Revision of “<Emphasis Type=Italic>Septonema ochraceum</Emphasis>” revealed three new species of <Emphasis Type=Italic>Venturiaceae</Emphasis> and <Emphasis Type=Italic>Herpotrichiellaceae</Emphasis>

Survey of seven strains determined as Septonema ochraceum (Dothideomycetes, inc. sed.) isolated from pine litter or obtained from public collections revealed three new species, Fusicladium cordae, F. sicilianum (Venturiaceae), Cladophialophora matsushimae (Herpotrichiellaceae) and a cryptic species morphologically identical to Devriesia americana (Teratosphaeriaceae), but phylogenetically distinct. Morphological survey and phylogenetic analysis using nucleotide sequence data from the nuclear ribosomal subunit genes indicate a close relationship within three species colonising pine litter needles, F. cordae, F. pini and F. ramoconidii. F. sicilianum is most related to F. rhodense. C. matsushimae represents a species belonging to one of the lineages of the polyphyletic genus Cladophialophora. None of the strains observed can be classified morphologically as S. ochraceum, of which the type material does not exist.     

Small colony variants of <Emphasis Type=Italic>Staphylococcus aureus</Emphasis> — <Emphasis Type=Italic>review</Emphasis>

Bacterial variants of Staphylococcus aureus called small colony variants (SCVs) originate by mutations in metabolic genes, resulting in emergence of auxotrophic bacterial subpopulations. These variants are not particularly virulent but are able to persist viable inside host cells. SCVs show their characteristic auxotrophic growth deficiency and depressed α-cytotoxin activity. Environmental pressure such as antibiotics, select for isogenic SCV cells that are frequently found coexisting with their parent wild-type strains in a mixed bacterial culture. SCV strains often grow on blood agar as non-pigmented or pinpoint pigmented colonies and their key biochemical tests are often non-reactive. Their altered metabolism or auxotrophism can result in long generation time and thus SCV phenotype, more often than not SCV can be overgrown by their wild-type counterparts and other competitive respiratory flora. This could affect laboratory detection. Thus, molecular methods, such as 16S rRNA partial sequencing or amplification of species-specific DNA targets (e.g. coagulase, nuclease) directly from clinical material or isolated bacterial colonies, become the method of choice. Patients at risk of infection by S. aureus SCVs include cystic fibrosis patients (CF), patients with skin and foreign-body related infections and osteomyelitis, as they suffer from chronic staphylococcal infections and are subject to long-term antibiotic therapy. Molecular evidence of SCV development has not been found except for some random mutations of the thymidylate synthase gene (thyA) described in SCV S. aureus strains of CF patients. These variants are able to bypass the antibiotic effect of folic acid antagonists such as sulfonamides and trimethoprim. Resistance to gentamicin and aminoglycosides in the hemin or menadione auxotrophic SCVs was hypothesized as being due to decreased influx of the drugs into cells as a result of decreased ATP production and decreased electrochemical gradient on cell membranes.     

High level accumulation of α-glucan in maize kernels by expressing the <Emphasis Type="Italic">gtfD</Emphasis> gene from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Glucosyltransferases (GTFs, EC.2.4.1.5) are bacterial enzymes that catalyze the polymerization of glucose residues from sucrose, leading to the production of high molecular weight glucan with α-1,3 /α-1,6 linkages. Such glucans, with many potential food and industrial applications, do not normally exist in higher plants. We fused a mutant form of the gtfD gene from Sreptococcus mutans with the maize (Zea mays L.) chloroplastic Brittle 1 transit peptide for amyloplast targeting. This construct, driven by the ubiquitin promoter, was introduced into maize by Agrobacterium-mediated transformation. We developed a novel HPLC-based method that enabled us differentially to distinguish transgene glucan from other endogenous polysaccharides in maize kernels. Using this method, we screened over 100 transgenic plants for the presence of GTF-produced glucan whose content varied between 0.8 and 14% of dry weight in the mature transgenic seeds. The mature transgenic plants were indistinguishable from wildtype plants in growth rate and morphology. Furthermore, starch granule size in the transgenic maize kernel was unaffected by the accumulation of the foreign polysaccharide. Mutation in Sh2, which encodes a subunit of ADP-glucose pyrophosphorylase, had no effect on glucan accumulation caused by gtfD expression. Our results indicated that high levels of novel carbohydrate polymer can be accumulated in crop plants through transgene technology.     

Response surface methodology for the optimization of <Emphasis Type=Italic>α</Emphasis>-amylase production by <Emphasis Type=Italic>Streptomyces</Emphasis> sp. ML12 using agricultural byproducts

Amylases constitute one of the most important groups of enzymes for commercial use. In the present study, production of α-amylase was optimized using a newly isolated actinobacterial strain from the coral reef environment of the Gulf of Mannar Biosphere Reserve, India. It was identified as Streptomyces sp. ML12 based on chemotaxonomy, cultural and morphological characteristics, carbon source utilization and 16S rRNA gene sequencing. Fermentation variables were selected in accordance with the Plackett-Burman design and were optimized by response surface methodology. Five significant variables (rice bran and wheat bran — both agricultural byproducts, sodium chloride, magnesium sulphate and incubation period) were selected for the optimization via central composite design. The optimal features were rice bran (5.5 g/100 mL), wheat bran (5.3 g/100 mL), sodium chloride (2.8 g/100 mL), magnesium sulphate (1.4 g/100 mL) and 8 days of incubation period. Optimization of the medium with the above tested features increased the amylase yield by 4.4-fold.     

High-level expression,purification and characterization of a recombinant medium-temperature <Emphasis Type=Italic>α</Emphasis>-amylase from <Emphasis Type=Italic>Bacillus subtilis</Emphasis>

Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml⁻¹) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg⁻¹ having optimal activity at pH 6.0 and 60°C.     

<Emphasis Type=Italic>Pseudomonas seleniipraecipitatus</Emphasis> sp. nov.: A Selenite Reducing γ-<Emphasis Type=Italic>Proteobacteria</Emphasis> Isolated from Soil

Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. This study examined the formation in culture of an unidentified bacterial oxidant and investigated the performance of this oxidant in a two-chambered MFC with a proton exchange membrane and an uncoated carbon cathode. DNA, FAME profile and characterization studies identified the microorganism that produced the oxidant as Burkholderia cenocepacia. The oxidant was produced by log phase cells, oxidized the dye 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), had a mass below 1 kD, was heat stable (121°C) and was soluble in ethanol. In a MFC with a 1000 Ω load and ABTS as a mediator, the oxidizer increased cell voltage 11 times higher than atmospheric oxygen and 2.9 times higher than that observed with ferricyanide in the cathode chamber. No increase in cell voltage was observed when no mediator was present. Organisms that produce and release oxidizers into the media may prove useful as bio-cathodes by improving the electrical output of MFCs.     

Polygenic control for fermentation of β-fructosides in the yeast <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>: New genes <Emphasis Type=Italic>SUC9</Emphasis> and <Emphasis Type=Italic>SUC10</Emphasis>

Using molecular karyotyping and genetic hybridization analysis, two new polymeric β-fructosidase genes, SUC9 and SUC10, were identified in the yeast Saccharomyces cerevisiae, which are located on chromosome XIV and on the chromosome XVI/XIII doublet, respectively. The genes are responsible for fermentation of sucrose and raffinose. The SUC gene genotypes of strains VKM Y-1831 and DBVPG 1340 are SUC2 SUC9 and suc2 ⁰ SUC10, respectively. suc2 ⁰ is a silent sequence. The scientific and applied significance of SUC genes is discussed.     

Isozymes of <Emphasis Type=Italic>α</Emphasis>-amylases from newly isolated <Emphasis Type=Italic>Bacillus thuringiensis</Emphasis> CKB19: Production from immobilized cells

The aim of this study was to produce two isozymes of α-amylase by immobilization of a newly isolated soil bacterium. The bacterium was identified as Bacillus thuringiensis CKB19 on the basis of its 16S rRNA profile. Enzyme production by free cells increased linearly with cell growth up to 34 h in starch containing enriched liquid media. The active bacterial cells were immobilized in Caalginate beads, and operational stability of the entrapped cell was optimized for amylase production. Enzyme production was optimal at an alginate concentration of 2 g% (w/v), calcium chloride concentration of 1 M, and with 300 beads (each bead contained 2 × 10⁷ cells)/250 mL flask. Amylase production by the immobilized cells was about 3 times higher than free cell fermentation after 34 h of incubation. It was observed that the immobilized bacterium secreted two different amylases (Am-I and Am-II) into the culture fluid. The molecular masses of Am-I and Am-II were 59.6 and 44.7 kd, respectively, and showed optimum activity at pH 5.0 and 9.0. Both amylases showed optimum activity at 40°C and were stable at the same temperature, with losses of only 10 and 20% (for Am I and Am II, respectively) of their original activities after 24 h of incubation. Further, both amylases were salt tolerant (up to 4 M NaCl) and hydrolyzed raw starchy foods into glucose. All these characteristics make this enzyme mixture suitable for use as a digestive aid and for the improvement of digestibility of animal feed ingredients.     

