Nitrogen-fixing cyanobacteria as gene delivery system for expressing mosquitocidal toxins of Bacillus thuringiensis ssp. israelensis

Classical biological control is the most successfuland promising way to replace chemical pesticides. Thesubspecies israelensis of Bacillusthuringiensis (Bti) is a safe and efficient agent tocontrol mosquito larvae and hence mosquito-bornediseases. One approach to overcome the low efficacyand short half-life in nature of current formulationsof Bti is by expressing the toxin genes in recombinantcyanobacteria as a delivery system. Attempts toexpress Bti toxin in cyanoabcteria have been carriedout during the last ten years. Toxicities of thetransgenic strains were however very low, even underregulation of strong promoters, too low to beeffective in vivo. Two Bti Cry proteins haverecently been co-expressed in the filamentousnitrogen-fixing cyanobacterium Anabaena PCC7120, resulting in clones with the highest toxicitiesand stabilities ever reached so far. However, toobtain a long-lasting preparation, it would be usefulto express Bti toxin genes in cyanobacterial strainsisolated from nature. This approach requiresdevelopment of a system for effective transformationinto such strains. Releasing such recombinant strainsto open environments is still a major obstacle inexploiting this biotechnology.     

Bacillus subtilis as a host for mosquitocidal toxins production

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.     

Hydrogen metabolism of three unicellular nitrogen-fixing cyanobacteria

Abstract The enzyme activities responsible for the evolution and consumption of hydrogen in three unicellular cyanobacteria were investigated. Gloeothece sp. 6909 and Cyanothece sp. 7822 performed an oxygen-tolerant nitrogen fixation, whereas the nitrogenase activity of Synechococcus sp. 7425 was much more sensitive to oxygen. While in Gloeothece the net hydrogen production during nitrogen fixation was relatively low due to recycling by an uptake hydrogenase, little hydrogen consumption was detected in Cyanothece and Synechococcu . On the other hand a reversible hydrogenase was demonstrated in the latter strains. However, only Cyanothece shows hydrogenase-catalysed hydrogen production in vivo under anaerobic conditions in the dark. It is suggested that hydrogen is a fermentation product, and that the physiological function of this reversible hydrogenase is the removal of excess reduction equivalents under such conditions.     

Characterization of four endophytic fungi as potential consolidated bioprocessing hosts for conversion of lignocellulose into advanced biofuels

Applied Microbiology and Biotechnology - Recently, several endophytic fungi have been demonstrated to produce volatile organic compounds (VOCs) with properties similar to fossil fuels, called...     

The phylogeny of unicellular, extremely halotolerant cyanobacteria

We examined the morphology, physiology, and 16S rRNA gene sequences of three culture collection strains and of ten novel isolates of unicellular cyanobacteria from hypersaline environments. The strains were morphologically diverse, with average cell widths ranging from 2.8 to 10.3 μm. There were single-celled, colonial, and baeocyte-forming strains. However, morphological traits were markedly variable with culture conditions. In contrast, all strains displayed extreme halotolerance (growing close to optimally at above 12% salinity); all were obligately marine, euryhaline, and moderately thermophilic; and all shared a suite of chemotaxonomic markers including phycobilins, carotenoids, and mycosporine-like amino acids. 16S rRNA gene sequence analysis indicated that the strains were related to each other. Sequence similarity analysis placed the strains in a monophyletic cluster (which we named the Halothece cluster) apart from all cultured or uncultured, not extremely halotolerant cyanobacteria whose 16S rRNA gene sequences are available in public nucleotide sequence databases. This represents the first case in which a phylogenetically coherent group of cyanobacteria can be defined on the basis of physiology. The Halothece cluster contained two subclusters that may be divergent at the generic level, one encompassing 12 strains (spanning 5% 16S rRNA gene sequence divergence and named the Euhalothece subcluster), and a single deep-branching isolate. Phenotypic characterization of the isolates, including morphological, physiological, and chemotaxonomic traits, did not distinguish these subclusters and only weakly suggested the existence of two separate clades, one encompassing strains of small cell size (cell width < 5 m) and another one encompassing strains of larger cell size. Received: 1 September 1997 / Accepted: 13 February 1998     

