLM-RACE assay. A significant down-regulation of mus309 was observed in dme-miR-314-3p overexpression strain (myo-gal4>UAS-miR-314-3p) as compared with that in parental strains (myo-gal4 and UAS-miR-314-3p) and in w1118. A significant increase in DNA damage including double strand breaks generation was observed in exposed myo-gal4>UAS-miR-314 and mus309 mutants as compared with that in parental strain and in unexposed control. A significant down-regulation of cell cycle regulation genes (CycA, CycB and cdc2) was observed in these exposed genotypes. Collectively, the study demonstrates that dme-miR-314-3p can mediate the downregulation of repair deficient gene mus309 leading to increased DNA damage and cell cycle arrest in exposed organism which may affect Cr(VI) mediated carcinogenesis.

The role of H4 flagella in Escherichia coli ST131 virulence.

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug resistant clone associated with urinary tract and bloodstream infections. Most ST131 strains exhibit resistance to multiple antibiotics and cause infections associated with limited treatment options. The largest sub-clonal ST131 lineage is resistant to fluoroquinolones, contains the type 1 fimbriae fimH30 allele and expresses an H4 flagella antigen. Flagella are motility organelles that contribute to UPEC colonisation of the upper urinary tract. In this study, we examined the specific role of H4 flagella in ST131 motility and interaction with host epithelial and immune cells. We show that the majority of H4-positive ST131 strains are motile and are enriched for flagella expression during static pellicle growth. We also tested the role of H4 flagella in ST131 through the construction of specific mutants, over-expression strains and isogenic mutants that expressed alternative H1 and H7 flagellar subtypes. Overall, our results revealed that H4, H1 and H7 flagella possess conserved phenotypes with regards to motility, epithelial cell adhesion, invasion and uptake by macrophages. In contrast, H4 flagella trigger enhanced induction of the anti-inflammatory cytokine IL-10 compared to H1 and H7 flagella, a property that may contribute to ST131 fitness in the urinary tract.

Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides.

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig.1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70% (from 13 to 22% dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27% (from 13 to 9.5% CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

Identification and characterization of a flavin-containing monooxygenase MoA and its function in a specific sophorolipid molecule metabolism in Starmerella bombicola.

The yeast Starmerella bombicola CGMCC 1576 can produce abundant sophorolipids (SLs) including almost equal proportion of acidic and lactonic SLs. In this study, a monooxygenase MoA responsible for the metabolism of a sophorolipid molecule, C18:2 diacetylated acidic sophorolipid (C18:2 DASL), was identified, through genomic analysis, protein modeling, and gene knocking out strategy. The yield and compositions of SLs produced by the deletion mutant ∆moA changed dramatically. In HPLC chromatogram, the UV absorption area of C18:2 DASL (one major acidic sophorolipid component) increased from 9.84 × 10(6) mAU × s to 34.26 × 10(6) mAU × s by an increase of 248.17% when oleic acid was used as hydrophobic carbon source. Moreover, when linoleic acid was used as hydrophobic carbon source, the content of C18:2 DASL component produced by the overexpressed strain Peno::moA decreased significantly compared with that of wild type and △moA. Furthermore, the MoA enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with a recombinant plasmid named pMAL-c2x-moA, and the purified enzyme was obtained through a maltose-binding protein (MBP) affinity chromatography column. The purified C18:2 DASL and C18:1 DASL were applied to be catalyzed by MoA enzyme, respectively; it turned to be that C18:1 DASL still remained in the MoA reaction system, but C18:2 DASL disappeared.

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps.

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.

AoRim15 is involved in conidial stress tolerance, conidiation and sclerotia formation in the filamentous fungus Aspergillus oryzae

The serine-threonine kinase Rim15p is a master regulator of stress signaling and is required for stress tolerance and sexual sporulation in the yeast Saccharomyces cerevisiae. However, in filamentous fungi that reproduce asexually via conidiation, the physiological function of Rim15p homologs has not been extensively analyzed. Here, we functionally characterized the protein homolog of Rim15p in the filamentous fungus Aspergillus oryzae, by deleting and overexpressing the corresponding Aorim15 gene and examining the role of this protein in stress tolerance and development. Deletion of Aorim15 resulted in an increase in the sensitivity of conidia to oxidative and heat stresses, whereas conidia of the Aorim15 overexpressing strain were more resistant to these stresses. These results indicated that AoRim15 functions in stress tolerance, similar to S. cerevisiae Rim15p. Phenotypic analysis revealed that conidiation was markedly reduced by overexpression of Aorim15 in A. oryzae, and was completely abolished in the deletion strain. In addition, the formation of sclerotia, which is another type of developmental structure in filamentous fungi, was decreased by the deletion of Aorim15, whereas Aorim15 overexpression increased the number of sclerotia. These results indicated that AoRim15 is a positive regulator of sclerotia formation and that overexpression of AoRim15 shifts the developmental balance from conidiation towards sclerotia formation. Collectively, we demonstrated that AoRim15 is involved in the stress tolerance of conidia and differentially regulates between the two developmental fates of conidiation and sclerotia formation.

Modification of photosynthetic electron transport and amino acid levels by overexpression of a circadian-related histidine kinase hik8 in Synechocystis sp. PCC 6803.

Cyanobacteria perform oxygenic photosynthesis, and the maintenance of photosynthetic electron transport chains is indispensable to their survival in various environmental conditions. Photosynthetic electron transport in cyanobacteria can be studied through genetic analysis because of the natural competence of cyanobacteria. We here show that a strain overexpressing hik8, a histidine kinase gene related to the circadian clock, exhibits an altered photosynthetic electron transport chain in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Respiratory activity was down-regulated under nitrogen-replete conditions. Photosynthetic activity was slightly lower in the hik8-overexpressing strain than in the wild-type after nitrogen depletion, and the values of photosynthetic parameters were altered by hik8 overexpression under nitrogen-replete and nitrogen-depleted conditions. Transcripts of genes encoding Photosystem I and II were increased by hik8 overexpression under nitrogen-replete conditions. Nitrogen starvation triggers increase in amino acids but the magnitude of the increase in several amino acids was diminished by hik8 overexpression. These genetic data indicate that Hik8 regulates the photosynthetic electron transport, which in turn alters primary metabolism during nitrogen starvation in this cyanobacterium.

Polyphosphates and Polyphosphatase Activity in the Yeast Saccharomyces cerevisiae during Overexpression of the DDP1 Gene.

The effects of overexpression of yeast diphosphoinositol polyphosphate phosphohydrolase (DDP1) having endopolyphosphatase activity on inorganic polyphosphate metabolism in Saccharomyces cerevisiae were studied. The endopolyphosphatase activity in the transformed strain significantly increased compared to the parent strain. This activity was observed with polyphosphates of different chain length, being suppressed by 2mM tripolyphosphate or ATP. The content of acid-soluble and acid-insoluble polyphosphates under DDP1 overexpression decreased by 9 and 28%, respectively. The average chain length of salt-soluble and alkali-soluble fractions did not change in the overexpressing strain, and that of acid-soluble polyphosphate increased under phosphate excess. At the initial stage of polyphosphate recovery after phosphorus starvation, the chain length of the acid-soluble fraction in transformed cells was lower compared to the recipient strain. This observation suggests the complex nature of DDP1 involvement in the regulation of polyphosphate content and chain length in yeasts.

Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation.

Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D β-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.

Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola.

Although sophorolipids (SLs) produced by S. bombicola are a real showcase for the industrialization of microbial biosurfactants, some important drawbacks are associated with this efficient biological process, e.g., the simultaneous production of acidic and lactonic SLs. Depending on the application, there is a requirement for the naturally produced mixture to be manipulated to give defined ratios of the components. Recently, the enzyme responsible for the lactonization of SLs was discovered. The discovery of the gene encoding this lactone esterase (sble) enabled the development of promising S. bombicola strains producing either solely lactonic (using a sble overexpression strain described in this paper: oe sble) or solely acidic SLs (using a sble deletion strain, which was recently described, but not characterized yet: Δsble). The new S. bombicola strains were used to investigate the production processes (fermentation and purification) of either lactonic or acidic SLs. The strains maintain the high inherent productivities of the wild-type or even perform slightly better and thus represent a realistic industrial opportunity. 100% acidic SLs with a mixed acetylation pattern were obtained for the Δsble strain, while the inherent capacity to selectively produce lactonic SLs was significantly increased (+42%) for the oe sble strain (99% lactonic SLs). Moreover, the regulatory effect of citrate on lactone SL formation for the wild-type was absent in this new strain, which indicates that it is more robust and better suited for the industrial production of lactonic SLs. Basic parameters were determined for the purified SLs, which confirm that the two new strains produce molecules with distinctive properties of which the application potential can now easily be investigated independently.

Dehydrogenase genes in the ectomycorrhizal fungus Tricholoma vaccinum: A role for Ald1 in mycorrhizal symbiosis.

Ectomycorrhizal symbiosis is important for forest ecosystem functioning with tree-fungal cooperation increasing performance and countering stress conditions. Aldehyde dehydrogenases (ALDHs) are key enzymes for detoxification and thus may play a role in stress response of the symbiotic association. With this focus, eight dehydrogenases, Ald1 through Ald7 and TyrA, of the ectomycorrhizal basidiomycete Tricholoma vaccinum were characterized and phylogenetically investigated. Functional analysis was performed through differential expression analysis by feeding different, environmentally important substances. A strong effect of indole-3-acetic acid (IAA) was identified, linking mycorrhiza formation and auxin signaling between the symbiosis partners. We investigated ald1 overexpressing strains for performance in mycorrhiza with the host tree spruce (Picea abies) and observed an increased width of the apoplast, accommodating the Hartig' net hyphae of the T. vaccinum over-expressing transformants. The results support a role for Ald1 in ectomycorrhiza formation and underline functional differentiation within fungal aldehyde dehydrogenases in the family 1 of ALDHs.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Changes in primary metabolism under light and dark conditions in response to overproduction of a response regulator RpaA in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

The study of the primary metabolism of cyanobacteria in response to light conditions is important for environmental biology because cyanobacteria are widely distributed among various ecological niches. Cyanobacteria uniquely possess circadian rhythms, with central oscillators consisting from three proteins, KaiA, KaiB, and KaiC. The two-component histidine kinase SasA/Hik8 and response regulator RpaA transduce the circadian signal from KaiABC to control gene expression. Here, we generated a strain overexpressing rpaA in a unicellular cyanobacterium Synechocystis sp. PCC 6803. The rpaA-overexpressing strain showed pleiotropic phenotypes, including slower growth, aberrant degradation of an RNA polymerase sigma factor SigE after the light-to-dark transition, and higher accumulation of sugar catabolic enzyme transcripts under dark conditions. Metabolome analysis revealed delayed glycogen degradation, decreased sugar phosphates and organic acids in the tricarboxylic acid cycle, and increased amino acids under dark conditions. The current results demonstrate that in this cyanobacterium, RpaA is a regulator of primary metabolism and involved in adaptation to changes in light conditions.

Functional role of oppA encoding an oligopeptide-binding protein from Lactobacillus salivarius Ren in bile tolerance.

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5% oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.

Production and secretion of naphthoquinones is mediated by the MFS transporter MFS1 in the entomopathogenic fungus Ophiocordyceps sp. BCC1869.

Naphthoquinones are deep red polyketide pigments produced by the ant-pathogenic fungus Ophiocordyceps sp. BCC1869. In culture, biosynthesis of these naphthoquinones remains at a low level during the first 20 days and reaches its maximum production level at approximately 50 days. The MFS transporter gene MFS1 was previously identified in Ophiocordyceps sp. BCC1869 from a subtractive EST library between the fungus grown under naphthoquinone-inductive and naphthoquinone-repressive conditions. We cloned and sequenced this transporter gene, which has an open reading frame of 1505 bp and three introns (48, 52, and 58 bp). Phylogenetic analysis showed this MFS transporter was tightly clustered with fungal riboflavin transporters. Functional analysis of this gene was performed by overexpression of MFS1 under the control of a strong, constitutive promoter. We successfully transformed the fungus with this overexpression plasmid using PEG-protoplast transformation, which generated nine transformants per µg of plasmid. RT-PCR indicated that the MFS1 expression level in the overexpressing strains increased 3- to 10-fold compared to the wild type. HPLC analysis of crude extracts of mutants and wild type demonstrated that four naphthoquinone derivatives, erythrostominone, epierythrostominol, deoxyerythrostominone, and deoxyerythrostominol, were the major naphthoquinones produced and excreted in staggering quantities (20- to 2300-fold) in 7-day old liquid cultures by the mutant C7, compared to the wild type. High resolution electrospray ionization mass spectrometry verified mass spectra of these purified metabolites. Three other naphthoquinone derivatives, whose structures have not been identified, were also detected in high amount in the mutant liquid cultures.