Purification and characterization of highly thermostable α-amylase from thermophilic <Emphasis Type=Italic>Alicyclobacillus acidocaldarius</Emphasis>

In this study, the production of extracellular thermostable α-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60°C. This extracellular α-amylase was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The α-amylase was purified to 8.138 fold homogeneity with a final recovery of 58% and a specific activity of 3,239 U/mg proteins. The purified α-amylase appeared as a single protein band on SDS-PAGE with a molecular mass of 94.5 kDa. Non-denaturing PAGE analysis showed one major band associated with enzyme activity, indicating the absence of isoenzymes. A TLC analysis showed maltose as major end product of the enzyme. The optimum assay temperature and pH for enzyme activity were 60°C and 6.0 respectively; however, the enzyme activity was stable over a wide range of pH and temperatures. The α-amylase retained its activity in the presence of the denaturing agents — SDS, Triton X-100, Tween-20, Tween-80, and was significantly inhibited by EDTA and urea. Calcium ions increased the enzyme activity, while Hg²⁺, Zn²⁺, and Co²⁺ had inhibitory effects. The K _m and V _max values were found to be 2.9 mg/mL and 7936 U/mL respectively.     

A new species of <Emphasis Type=Italic>Chamaecrista</Emphasis> sect. <Emphasis Type=Italic>Chamaecrista</Emphasis> ser. <Emphasis Type=Italic>Flexuosae</Emphasis> (Leguminosae,Caesalpinioideae) from Serra do Cipó, Minas Gerais,Brazil

A new species, Chamaecrista truncata, from southeastern Brazil, is described, illustrated and compared to its putative closest relative, C. parvistipula. The new species belongs to Chamaecrista sect. Chamaecrista ser. Flexuosae which is characterized by asymmetrical leaflets with palmate venation, quadrangular stems and axillary peduncles. Additionally, the venation pattern of the leaflets and the different types of stipules observed within this series are shown.     

Identification of a <Emphasis Type=Italic>Rice stripe necrosis virus</Emphasis> resistance locus and yield component QTLs using <Emphasis Type=Italic>Oryza sativa</Emphasis> × <Emphasis Type=Italic>O. glaberrima</Emphasis> introgression lines

Background  

Developing new population types based on interspecific introgressions has been suggested by several authors to facilitate the discovery of novel allelic sources for traits of agronomic importance. Chromosome segment substitution lines from interspecific crosses represent a powerful and useful genetic resource for QTL detection and breeding programs.     

NaCl-induced photoinhibition and recovery of the photosynthetic activity of a <Emphasis Type=Italic>katG</Emphasis><Superscript>−</Superscript> mutant of cyanobacterium <Emphasis Type=Italic>Synechocystis</Emphasis> sp. PCC 6803

The joint effects of 0.5 M NaCl and light of different intensities on the activity of the photosynthetic apparatus and ATP content in cells of the katG ⁻ mutant of cyanobacterium Synechocystis sp. PCC 6803 have been studied. The mutant demonstrated a higher photoinhibition rate and a slower rate of recovery compared with the wild type, as shown by measurements of the CO₂-dependent O₂ production and delayed fluorescence of Chl a. The presence of 0.5 M NaCl in the incubation medium caused equal photoinhibition of the photosynthetic apparatus at I = 1200 μE m⁻² s⁻¹ in the mutant and wild-type cells. At I = 2400 μE m⁻² s⁻¹, we observed stronger inhibition and slower recovery of the photosynthetic apparatus in the katG ⁻ mutant than in wild-type cells. The data obtained evidence an important role of catalase-peroxidase in the system of reparation of the photosynthetic apparatus damaged by high-intensity light, especially at the background of NaCl stress.     

Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by <Emphasis Type=Italic>Humicola grisea</Emphasis> var. <Emphasis Type=Italic>thermoidea</Emphasis> grown on sugarcane bagasse

The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.