Dormancy of cells and organisms – strategies for survival and preservation

Cyanobacteria Dormancy Forms in an Aquatic environment

Ora Hadas, Assaf Sukenik, Ruth Kaplan-LeviDiti Viner-Mozzini, Merav Hadary

Kinneret Limnological LaboratoryIsrael 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 akinetesAkinetes induction in –P –K medium

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Fluorescence emission spectra of Aphanizomenon cultures and akinetes

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trichomes

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Variations in fluorescence response of vegetative cells and akinetes of A. ovalisporumSpectral 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.

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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 conditionsAkinetes induction in –P –K medium vs –K +P medium

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Growth and akinete formation under different K concentrations

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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)

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% germination (STD)

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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 fosmids2. 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 29413Genome 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 SeqManTMII 5.03, DNASTAR package .Anabaena variabillis ATCC 29413 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 – http://est.molgen.mpg.de/Sleeping Beauty/A. variabillis ATCC 29413 genome - ftp://ftp.ncbi.nih.gov/genomes/Bacteria/

Gene Function Fosmid

AvaK Akinete marker sbfos01-3p12, sbfos02-6e7

HetR protease with DNA binding activity sbfos02-9m21,sbfos02-7e10

DevR two-component system, regulatory protein sbfos01-3d6, sbfos02-8h10

HepA ABC transporter sbfos01-9d6

CphA Cyanophycin synthetase sbfos01-4l16

CphB Cyanophycinase sbfos01-4l16

Kdp operon K+ transporting ATPase sbfos01-3f13

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: