Enantioselective epoxidation of linolenic acid catalysed by cytochrome P450BM3 from Bacillus megaterium

Cytochrome P450BM3, from Bacillus megaterium, catalyses the epoxidation of linolenic acid 1 yielding 15,16-epoxyoctadeca-9,12-dienoic acid 2 with complete regio- and moderate enantio-selectivity (60% ee). The absolute configuration of the product is tentatively assigned as 15(R),16(S)-. The Michaelis–Menten parameters kcat and Km for the reaction were determined to be 3126 ± 226 min−1 and 24 ± 6 μM respectively.

Some novel techniques of parameter estimation for the dynamical models in biological systems

Inverse problems based on using experimental data to estimate unknown parameters of a system often arise in biological and chaotic systems. In this paper, we consider parameter estimation in systems biology involving linear and non-linear complex dynamical models, including the Michaelis–Menten enzyme kinetic system, a dynamical model of competence induction in Bacillus subtilis bacteria and a model of feedback bypass in B. subtilis bacteria. We propose some novel techniques for inverse problems. Firstly, we establish an approximation of a non-linear differential algebraic equation that corresponds to the given biological systems. Secondly, we use the Picard contraction mapping, collage methods and numerical integration techniques to convert the parameter estimation into a minimization problem of the parameters. We propose two optimization techniques: a grid approximation method and a modified hybrid Nelder–Mead simplex search and particle swarm optimization (MH-NMSS-PSO) for non-linear parameter estimation. The two techniques are used for parameter estimation in a model of competence induction in B. subtilis bacteria with noisy data. The MH-NMSS-PSO scheme is applied to a dynamical model of competence induction in B. subtilis bacteria based on experimental data and the model for feedback bypass. Numerical results demonstrate the effectiveness of our approach.

Metabolic and ecological study of environmental pentose utilizing bacteria (E-PUB)

Lignocellulosic materials, such as sugar cane bagasse, a waste product of the sugarcane processing industry, agricultural residues and herbaceous crops, may serve as an abundant and comparatively cheap feedstock for largescale industrial fermentation, resulting in the production of marketable end-products. However, the complex structure of lignocellulosic materials, the presence of various hexose and pentose sugars in the hemicellulose component, and the presence of various compounds that inhibit the organisms selected for the fermentation process, all constitute barriers that add to the production costs and make full scale industrial production economically less feasible. The work presented in this thesis was conducted in order to screen microorganisms for ability to utilize pentose sugars derived from the sugar mill industrial waste. A large number of individual bacterial strains were investigated from hemi-cellulose rich material collected at the Proserpine and Maryborough sugar mills, notably soil samples from the mill sites. The research conducted to isolation of six pentose-capable Gram-positive organisms from the actinomycetes group by using pentose as a sole carbon source in the cultivation process. The isolates were identified as Corynebacterium glutamicum, Actinomyces odontolyticus, Nocardia elegans, and Propionibacterium freudenreichii all of which were isolated from the hemicellulose-enriched soil. Pentose degrading microbes are very rare in the environment, so this was a significant discovery. Previous research indicated that microbes could degrade pentose after genetic modification but the microbes discovered in this research were able to naturally utilize pentose. Six isolates, identified as four different genera, were investigated for their ability to utilize single sugars as substrates (glucose, xylose, arabinose or ribose), and also dual sugars as substrates (a hexose plus a pentose). The results demonstrated that C. glutamicum, A. odontolyticus, N. elegans, and P. freudenreichii were pentose-capable (able to grow using xylose or other pentose sugar), and also showed diauxie growth characteristics during the dual-sugar (glucose, in combination with xylose, arabinose or ribose) carbon source tests. In addition, it was shown that the isolates displayed very small differences in growth rates when grown on dual sugars as compared to single sugars, whether pentose or hexose in nature. The anabolic characteristics of C. glutamicum, A. odontolyticus, N. elegans and P. freudenreichii were subsequently investigated by qualitative analysis of their end-products, using high performance liquid chromatography (HPLC). All of the organisms produced arginine and cysteine after utilization of the pentose substrates alone. In addition, P. freudenreichii produced alanine and glycine. The end-product profile arising from culture with dual carbon sources was also tested. Interestingly, this time the product was different. All of them produced the amino acid glycine, when grown on a combination substrate-mix of glucose with xylose, and also glucose with arabinose. Only N. elegans was able to break down ribose, either singly or in combination with glucose, and the end-product of metabolism of the glucose plus ribose substrate combination was glutamic acid. The ecological analysis of microbial abundance in sugar mill waste was performed using denaturing gradient gel electrophoresis (DGGE) and also the metagenomic microarray PhyloChip method. Eleven solid samples and seven liquid samples were investigated. A very complex bacterial ecosystem was demonstrated in the seven liquid samples after testing with the PhyloChip method. It was also shown that bagasse leachate was the most different, compared to all of the other samples, by virtue of its richness in variety of taxa and the complexity of its bacterial community. The bacterial community in solid samples from Proserpine, Mackay and Maryborough sugar mills showed huge diversity. The information found from 16S rDNA sequencing results was that the bacterial genera Brevibacillus, Rhodospirillaceae, Bacillus, Vibrio and Pseudomonas were present in greatest abundance. In addition, Corynebacterium was also found in the soil samples. The metagenomic studies of the sugar mill samples demonstrate two important outcomes: firstly that the bagasse leachate, as potentially the most pentose-rich sample tested, had the most complex and diverse bacterial community; and secondly that the pentose-capable isolates that were initially discovered at the beginning of this study, were not amongst the most abundant taxonomic groups discovered in the sugar mill samples, and in fact were, as suspected, very rare. As a bioprospecting exercise, therefore, the study has discovered organisms that are naturally present, but in very small numbers, in the appropriate natural environment. This has implications for the industrial application of E-PUB, in that a seeding process using a starter culture will be necessary for industrial purposes, rather than simply assuming that natural fermentation might occur.