The Zn(II)2Cys6 putative transcription factor is involved in the regulation of leucinostatin production and pathogenicity of the nematophagous fungus Paecilomyces lilacinus

Zn(II)2Cys6 transcription factor genes encode transcription regulators that manage the infection potential and production of secondary metabolites such as toxins in fungi. In this study, a gene named rolP that encodes a putative Zn(II)2Cys6 transcription factor regulating leucinostatin production in the filamentous fungus Paecilomyces lilacinus was characterized by a gene knockout approach. The deduced proprotein consists of 705 amino acids and is highly homologous to the Zn(II)2Cys6 transcription factor of the entomopathogenic fungus Metarhizium brunneum. Predictive analysis of the secondary structure of the proportion showed that it has two domains. Paecilomyces lilacinus can produce nematotoxins leucinostatins A and B. Deletion of rolP from the P. lilacinus wild-type strain Pl36-1 leads to the absence of leucinostatin A, while a large increase in the leucinostatin A level was achieved in the rolP overexpression strain Ov-Pl36-1. During the process of nematode infection, rolP showed high expression levels at 48–72 h and peaked at 48 h. Bioassay tests confirmed the requirement of rolP for leucinostatin production. However, the root-knot second-stage juvenile-toxicity was minimized to 35.3% in ΔrolP; toxicity was 97.2% with Ov-Pl36-1 compared with 68.4% with the wild type strain Pl36-1. Interestingly, neither knockout nor overexpression of the rolP gene affected the growth or sporulation of P. lilacinus. Additionally, fungal nutrition and acidic media might stimulate the rolP activity of the wild type strain against nematodes. These findings suggested that the rolP gene is required for the induction and production of leucinostatins and is considered a leucinostatin regulatory gene in P. lilacinus.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

Host Cytosolic Glutathione Sensing by a Membrane Histidine Kinase Activates the Type VI Secretion System in an Intracellular Bacterium.

Type VI secretion systems (T6SSs) are major virulence mechanisms in many Gram-negative bacteria, but the physiological signals that activate them are not well understood. The T6SS1 of Burkholderia pseudomallei is essential for pathogenesis in mammalian hosts and is only expressed when the bacterium is intracellular. We found that signals for T6SS1 activation reside in the host cytosol. Through site-directed mutagenesis and biochemical studies, we identified low molecular weight thiols, particularly glutathione, as the signal sensed by a periplasmic cysteine residue (C62) on the histidine kinase sensor VirA. Upon glutathione exposure, dimeric VirA is converted to monomers via reduction at C62. When glutathione in the host was depleted, T6SS1 expression was abrogated, and bacteria could no longer induce multinucleate giant cell formation, the hallmark of T6SS1 function. Therefore, intracellular bacteria exploit the abundance of glutathione in host cytosol as a signal for expression of virulence at the appropriate time and place.

Expression of RCK2 MAPKAP (MAPK-activated protein kinase) rescues yeast cells sensitivity to osmotic stress.

BackgroundSaccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars during beverage or bioethanol fermentations. These fermentations are characterised by high osmotic stress on a yeast cell, with selected brewing fermentations beginning at 20–25% fermentable sugars and bioethanol fermentations at 13% fermentable sugars.ResultsRCK2 encodes for a MAPKAP (MAPK-activated protein kinase) enzyme and was identified on a locus by QTL analysis in yeast cells under osmotic stress, RCK2 expression was placed under a tetracycline regulatable vector and rescued glucose, sorbitol or glycerol induced osmotic stress in an rck2 null strain. A strain overexpressing RCK2 had significantly faster fermentation rates when compared with the empty vector control strain.ConclusionsPresence of RCK2 increased rates of glucose utilisation (~40 g glucose in first 8 h) during a 15% glucose fermentation and concurrent production of ethanol when compared with empty vector controls. Tolerance to osmotic stress using the tetracycline regulatable vectors could be turned off with the addition of tetracycline returning a rck2 null strain back to osmotic sensitivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0276-7) contains supplementary material, which is available to authorized users.