Molecular characterization of the <Emphasis Type="Italic">Aspergillus fumigatus NCS-1</Emphasis> homologue,NcsA

Here, we characterize the Aspergillus fumigatus homologue ncsA Neuronal Calcium Sensor. We showed that ncsA is not an essential gene and ?ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ?ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm and its cellular localization is not affected by the cellular response to either calcium chloride or EGTA. The ?ncsA mutant strain is more sensitive to voriconazole, itraconazole, and amphotericin. Polar growth in the ΔncsA mutant was also considerably more affected by lovastatin than in the wild type strain. The Spitzenkörper can be visualized in both strains and although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the ?ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the ΔncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

The Role of <Emphasis Type="Italic">sho1</Emphasis> in Polarized Growth of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Aspergillus fumigatus is an opportunistic pathogen that may cause severe invasive disease in immunocompromised patients. The filamentous fungi undergo polarized growth, searching for nutrients in the environment and causing invasive growth in tissue. Sho1 is a sensor of the high osmolarity glycerol pathway, and the sho1 mutant showed a decrease in growth rate. We found that sho1 is involved in the polarized growth of A. fumigatus. The sho1 mutation resulted in extended isotropic growth of germinating conidia followed by multiple germ tubes and wide hyphae with short intercalary cells by calcofluor white staining. The mechanism by which sho1 gene affected polarized growth is investigated. A reduced number of apical vesicles with greater dispersion were observed by transmission electron microscopy in the Spitzenkörper body of the sho1 mutant. Actin patches were distributed randomly at low density at early stages of mutant strain fungal development and reaggregated to the hyphal tip of later stages when long filamentous fungi formed. Actin patches located at the tip of polarized wild-type cells. RNA levels of polarized growth-related genes Rho GTPases were detected by real-time PCR. The sho1 gene did not affect the RNA expression when strains were cultured at 37°C for 6 h. At 17 h, the RNA expression of rho1, rho3 and CDC42 in the sho1 mutant were 0.18-, 0.18- and 0.33-fold of that in the wild type. The sho1 gene affected the polarized growth through affecting the expression of Rho GTPases, the distribution of actin cytoskeleton, vesicle quantity and distribution.     

Co-expression of Beta-Glucosidase and Laccase in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> by Random Insertion with Enhanced Filter Paper Activity

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P _pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P _gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.     

Characterization and Proteomic Analysis of the <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 <Emphasis Type="Italic">xenB</Emphasis> Knockout Mutant Under RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Stress

Pseudomonas sp. HK-6 is able to utilize RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) as its sole nitrogen source. The role of the xenB gene, encoding xenobiotic reductase B, was investigated using HK-6 xenB knockout mutants. The xenB mutant degraded RDX to a level that was 10-fold less than that obtained with the wild-type HK-6 strain. After 60 days of culture with 25 or 50 μM RDX, no residual RDX was detected in the supernatants of the wild-type aerobically grown cultures, whereas approximately 90 % of the RDX remained in the xenB mutant cultures. The xenB mutant bacteria exhibited a 10²–10⁴-fold decrease in survival rate compared to the wild-type. The expression of DnaK and GroEL proteins, two typical stress shock proteins (SSPs), in the xenB mutant increased after immediate exposure to RDX, yet dramatically decreased after 4 h of exposure. In addition, DnaK and GroEL were more highly expressed in the cultures with 25 μM RDX in the medium but showed low expression in the cultures with 50 or 75 μM RDX. The expression levels of the dnaK and groEL genes measured by RT-qPCR were also much lower in the xenB genetic background. Analyses of the proteomes of the HK-6 and xenB mutant cells grown under conditions of RDX stress showed increased induction of several proteins, such as Alg8, alginate biosynthesis sensor histidine kinase, and OprH in the xenB mutants when compared to wild-type. However, many proteins, including two SSPs (DnaK and GroEL) and proteins involved in metabolism, exhibited lower expression levels in the xenB mutant than in the wild-type HK-6 strain. The xenB knockout mutation leads to reduced RDX degradation ability, which renders the mutant more sensitive to RDX stress and results in a lower survival rate and an altered proteomic profile under RDX stress.     

