"Endomicrobia" and Other Bacteria Associated with the Hindgut of Dermolepida albohirtum Larvae

Symbiotic bacteria residing in the hindgut chambers of scarab beetle larvae may be useful in paratransgenic approaches to reduce larval root-feeding activities on agricultural crops. We compared the bacterial community profiles associated with the hindgut walls of individual Dermolepida albohirtum third-instar larvae over 2 years and those associated with their plant root food source among different geographic regions. Denaturing gradient gel electrophoresis analysis was used with universal and Actinobacteria-specific 16S rRNA primers to reveal a number of taxa that were found consistently in all D. albohirtum larvae but not in samples from their food source, sugarcane roots. These taxa included representatives from the "Endomicrobia," Firmicutes, Proteobacteria, and Actinobacteria and were related to previously described bacteria from the intestines of other scarab larvae and termites. These universally distributed taxa have the potential to form vertically transmitted symbiotic associations with these insects

RNAi-mediated abrogation of trehalase expression does not affect trehalase activity in sugarcane

To engineer trehalose metabolism in sugarcane (Saccharum spp. hybrids) two transgenes were introduced to the genome: trehalose-6-phosphate synthase- phosphatase (TPSP), to increase trehalose biosynthesis and an RNAi transgene specific for trehalase, to abrogate trehalose catabolism. In RNAi-expressing lines trehalase expression was abrogated in many plants however no decrease in trehalase activity was observed. In TPSP lines trehalase activity was significantly higher. No events of co-integration of TPSP and RNAi transgenes were observed. We suggest trehalase activity is essential to mitigate embryonic lethal effects of trehalose metabolism and discuss the implications for engineering trehalose metabolism

Metabolic engineering of plants for the synthesis of polyhydroxyalkanaotes

Synthesis of polyhydroxyalkanoates (PHAs) in crop plants is viewed as an attractive approach for the production of this family of biodegradable plastics in large quantities and at low costs. Synthesis of PHAs containing various monomers has so far been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHAs in crop plants remains a challenging project. PHA synthesis at a high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth in some plants. The challenges for the future are to succeed in the synthesis of PHA copolymer with a narrow range of monomer composition, at levels that do not compromise plant productivity, and in creating methods for efficient and economical extraction of polymer from plants. These goals will undoubtedly require a deeper understanding of plant biochemical pathways as well as advances in biorefinery

Isolation and characterisation of genes encoding thermoactive and thermostable dextranases from two thermotolerant soil bacteria

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Assessment of Gut Bacteria for a Paratransgenic Approach To Control Dermolepida albohirtum Larvae▿

Bacteria from the hindguts of Dermolepida albohirtum larvae were assessed for their potential to be used in paratransgenic strategies that target scarab pests of sugarcane. Bacteria isolated in pure culture from the hindguts of D. albohirtum larvae were from the Proteobacteria, Firmicutes, and Actinobacteria phyla and matched closely with taxa from intestinal and rhizosphere environments. However, these isolates were not the most common gut-associated bacteria identified in denaturing gradient gel electrophoresis (DGGE) hindgut profiles. Subsequently, eight species of gut bacteria were fed to larvae, and RNA-based DGGE analysis of 16S rRNA was used to detect the persistence of these isolates in the hindgut environment. One of these isolates (Da-11) remained metabolically active in the hindgut for 19 days postconsumption. Da-11 most likely forms a new genus within the Burkholderiales order, along with taxa independently identified from larvae of the European scarab pest, Melolontha melolontha. Using the EZ::Tn5 transposon system, a kanamycin resistance gene was inserted into the chromosome of Da-11, thus establishing a stable transformation technique for this species. A second feeding trial that included inoculating approximately 400 transgenic Da-11 cells onto a food source resulted in a density of 1 × 106 transgenic Da-11 cells/ml in the hindguts of larvae at 9 days postconsumption. These populations were maintained in the hindgut for at least another 12 days. The successful isolation, genetic transformation, and establishment of transgenic Da-11 cells in the hindguts of D. albohirtum larvae fulfill fundamental requirements for the future development of a paratransgenic approach to control scarab pests of sugarcane

RNAi-mediated abrogation of trehalase expression does not affect trehalase activity in sugarcane

Article discussing how RNAi-mediated abrogation of trehalase expression does not affect trehalase activity in sugarcane

The N-terminal presequence from F1-ATPase β-subunit of Nicotiana plumbaginifolia efficiently targets green fluorescent fusion protein to the mitochondria in diverse commercial crops

Approximately 10-15% of plant nuclear genes appear to encode mitochondrial proteins that are directed to mitochondria by specific targeting signals. Reports on the heterologous function of these targeting signals are generally limited to one or a few species, with an emphasis on model plants such as tobacco and Arabidopsis. Given their sequence diversity and their insufficient testing in commercially important crops (including monocotyledonous crops), the extent to which these signals can be relied on for biotechnological purposes across species remains to be established. This study provides the experimental verification of a mitochondrial signal that is functional across diverse crop species, including five monocots (sugarcane, wheat, corn, sorghum and onion) and seven dicots ( cucumber, cauliflower, tomato, capsicum, pumpkin, coriander and sunflower). In all 12 crops, transient assays following microprojectile bombardment showed that the N-terminal mitochondrial presequence from F-1-ATPase beta-subunit (ATPase-beta) of Nicotiana plumbaginifolia Viv. targeted green fluorescent fusion protein to the mitochondria. The transient assay results in sugarcane were confirmed in stably transformed root cells. The ATPase-beta signal should be a useful metabolic engineering tool for directing recombinant proteins to the mitochondrial matrix in diverse plant species of commercial interest

A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity

Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50°C after exposure to 68°C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62°C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T opt 60°C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling