Work package 1 (WP1) Topic: Cyanobacterial Dormant Forms in an Aquatic environment Objective: Establishment of dormant stage/ Induction of akinetes in cyanobacteria TaskDeliverable #month 1. Determine conditions for the induction of akinetes D49 (M9) 2. Physiological processes involved in the induction of the dormant stage D830 (M30) 3. Compounds and metabolites associated with the dormant stage D830 (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 D735 (M35) 6. Summary and Report(M36)

Akinete formation – triggers and processes ObservationOrganismSource 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 Akinete differentiation commences when Qp fell to a critical concentration (  gP cell -1 ). Akinetes developed at low temperature °C during the late exponential or stationary phase of growth. Anabaena spp. Li et al 1997 Akinetes were not formed at high temperature >30 °C. 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 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, O 2 (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?) O 2 evolution/consumption Reduced energy production? To collect adequate material for further genomic, proteomic and metabolomic studies.

Genetic regulation of Akinete GeneFunctionOrganismSource 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 Wolk et al 1994 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 Leganes et al., 1994 hetR mutant forms large akinete-like chilling-resistance cells following P starvation Nostoc punctiforme Wong & Meeks 2002 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 Campbell et al 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 Zhou & Wolk 2002 argL Akinetes of argL mutant failed to germinate and lacked granules of cyanophycin. Nostoc ellipsosporum 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