Microbial combinatorics: a simplified approach for isolating insecticidal bacteria

Bacteria can control pest insects that damage food crops, vector diseases and defoliate trees. Conventionally, isolation of these bacteria has been from soil and sporadically from dead insects. A simplified approach for isolating insecticidal bacteria from soil using the target insect as the selective agent was employed in this study. Instead of isolating single strains of bacteria from soil and testing each individual strain for insect toxicity, mixtures of bacteria present in each soil sample were tested together directly for toxicity using Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) as a model insect. Thirty-five soil suspensions or bacterial suspensions of the 40 suspensions tested killed at least one M. sexta larva. All but one bacterial culture isolated from dead larvae and retested for toxicity, killed at least one M. sexta larva. Nineteen bacterial strains isolated from larvae killed in the first test, were identical to the bacteria fed to the retested larvae. Of the 19 strains isolated, 14 were identified by 16S rDNA sequencing as belonging to the Bacillus cereus group including three strains that formed crystals that were identified as B. thuringiensis. Of the three other spore-forming strains, two were identified as psychrotrophic B. weihenstephanensis and the third as Lysinibacillus fusiformis. Two others were identified as Enterococcus faecalis. This approach, microbial combinatorics, reduces the number of insects necessary for toxicity screening and associated time and resources compared to conventional methods that first isolate bacteria and then individually test for toxicity as well as a means of discovery of new pathogens using the insect as the selective agent.     

Protection of cells from salinity stress by extracellular polymeric substances in diatom biofilms

Diatom biofilms are abundant in the marine environment. It is assumed (but untested) that extracellular polymeric substances (EPS), produced by diatoms, enable cells to cope with fluctuating salinity. To determine the protective role of EPS, Cylindrotheca closterium was grown in xanthan gum at salinities of 35, 50, 70 and 90 ppt. A xanthan matrix significantly increased cell viability (determined by SYTOX-Green), growth rate and population density by up to 300, 2,300 and 200%, respectively. Diatoms grown in 0.75% w/v xanthan, subjected to acute salinity shock treatments (at salinities 17.5, 50, 70 and 90 ppt) maintained photosynthetic capacity, F_q′/F_m′, within 4% of pre-shock values, whereas F_q′/F_m′ in cells grown without xanthan declined by up to 64% with hypersaline shock. Biofilms that developed in xanthan at standard salinity helped cells to maintain function during salinity shock. These results provide evidence of the benefits of living in an EPS matrix for biofilm diatoms.     

Iodine binding and peroxidase activity in the endostyle of Salpa fusiformis,Thalia democratica,Dolioletta gegenbauri and Doliolum nationalis (Tunicata,Thaliacea)

Summary The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electronmicroscopic levels. To reveal the primary binding site for iodine, autoradiography was carried out on endostylar tissue from animals that had been incubated with high activity ¹²⁵I^- over a short period of time. The specific iodine binding enzyme, a peroxidase, was traced by its reaction with DAB. In accordance with previous findings, the iodinebinding cells proved to be the same as those containing the peroxidase. There were also strong indications of a secondary uptake of iodinated compounds and subsequent release into the body fluid. Together with the ultrastructural features, the data provided strong evidence indicating that these cells constitute a protothyroid region, which partly functions as an endocrine organ, possibly homologous with the vertebrate thyroid gland. Since the number of zones varied between the species, the numeration of the protothyroid region also varied. However, in all the examined endostyles, the protothyroid region was seen to be situated dorsolaterally to the glandular regions of the endostyle concerned with food capture.     

Hironoia fusiformis gen. et sp. nov.; a cornalean fruit from the Kamikitaba locality (Upper Cretaceous,Lower Coniacian) in northeastern Japan

The application of sieving techniques to bulk samples from the Ashizawa Formation, Futaba Group (Lower Coniacian) of northeastern Honshu, Japan, has yielded well-preserved mesofossil assemblages comparable with those recently described from eastern North America, Europe, and central Asia. Among the most abundant and distinctive components of these assemblages are fusiform fruits that are assigned here to a new genus and species, Hironoia fusiformis gen. et sp. nov. The fruits developed from an epigynous ovary with three to four locules. Each locule bears one seed and has a distinctive dorsal germination valve. These features of the fruit, along with the adnate calyx, indicate an affinity to extant Cornales and specifically the Cornaceae sensu lato. The recognition of an unequivocal cornalean fruit in the Early Coniacian–Early Santonian of Japan provides the earliest record of this group in the fossil record. It also establishes a minimum age for the early divergence of the asterid clade, a major group of living angiosperms comprising more than a third of all species of extant flowering plants. Electronic Publication     

Differences in growth,total lipid content and fatty acid composition among 60 clones of <Emphasis Type="Italic">Cylindrotheca fusiformis</Emphasis>

Differences between clones of the diatom Cylindrotheca fusiformi were studied with respect to growth rate, total lipid content and fatty acid composition. Sixty clones were isolated and cultivated under batch conditions. All clones were grown under identical conditions (temperature 22±1°C, light intensity 100 μmol photon m⁻² s⁻¹, salinity 28, F/2 medium) and were harvested in the late exponential growth phase for lipid and fatty acid analysis. The results show a wide variation in growth, total lipid content and fatty acid profiles among clones (p<0.05). The major fatty acids in the 60 clones were 14:0 (4.6–9.1%), 16:0 (18.2–32.0%), 16:1n-7 (21.6–33.1%), 20:4n-6 (4.1–13.5%) and 20:5n-3 (6.2–17.2%), with the highest proportion of 20:4n-6 in clone CF13 (13.5%), and the highest proportion of 20:5n-3 in clone CF5 (17.2%). The results support the view that some microalgal fatty acid variation is not restricted to interspecific variation and external factors, but also varies from clone to clone within the same species.     

Role of silicon in diatom metabolism

Levels of the cyclic nucleotides, cAMP and cGMP, were determined in four species of pennate diatoms; changes in their levels and ratios were monitored in silicon-starved and light-dark synchronized cultures of Cylindrotheca fusiformis. Content of both cAMP and cGMP changed during the cell cycles: when silicate was added to starved cultures, cAMP, cGMP and DNA levels rose rapidly; cAMP and cGMP declined before DNA synthesis was complete and continued to fall during the events leading to cell separation. In unstarved synchronies, net synthesis of DNA continued until cell separation; 1 h before cell separation cAMP levels fell while those of cGMP rose. The results support the proposal that cAMP and cGMP may play a part in the process of cell division in the diatom, possibly involving silicon.     

转化异丁香酚生成香草醛纺锤芽孢杆菌的筛选

以底物异丁香酚为唯一碳源从七壤中筛选获得了一株能高效转化异丁香酚生成香草醛的芽孢杆菌。根据生理生化特性及16SrRNA序列分析鉴定其属于纺锤芽孢杆菌（Bacillus fusiformis）,初步试验表明该菌能转化2％异丁香酚生成4．20g/L香草醚。     

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28–31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.     

OPTIMAL GROWTH CONDITIONS AND ANTIOXIDATIVE ACTIVITIES OF CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)1

We isolated the unialgal strain of Cylindotheca closterium (Ehrenb.) Reimann et J. C. Lewin and produced an axenic strain using an antibiotic cocktail of enriched f/2 artificial seawater medium. The optimal growth conditions were estimated under 27 different combinations of temperature, salinity, and nutrients, and mass culture was performed based on the best specific growth conditions. Its antioxidant activities were determined from the extracts of methanol, water, and enzymes (proteases and carbohydrases). The maximum specific growth rate (μ_max) varied from 0.63 to 0.97 · d^?1. The maximum cell density was 7.20 × 10⁴ cells · mL^?1, while the μ_max was 0.82 · d^?1 in culture conditions of 20°C, 30 psu (practical salinity unit), and “F” nutrient concentrations on day 10 of the culture period. The scavenging rates for 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical were 72.5% and 69.4% from Viscozyme and methanol extracts, respectively. The enzymatic extracts of C. closterium prepared by the hydrolyses of Amyloglucosidase (AMG) and Viscozyme showed 45.8% and 45.5% nitric‐oxide‐scavenging rates, slightly lower than the activity of alpha‐tocopherol (α‐tocopherol) but similar to butylated hydroxytoluene (BHT). The extract from methanol and water showed 44.8% and 44.4% scavenging rates, statistically similar with BHT. The metal‐chelating activities of the Kojizyme, Alcalase, methanol, Viscozyme, and Neutrase extracts were 67.1, 53.9, 53.2, 52.1, and 50.2 %, respectively, five to six times higher than the commercial antioxidants. The AMG, Viscozyme, and Neutrase extracts showed a remarkable linoleic acid peroxidation inhibition, which was higher than BHT and statistically similar with α‐tocopherol.