The role of systems biology in developing non-model cyanobacteria as hosts for chemical production

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Evaluation on potential of wild hosts as trap plants for managing gramineous stemborers in maize based-agroecosystem

As part of habitat management system to control cereal stemborers, various wild hosts used as trap plants were studied during the dry season from November 2003 to March 2004 at Melkassa, central Ethiopia. Five wild hosts of the family Poaceae [Pennisetum purpurum (Schumach), Sorghum vulgare variety sudanense (Pers.), Panicum coloratum L., Sorghum arundinaceum Stapf, and Hyperrhania rufa (Nees)] were evaluated as trap plants in maize, Zea mays L.,-based agroecosystem. The results of the study showed that maize plots surrounded by all tested wild hosts had significantly lower mean percentage of foliage damage and stemborer density than maize monocrop plots 15 m away from the treatment blocks. Interestingly, mean foliar damage and stemborer density between maize plots surrounded by wild hosts and maize monocrop plots within the treatment blocks was not significant. Percentage of tunneled stalks was significantly greater in maize monocrop plots 15 m away from the treatment blocks than in maize plots surrounded by all tested wild host plant species. Moreover, the highest mean percentage of parasitism (62%) of Chilo partellus (Swinhoe) by Cotesia flavipes (Cameron) was recorded in maize plots surrounded by P. purpureum. Therefore, the findings revealed that these wild hosts have considerable merit to be used as trap plants in the development of strategies for managing cereal stemborers in maize crops.     

Isolation of the carotenoid-containing cell wall of three unicellular cyanobacteria.

A carotenoid-containing membrane fraction devoid of chlorophyll and phycobiliproteins was isolated from three unicellular cyanobacteria, Synechococcus sp., Synechococcus leopoliensis UTEX 625, and Anacystis nidulans R-2, by aqueous-phase separation, hydrophobic chromatography, and differential centrifugation. The presence of 2-keto-3-deoxyoctonate, muramic acid, and diaminopimelic acid suggests that the preparation is highly enriched in cell wall. Electron micrographs of thin sections of this material showed C-shaped membrane profiles similar to those seen in other gram-negative cell wall preparations. The inactivation of cyanophage AS-1 by this fraction confirmed its identity as cell wall. The cell wall contained approximately equal weights of total carbohydrate and protein. Absorption maxima at 434, 452, and 488 nm indicated the presence of carotenoids. These were in the outer membrane and were not due to contaminating cytoplasmic or thylakoid membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed a broad band of approximately 50,000 molecular weight which contained 35% of the total outer membrane protein. This band was resolved into at least two components running at approximately 50,000 and 52,000 molecular weight. The smaller of these polypeptides was a glycoprotein. The polypeptide components were unaffected by protease or detergent treatment in either whole cells or isolated cell wall preparations, indicating that the polypeptide components were not exposed to the surface or easily removed from the hydrophobic environment.     

Metabolic pathways for photobiological hydrogen production by nitrogenase- and hydrogenase-containing unicellular cyanobacteria Cyanothece

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.     

Relationship between unicellular cyanobacteria established by indirect immunofluorescence of intact cells

Antisera prepared against intact, viable cells were used to show the applicability of a serological approach to detect relationships between unicellular cyanobacteria. Antisera were raised against eight unicellular cyanobacteria and two chlorophycean unicellular organisms. The staining reactivity of each antiserum was tested by the fixed indirect immunofluorescence assay against the different organisms, and each organism was tested for its reactivity with all of the different antisera. Absorption of antisera with the appropriate heterologous antigens was used to further characterize the relationship betweenAnacystis nidulans andSynechococcus cedrorum, and also the relationship between two subcultures of an isolate distinguished by morphological features. Absorption of antiserum was also used for the removal of antibodies to contaminating bacteria. The approaches used are suggested as a useful tool for determining relationships between unicellular cyanobacteria.     

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.     

Occurrence of facultative anoxygenic photosynthesis among filamentous and unicellular cyanobacteria.