High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Repair of cisplatin-DNA adducts in mutants for genes controlling spontaneous and induced mutagenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast

Sensitivity to the lethal action of the anticancer substance cisplatin was studied in the yeast mutants him1, hsm2, hsm3, and hsm6, deficient for repair of spontaneous and induced mutations. The him1 and hsm3 mutants were as resistant to the agent under study as the wild-type strain. The survival of the double mutant rad2 hsm3 was higher than that of the single mutant rad2. The hsm2 and hsm6 mutants were more cisplatin-sensitive than the wild type. Cisplatin was shown to have high mutagenic and recombinogenic effects on yeast cells.     

Synthesis of exo-β-glucosaminidase BY FUNGUS <Emphasis Type="Italic">Penicillium</Emphasis> sp. IB-37-2

A new strain Penicillium sp. IB-37-2, which actively hydrolyzes chitosan (SD ～80–85%) but possesses low activity against colloidal chitin, was isolated. The fungus was observed to have a high level chitosanase biosynthesis (1.5–3.0 U/mL) during submerged cultivation at 28°C, with a pH of 3.5–7.0 and 220 rpm in nutrient media containing chitosan or chitin from shells of crabs. Purification of the chitosanase enzyme complex from Penicillium sp. IB-37-2 by ultrafiltration and hydrophobic chromatography, followed by denaturing electrophoresis, revealed two predominant proteins with molecular weights of 89 and 41 kDa. The purified enzyme complex demonstrated maximal activity (maximal rate of hydrolysis of dissolved chitosan) and stability at 50–55°C and a pH of 3.5–4.0. The enzyme preparation also hydrolyzed laminarin, β-(1,3)-(1,4)-glycan, and colloidal chitin. Exohydrolysis of chitosan by the preparation isolated from Penicillium sp. IB-37-2 resulted in the formation of single product, D-glucosamine.     

Non-capsulated mutants of a chemical-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strain

Objectives

To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae.

Results

The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4 × 10⁵ CFU μg^?1 DNA, which is 10 times higher than that of the wild strain. 52.2 g 1,3-propanediol L^?1, 30.7 g 2,3-butanediol L^?1, and 175.9 g 2-ketogluconic acid L^?1 were produced by non-capsulated mutants, which were 10–40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8–2.2 cP.

Conclusions

Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.

     

<Emphasis Type="Italic">N</Emphasis>-Terminal seven-amino-acid extension simultaneously improves the pH stability,optimal temperature,thermostability and catalytic efficiency of chitosanase CsnA

Objective

To determine the effects of the extra N-terminal seven-amino-acid sequence on the function of chitosanase CsnA.

Results

Sequence and structure analysis indicated that the mature CsnA contains a seven-amino-acid extension in a disordered form at the N-terminus. To determine the function of this sequence, both mature CsnA and its N-terminus-truncated mutant, CsnAΔN, were expressed in Escherichia coli and characterized. Compared with CsnAΔN, CsnA exhibited a 15 °C higher temperature optimum, enhanced pH stability, thermostability and catalytic efficiency. The underlying mechanisms responsible for these changes were analyzed by circular dichroism (CD) spectroscopy. CD analysis revealed that the deletion of the N-terminal sequence resulted in a decrease in the T_m of 4.3 °C and this sequence altered the secondary structure of the enzyme.

Conclusions

The N-terminal sequence is essential for the stability and activity of chitosanase CsnA.

