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: c-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) Akinete formation – triggers and processes Observation Organism Source Generation of a nitrogen starvation signal resulting from progressive loss of the activity of nitrogenase and glutamine synthetase during diazotrophic growth Anabaena doliolum Rao et al, 1987 Akinetes differentiate upon P limitation but not under N, DIN, Fe, trace elements or light limitation. Anabaena cincinalis Van Dok & Hart 1996 Anabaena spp. Li et al 1997 The formation of akinetes in C .raciborskii can be triggered by an initial temperature shock and phosphorus is a necessary requirement to allow further growth and full development of akinetes. Cylindrosper mopsis raciborskii Moore et al 2003 Akinete differentiation was affected by the frequency of temperature fluctuations. Higher akinete frequency with multiple diurnal temperature fluctuations. Cylindrosper mopsis raciborskii Moore et al 2005 Akinete differentiation commences when Qp fell to a critical concentration (0.30.45 mgP cell-1). Akinetes developed at low temperature 10 -15 °C during the late exponential or stationary phase of growth. Akinetes were not formed at high temperature >30 °C. Four fold increase in light intensity imposed substantial increase in akinete concentration. # 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) 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. Genetic regulation of Akinete Gene Function Organism hepA Involved in normal deposition of the envelope in both heterocysts and akinetes. Essential for heterocyst formation, mutation in hepA result in abnormal akinete envelope. Anabaena variabilis hetR An intact hetR gene is required for akinete differentiation, and hetR is actively expressed in akinetes. Mutation in hetR blocks both hetrocyst and akinete differentiation. Nostoc ellipsosporum hetR mutant forms large akinete-like chilling-resistance cells following P starvation Nostoc punctiforme devR Over expression of devR, encoding a response regulatory protein that is likely involved in heterocyst polysaccharide synthesis, results in enhanced akinete differentiation. Nostoc punctiforme avaK Encodes for a protein enriched in akinetes. AvaK has no obvious structural motifs, no sequence similarity to known genes. No specific role in differentiation of akinete function was assigned. Anabaena variabilis argL Akinetes of argL mutant failed to germinate and lacked granules of cyanophycin. Nostoc ellipsosporum Source Wolk et al 1994 Leganes et al., 1994 Wong & Meeks 2002 Campbell et al. 1996 Zhou & Wolk 2002 Leganes et al 1998 # D5, # D6, # D7 Development of molecular tools for cyanobacteria • Genes that are expressed • Subtractive c-DNA libraries from A. ovalisporum Akinetes as compared to a culture producing akinetes and to a culture without akinete production • Expression studies • Identification of proteins found specifically in akinetes • Metabolomic profiling for carbohydrates associated with dormancy