Dormancy of cells and organisms – strategies for survival and preservation Cyanobacteria Dormancy Forms in an Aquatic environment Ora Hadas, Assaf Sukenik, Ruth Kaplan-Levi Diti Viner-Mozzini, Merav Hadary Kinneret Limnological Laboratory Israel Oceanographic & Limnological Research

Task: Determine conditions for the induction of akinetes  K deficiency triggers akinete formation in a yet unexplained process.  50% deficiency in K triggers the formation of akinetes but only slightly affect growth  P deficiency and high light have additional effect on the formation of akinetes K>HL>P

Task: Physiological processes involved in the induction of the dormant stage  Young akinetes maintain photosynthetic capacity at a similar manner as found for their adjacent vegetative cells in filaments grown in akinete- inducing medium.  Mature akinetes maintain residual photosynthetic activity.  Some components of the photosynthetic apparatus appear to remain intact in akinetes.  In mature akinetes Photosystem I (PSI) and Photosystem II (PSII) complexes are kept apparently at a slightly higher molar ratio then in vegetative young cells (less PSII).  The phycobilisome pool is reduced in akinetes and disattached from the core antenna complexes. Sukenik A., Beardall J. and Hadas O. (2006) Photosynthetic characterization of developing and mature akinetes of Aphanizomenon ovalisporum (Cyanoprokaryota). J. Phycol. (accepted)

Red fluorescence is lost in mature akinetes Akinetes induction in –P –K medium

Fluorescence emission spectra of Aphanizomenon cultures and akinetes

620 nm655 nm670 nm690 nm720 nm Exponentially grown trichomes Akinete- induced trichomes Isolated Akinetes Variations in fluorescence response of vegetative cells and akinetes of A. ovalisporum Spectral Laser Confocal Scanning Microscopy (LCSM) study Individual images taken from a lambda scan with 31 steps of 5 nm bandwidth between 600 and 750 nm. Photographs shown are images collected at specific fluorescence emission wavelength representing background emission at 620 and 720 nm, phycocyanin emission at 655 nm, allophycocyanin emission at 670 n, and chlorophyll at 690 nm.

Typical fluorescence emission spectra of vegetative cells and akinetes of A. ovalisporum - A Spectral Laser Confocal Scanning Microscopy (LCSM) study A - exponentially grown vegetative cell B - vegetative cell in akinete-induced culture, C - trichome attached akinete in akinete- induced culture, D - isolated fluoresce akinete, E - isolated non-fluoresce akinete.

Principal component analysis (PCA) of fluorescence emission spectra data of vegetative cells and akinetes of A. ovalisporum Exponentially grown vegetative cells Akinete in exponentially grown cells Highly fluoresce akinete Non- fluoresce akinete

Is the loss of red fluorescence in mature akinetes related to growth conditions Akinetes induction in –P –K medium vs –K +P medium

Growth conditions BG11 (control) 0.23 mM K BG mM K BG mM K BG mM K BG11 K depleted Doubling time (d) No growth Growth and akinete formation under different K concentrations

Task: Determine environmental variables/stimuli responsible for germination of akinetes  Induction and isolation of akinetes  Germination experiments in multi-well plates  Experimental parameters: P concentration, pH, temperature (10 – 30 C), light intensity and quality, L/D regime Preliminary results (Effect of P concentration on germination rate) P  M % germination (STD) 019 (6)14 (3)34 (10)

Genomic Library construction Two cultures of Aphanizomenon ovalisporum were provided to Richard Reinhardt MPI Molecular Genetics Berlin-Dahlem, to create genomic libraries: 1. KLL strain grown in rich medium (BG11) 2. HUJI strain grown in rich medium (BG11) The genomic libraries were cloned into pCC1Fos. The number of clones per culture: 1. 13,056 fosmids 2. 15,360 fosmids A total of 3481 sequencing reads were made from both library fosmids, using primers T7 and M13(-28) Task: Development of molecular tools to study the development and germination of akinetes - Progress, obstacles and plans

Anabaena variabillis ATCC Genome size ~ 6.4 Mbp Aphanizomenon ovalisporum ~ 3500 sequencing fragments from the genomic libraries Contig assembly of Aphanizomenon ovalisporum genome The sequencing results of the Aphanizomenon ovalisporum genomic libraries were submitted to the assemble program SeqMan TM II 5.03, DNASTAR package. Anabaena variabillis ATCC genome was included in the contig assembly, as both Cyanobacteria are members of the Nostoceae family, it is expected to share some similarities in various gene loci. A. ovalisporum nucleotides database – Beauty/ Beauty/ A. variabillis ATCC genome - ftp://ftp.ncbi.nih.gov/genomes/Bacteria/ftp://ftp.ncbi.nih.gov/genomes/Bacteria/

GeneFunctionFosmid AvaKAkinete markersbfos01-3p12, sbfos02-6e7 HetRprotease with DNA binding activitysbfos02-9m21,sbfos02-7e10 DevRtwo-component system, regulatory proteinsbfos01-3d6, sbfos02-8h10 HepAABC transportersbfos01-9d6 CphACyanophycin synthetasesbfos01-4l16 CphBCyanophycinasesbfos01-4l16 Kdp operonK+ transporting ATPasesbfos01-3f13 Candidate genes in akinetes differentiation: Comparison between Anabaena genome and Aphanizomenon contigs led to the selection of nine fosmids that may contain candidate genes loci. Working hypothesis: Genome sequence of target gene loci may reveal other genetic units that function in a coordinate manner under the same transcriptional control as the target genes e.g. genes associated with the akinete’s development/germination processes. Selected fosmids were provided by M. Kube, from MPI Molecular Genetics Berlin-Dahlem

Following up several candidate genes: cloning, sequencing, expression, etc. Total RNA extraction and mRNA isolation from various developmental stages (Exponentially grown culture, akinete induced culture, mature isolated akinetes, germinating akinetes). In cooperation with MPI Preparation of normalized combined cDNA libraries (by MPI) to be further used for microarray slides. ESTs of a cDNA library Microarray analysis to test genes expression from different developmental stages. Candidate genes selected from the microarray experiments will be further analyzed by Real-Time PCR. Genes sequences will be deposited in SB gene database and analyzed by bioinformatic tools (i.e. protein structure, comparison to genes expressed in other organisms, etc). Establishment of a transformation system, in order to follow gene expression in vivo, by GFP (green fluorescence protein) fusions. Working Plan: