Project description:Transcriptional changes were monitored in roots of the barley cultivar Regina following inoculation with zoospores of the nonadapted plasmodiophorid virus vector Polymyxa betae using the Affymetrix Barley1 GeneChip®. Barley cv. Regina seeds were imbibed in distilled water over night and then transferred on to moist heat treated (90°C over night) sand in plastic trays that were sealed. Trays were incubated at room temperature for 4 days to allow the seeds to germinate to Zadocks stage 07 of the decimal code for the growth stages of cereals (Tottman et al., 1979 Annals of Applied Biology, 93: 221-234; Zadocks et al., 1974 Weed Research, 14: 415-421). 110 barley (cv. Regina) seedling (Zadocks stage 07) were placed into a 90 mm diameter plastic Petri dish (3 Petri dishes per treatment) and 60 mL of P. betae zoospore suspension added (1 x 10 6 spores mL-1). Control plants were treated exactly the same way except zoospore-free buffer (0.1 g L-1 Phostrogen, 0.5% Bovine serum albumen) was added. Zoospore challenge (and control) occurred at an ambient temperature of 22°C. Roots were excised from ten plants per treatment at ten time points (15’, 30’, 45’ 1h, 2h, 3h, 4h, 5h, 6h, 7h) and frozen in liquid nitrogen. Three independent biological replicate experiments were done. Total RNA was isolated from each sample using TRI-reagent following the manufacturer’s instructions (Invitrogen) and then treated with DNase I (Ambion, Texas, USA) according to the manufacturer’s protocol. RNA samples for microarray hybridisation were further purified using RNeasy Mini Spin column purification (Qiagen, Hilden, Germany). The integrity of the RNA samples was confirmed using the BioAnalyzer 2100 (Agilent Technologies). Affymetrix GeneChip processing, including RNA quality control, microarray hybridisation and data acquisition was performed through contract research services by Geneservice Ltd (www.geneservice.co.uk). A total of six hybridisations were performed. Publication: European Journal of Plant Pathology 123(1):5-15.<br> ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Graham McGrann. The equivalent experiment is BB75 at PLEXdb.]
Project description:Paenibacillus polymyxa is a root-associated plant growth-promoting rhizobacterium. It was reported that many strains of P. polymyxa naturally exhibited the phenotypic variation. In the phase variation, the characteristics of the wild-type ‘B’ and the variant ‘F’ are very different in sporulation formation, motility, antibiotic ability and so on. For better understanding of the actual physiological changes, we performed RNA-seq analyses of P. polymyxa E681 to compare genome wide patterns of gene expression. As a result, we obtained 1,062 differentially expressed genes related to flagellar assembly and transport systems.
Project description:Due to the high capacity of their secretion machinery Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology. However, to date strains from only a few Bacillus species are used for enzyme production at the industrial scale. In this work, we introduce with Paenibacillus polymyxa DSM 292 a member of a different genus as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate efficient heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A was increased by optimizing the expression medium and testing various promoter sequences on the expression plasmid pBACOV. To identify promising new promoter sequences from the genome of P. polymyxa itself, quantitative mass spectrometry was used to analyze the secretome. The most strongly secreted host proteins were identified and the promoters regulating the expression of the corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from B. subtilis and B. megaterium. The best result was achieved with the promoter of the hypothetical protein PPOLYM_03468 from P. polymyxa, which in combination with the improved expression medium enabled the production of 5,475 U/l Cel8A which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.
Project description:Polymyxa betae belongs to the Plasmodiophorida (Phytomyxea, Rhizaria). Here, we report the first draft genome sequence of a member of the Polymyxa genus, which includes two obligate root endoparasite species, vectors of important soilborne plant viruses. The genome assembly was represented by 1,001 contigs, with a cumulated length of 27,085,946?bp.
Project description:This project investigated the protein expression pattern in P. polymyxa at different growth stages. The proteomics provided insights into responses of P. polymyxa to isobutanol and revealed a significant metabolic shift caused by alcohol production. These findings were used to evaluate the bottlenecks and further optimize the pathways towards improving product titers.