RNAi for insect-proof plants

RNA interference induced in insects after ingestion of plant-expressed hairpin RNA offers promise for managing devastating crop pests

A suite of novel promoters and terminators for plant biotechnology

The gene regulation signals from subterranean clover stunt virus (SCSV) were investigated for their expression in dicot plants. The SCSV genome has at least eight circular DNA molecules. Each circular DNA component contains a promoter element, a single open reading frame and a terminator. The promoters from seven of the segments were examined for their strength and tissue specificity in transgenic tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and cotton (Gossypium hirsutum L.) using a GUS reporter gene assay system. While the promoters of many of the segments were poorly expressed, promoters derived from segments 4 and 7 were shown to direct high levels of expression in various plant tissues and organs. The segment 1 promoter directs predominantly callus-specific expression and, when used to control a selectable marker gene, facilitated the transformation of all three species (tobacco, potato and cotton). From the results, a suite of plant expression vectors (pPLEX) derived from the SCSV genome were constructed and used here to produce herbicide- and insect-resistant cotton, demonstrating their utility in the expression of foreign genes in dicot crop species and their potential for use in agricultural biotechnology.

DDT resistance and transformation by different microbial strains isolated from DDT-contaminated soils and compost materials

Bioremediation is a potential option to treat 1, 1, 1-trichloro-2, 2 bis (4-chlorophenyl) ethane (DDT) contaminated sites. In areas where suitable microbes are not present, the use of DDT resistant microbial inoculants may be necessary. It is vital that such inoculants do not produce recalcitrant breakdown products e.g. 1, 1-dichloro-2, 2-bis (4-chlorophenyl) ethylene (DDE). Therefore, this work aimed to screen DDT-contaminated soil and compost materials for the presence of DDT-resistant microbes for use as potential inoculants. Four compost amended soils, contaminated with different concentrations of DDT, were used to isolate DDT-resistant microbes in media containing 150 mg I -1 DDT at three temperatures (25, 37 and 55°C). In all soils, bacteria were more sensitive to DDT than actinomycetes and fungi. Bacteria isolated at 55°C from any source were the most DDT sensitive. However DDT-resistant bacterial strains showed more promise in degrading DDT than isolated fungal strains, as 1, 1-dichloro 2, 2-bis (4-chlorophenyl) ethane (DDD) was a major bacterial transformation product, while fungi tended to produce more DDE. Further studies on selected bacterial isolates found that the most promising bacterial strain (Bacillus sp. BHD-4) could remove 51% of DDT from liquid culture after 7 days growth. Of the amount transformed, 6% was found as DDD and 3% as DDE suggesting that further transformation of DDT and its metabolites occurred.

Quality assurance of aerial applications of larvicides for mosquito control : effects of granule and catch tray size on field monitoring programs

Aerial applications of granular insecticides are preferable because they can effectively penetrate vegetation, there is less drift, and no loss of product due to evaporation. We aimed to 1) assess the field efficacy ofVectoBac G to control Aedes vigilax (Skuse) in saltmarsh pools, 2) develop a stochastic-modeling procedure to monitor application quality, and 3) assess the distribution of VectoBac G after an aerial application. Because ground-based studies with Ae. vigilax immatures found that VectoBac G provided effective control below the recommended label rate of 7 kg/ha, we trialed a nominated aerial rate of 5 kg/ha as a case study. Our distribution pattern modeling method indicated that the variability in the number of VectoBac G particles captured in catch-trays was greater than expected for 5 kg/ha and that the widely accepted contour mapping approach to visualize the deposition pattern provided spurious results and therefore was not statistically appropriate. Based on the results of distribution pattern modeling, we calculated the catch tray size required to analyze the distribution of aerially applied granular formulations. The minimum catch tray size for products with large granules was 4 m2 for Altosid pellets and 2 m2 for VectoBac G. In contrast, the minimum catch-tray size for Altosid XRG, Aquabac G, and Altosand, with smaller granule sizes, was 1 m2. Little gain in precision would be made by increasing the catch-tray size further, when the increased workload and infrastructure is considered. Our improved methods for monitoring the distribution pattern of aerially applied granular insecticides can be adapted for use by both public health and agricultural contractors.

Entomopathogens for control of Hypsipyla spp

Published information on the incidence of pathogens in the field and laboratory infections of Hypsipyla spp. with entomopathogens is reviewed. In addition, some preliminary results of field collections from Ghana and Costa Rica are presented. Fungal pathogens from the Deuteromycetes have been isolated from both H. robusta Moore and H. grandella Zeller. Mermithid nematodes, Hexamermis spp., have been frequently isolated from larvae in the field and incidence of infection with these pathogens can reach significant levels. Microsporidia have been found in cadavers of larvae collected in the field but none have been identified so far. A number of pathogens of other Lepidoptera have been shown to be infectious to H. grandella , including Bacillus thuringiensis , Deuteromycete fungi and a nucleopolyhedrovirus (NPV) from Autographa californica . Hypsipyla spp. are difficult targets for microbial control, since the larvae are cryptic, occur at low density and occur sporadically. In addition, there is a low damage threshold, the plant is susceptible for a number of years and the susceptible part of the plant will rapidly outgrow any surface application. Key features of the biology of entomopathogens with relevance to the control of low density and cryptic pests are discussed. In the light of this experience, we discuss strategies to improve the possibilities of microbial control of this pest and suggest areas for research.

Wound healing from the outback: novel wound healing therapeutics from native Australian plants

Introduction Chronic wounds are an area of major concern. The on-going and in-direct costs are substantial, reaching far beyond the costs of the hospitalization and associated care. As a result, pharmacological therapies have been developed to address treatment insufficiencies, however, the availability of drugs capable of promoting the wound repair process still remain limited. The wound healing properties of various herbal plants is well recognised amongst indigenous Australians. Hence, based on traditional accounts, we evaluated the wound healing potential of two Australian native plants. Methods Bioactive compounds were methanol extracted from dried plant leaves that were commercially sourced. Primary keratinocyte (Kc) and fibroblast (Fib) cells (denoted as Kc269, Kc274, Kc275, Kc276 and Fib274) obtained from surgical discarded tissue were cultured in 48-well plates and incubated (37⁰C, 5% CO2) overnight. The growth media was discarded and replaced with fresh growth media plus various concentrations (15.12 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL) of the plant extracts. Cellular responses were measured using the alamarBlue® assay and the CyQUANT® assay. Plant extracts in the aqueous phase were prepared by boiling whole leaves in water and taking aqueous phase samples at various (1, 2 , 5 minutes boiling) time points. Plant leaves were either added before the water was boiled (cold boiled) or after the water was boiled (hot boiled). The final concentrations of the aqueous plant extracts were 3.3 ng/mL (± 0.3 ng/mL) per sample. The antimicrobial properties of the plant extracts were tested using the well diffusion assay method against Staphylococcus aureus, Klebsiella pnuemoniae and methicillin resistant S. aureus and Bacillus cereus. Results Assay results from the almarBlue® and CYQUANT® assays indicated that extracts from both native plants at various time points (0, 24 and 48 hours) and concentrations (31.25 mg/mL, 62.5 mg/mL, and 125 mg/mL) were significantly higher (n=3, p=0.03 for Kc269, p=0.04 for Kc274, p=0.02 for Fib274, p=0.04 for Kc275 and p=0.001 for Kc276) compared with the untreated controls. Neither plant extract demonstrated cytotoxic effects. Significant antimicrobial activity against methicillin resistant Staphylococcus aureus (p=0.0009 for hot boiled plant A, n=2, p=0.034 for cold boiled plant A, n=2) K. pnuemoniae (p=0.0009 for hot boiled plant A, n=2, p=0.002 for cold boiled plant A, n=2) and B. cereus (p=0.0009 for hot boiled plant A, n=2, p=0.003 for cold boiled plant A, n=2) was observed at concentrations of 3.2 ng/mL for plant A and 3.4 ng/mL for plant B. Conclusion Both native plants contain bioactive compounds that increase cellular metabolic rates and total nucleic acid content. Neither plant was shown to be cytotoxic. Furthermore, both exhibited significant antimicrobial activity.

Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Antimicrobial effects of ethanolic extracts from Cuscuta reflexa Roxb. (Convolvulaceae).

Ethanolic whole plant extracts obtained from Cuscuta reflexa Roxb were screened against Gram positive (Bacillus subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli and Salmonella typhi) bacteria to evaluate their antimicrobial activity. Of the four concentrations of plant extract tested (200 µg/mL, 300 µg/mL, 400 µg/mL or 500 µg/mL), 500 µg/mL elicited the greatest zones of bacterial inhibition across three of the bacteria. In contrast, the growth of Salmonella typhi was not halted regardless of extract concentration. At 200 µg/mL, only the growth of E. coli was inhibited. Overall, although the greatest antimicrobial activity was demonstrated to be against E. coli at a concentration of 500 µg/mL (24.6±0.24), upon comparison to the other bacteria, both B. cereus and S. aureus educed similar zones of inhibition upon comparison to their positive antibiotic control.