Transcript Slide 1
Work package 1 (WP1) Topic: Cyanobacterial Dormant Forms in an Aquatic environment Objective: Establishment of dormant stage/ Induction of akinetes in cyanobacteria Task Deliverable # month 1. Determine conditions for the induction of akinetes D4 9 (M9) 2. Physiological processes involved in the induction of the dormant stage D8 30 (M30) 3. Compounds and metabolites associated with the dormant stage D8 30 (M30) 4. Development of molecular tools: DNA libraries. Gene expression during akinete formation (avaK, hetR) D5 D6 18 (M18) 27 (M27) 5. Quantitative analyses of the expression of specific genes by QPCR D7 35 (M35) 6. Summary and Report (M36) # D4 and # D8 Basic studies to reveal conditions and mechanisms involved in induction of akinetes formation, dormancy and desiccation: Nutrient Depletion (P), Light, Temp, O2 (hypoxia), K Specialized envelopes Storage of metabolites [carbohydrates, cyanophycin (N)] – enzymes involved, e.g cyanophycine synthase Tolerance to stress: heat shock proteins/chaperones-function in maintaining structural integrity of biomolecules. Tryptophan, aspartate, aspargin, phenyl-alanine and serine triggered akinete formation. Mature akinetes Respiration and Photosynthetic apparatus (is there pigments production?) O2 evolution/consumption Reduced energy production? • To collect adequate material for further genomic, proteomic and metabolomic studies. Research Objective: To elucidate the molecular mechanism of akinete differentiation in the cyanobacteria Aphanizomenon ovalisporum. Research plan: • Characterize morphologically the developmental stages from a vegetative cell to a mature akinete. • Test for environmental variations that can signal for akinete differentiation, such as: 1. K starvation 2. Different light/dark regimens (circadian clock) 3. Different light wavelengths (Phytochrome signaling pathway) 4. Temperature • Mutagenesis screen – looking for none akinetes forming filaments in a K depleted media. • Establish transformation of Aphanizomenon ovalisporum • Test for the expression of candidate genes in akinete cells by PCR, in situ hybridization and fusion proteins (i.e. GFP) Candidate genes in akinetes differentiation: AvaK – Akinete marker gene with an unknown function found in A.variabilis (Zhou and Wolk, 2002) Signal transduction: hetR - A protease showing DNA binding activity. hetR is required for akinete differentiation, and is actively expressed in akinetes (Leganés et al., 1994). devR - Introduction of a plasmid containing wild-type devR into cells of Nostoc sp. strain 29133 results in a stimulation of akinete formation (Campbell et al.,1996). devR is a member of the two-component system, regulatory protein family. Involved in signal transduction, that functions to detect and respond to environmental changes. kdp operon – K+ dependent ATPase Cell wall synthesis: hepA - An ABC transporter required for the normal deposition of akinetes and heterocyst envelope polysaccharide (Leganés, 1994 found in A. variabilis ATCC 29413). Cyanophysin synthesis and degradation: cphA – Cyanophycin synthetase cphB - Cyanophycinase (exopeptidase hydrolyzing cyanophycin)