     

Inactivation of <Emphasis Type="Italic">RAD52</Emphasis> and <Emphasis Type="Italic">HDF1</Emphasis> DNA repair genes leads to premature chronological aging and cellular instability

The present study aims to investigate the role of radiation sensitive 52 (RAD52) and high-affinity DNA binding factor 1 (HDF1) DNA repair genes on the life span of budding yeasts during chronological aging. Wild type (wt) and rad52, hdf1, and rad52 hdf1 mutant Saccharomyces cerevisiae strains were used. Chronological aging and survival assays were studied by clonogenic assay and drop test. DNA damage was analyzed by electrophoresis after phenol extraction. Mutant analysis, colony forming units and the index of respiratory competence were studied by growing on dextrose and glycerol plates as a carbon source. Rad52 and rad52 hdf1 mutants showed a gradual decrease in surviving fraction in relation to wt and hdf1 mutant during aging. Genomic DNA was spontaneously more degraded during aging, mainly in rad52 mutants. This strain showed an increased percentage of revertant colonies. Moreover, all mutants showed a decrease in the index of respiratory competence during aging. The inactivation of RAD52 leads to premature chronological aging with an increase in DNA degradation and mutation frequency. In addition, RAD52 and HDF1 contribute to maintain the metabolic state, in a different way, during chronological aging. The results obtained could have important implications in the chronobiology of aging.     

In vitro assessment of antimicrobial activity of aqueous and alcoholic extracts of moss <Emphasis Type="Italic">Atrichum undulatum</Emphasis> (Hedw.) P. Beauv.

Bryophytes, the shade loving plants, have tremendous medicinal properties. The aqueous and alcoholic extracts of Atrichum undulatum (Hedw.) P. Beauv. were analysed for antimicrobial properties against the fungi Aspergillus fumigatus and Fusarium oxysporum and the bacteria Escherichia coli, Bacillus mycoides, Proteus mirabilis, Staphylococcus aureus and Salmonella typhi. The study is an attempt to investigate the medicinal properties of Atrichum undulatum (Hedw.) P. Beauv. using disc-diffusion method. No inhibition was observed against A. fumigatus and P. mirabilis. For bacteria S. typhi and E. coli (20 and 15 mm), aqueous and alcoholic extracts of Atrichum showed significant inhibition. However, alcoholic extract was found remarkably effective against bacteria rather than aqueous extract.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Metabolic engineering of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and in silico investigation for enhanced 2,3-butanediol production

Objectives

To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated.

Results

Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation.

Conclusions

K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

     

Enhanced biosynthesis of phenazine-1-carboxamide by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> strains using statistical experimental designs

Phenazine-1-carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and  9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett–Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p?<?0.05). Response surface methodology (RSM) based on the central composite design (CCD) was adopted to determine these factors optimal levels and their interactive effects between culture components for PCN production. The predicted maximum PCN production was 9002 mg/L, whereas an actual PCN production of 9174 mg/L was recorded in the validation experiments using the optimal medium containing glycerol 37.08 mL/L, tryptone 20.00 g/L, and soy peptone 25.03 g/L, which was nearly threefolds higher than without optimization and 20-folds higher than the wild-type strain. In conclusion, the results revealed that P. chlororaphis display a high potential for industrial-scale production for phenazine biopesticides.     

Application of the LAMP assay for the detection of the Argentine ant, <Emphasis Type="Italic">Linepithema humile</Emphasis> (Hymenoptera: Formicidae), from captures of pan traps

We applied the loop-mediated isothermal amplification (LAMP) assay to monitor invasions of Linepithema humile (Mayr), the Argentine ant, a notorious invasive insect worldwide. Species-specific LAMP primers were designed on the basis of the partial sequence of the cytochrome c oxidase subunit I region of L. humile. The species specificity and sensitivity of these primers were determined in the laboratory and considered adequate for practical use. We also confirmed that the assay successfully detected L. humile from captures of pan traps, which contained L. humile and several non-target ant species. The assay detected the target species even when the captures contained only a leg or an antenna. Since the LAMP assay is simple and rapid, this assay will contribute to the early detection and accurate identification of L. humile.     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.