Eleven of 21 cyanobacteria strains examined are capable of facultative anoxygenic photosynthesis, as shown by their ability to photoassimilate CO2 in the presence of Na2S, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 703-nm light. These include different cyanobacterial types (filamentous and unicellular) of different growth histories (aerobic, anaerobic, and marine and freshwater). Oscillatoria limnetica, Aphanothece halophytica (7418), and Lyngbya (7104) have different optimal concentrations of Na2S permitting CO2 photoassimilation, above which the rate decreases: 3.5, 0.7, and 0.1 mM, respectively. In A. halophytica, for each CO2 molecule photoassimilated two sulfide molecules are oxidized to elemental sulfur, which is excreted from the cells.The ecological and evolutionary significance of anoxygenic photosynthesis in the cyanobacteria is discussed.     

Diversity of diazotrophic unicellular cyanobacteria in the tropical North Atlantic Ocean

We present data on the genetic diversity and phylogenetic affinities of N2-fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene (nifH) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom Climacodium frauenfeldianum. These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins.     

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.     

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).     

Cyclic appearance of aerobic nitrogenase activity during synchronous growth of unicellular cyanobacteria

The aerobic synchronous growth of marine unicellular cyanobacteriaSynechococcus spp., with molecular nitrogen as the sole nitrogen growth nutrient source, was experimentally demonstrated. Cell division synchrony was induced by withholding aeration and illumination for 20 h. Three distinct cell division cycles of approximately 20 h each were observed. During the first cell division cycle, high degrees of synchrony of 78%–86% were observed in the series of cultures studied. The aerobic nitrogenase activity appeared shortly while the cell division was completed and the cell size was still small, and it disappeared after the cells were enlarging photosynthetically. Thus, three distinct cycles in aerobic nitrogenase activity were also observed having approximately the same 20-h interval as the cell division cycle. The peak aerobic nitrogenase activity was determined to be as high as 840–1220 nmol C₂H₂ reduced per milligram dry weight per hour.     

Maintenance of broad host range vector pKT230 in marine unicellular cyanobacteria

Clinical isolate of Vibrio mimicus were examined for production of cell-associated hemagglutinin (HA) and pili and for adherence to formalin-fixed human intestinal mucosa. V. mimicus grown on CFA agar for 3 h at 37 degrees C possessed HA and adhered better to the mucus layer than to the epithelial cell surface. A significant correlation was found between the HA titers and adherence ability to the epithelial cell surface of villi (P less than 0.05); adherence to the ileal lymphoid follicle-associated epithelium occurred at higher levels. In contrast, V. mimicus grown on CFA agar for 20 h at 37 degrees C exhibited lower levels of HA and reduced adherence ability. The production of pili was more pronounced after 20 h of incubation than after 3 h of incubation. In comparison with V. cholerae 01 and V. cholerae non-01 cultured under similar conditions, V. mimicus showed inferior adherence, but with similar HA production or piliation.     

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge

This communication presents a short outline of the current knowledge on the molecular basis of P_II signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P_II signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P_II signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P_II phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P_II (P_II-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P_II-P dephosphorylation, novel insights into the signals, to which P_II-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P_II-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P_II-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P_II-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.     

Increased toxicity of modified mosquitocidal binary toxins of Bacillus sphaericus expressed in Escherichia coli

The binary mosquitocidal genes of 51-kDa and 42-kDa proteins isolated from Bacillus sphaericus 1593 have been expressed at moderate levels in Escherichia coli employing the pQE expression system. The expressed proteins are readily visible in Coomassie-blue-stained protein gels. The recombinant E. coli cells expressing toxic proteins were toxic towards Culex larvae. During the assembly of crystals in B. sphaericus, the 42-kDa toxin is first cleaved at the N-terminal end by a specific B. sphaericus protease. To express the toxins in E. coli the B.sphaericus specific protease-recognition site was deleted at the N-terminal end of the 42-kDa toxin, thereby mimicking the structure of the toxin as present in the crystal. This modification resulted in a twofold increase in the toxicity of the E. coli cells expressing the modified 42-kDa toxin as a constituent of the binary toxin. Our results demonstrate the utility of this modification for heterologous expression of the binary toxin genes from B. sphaericus. Received: 18 July 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997