Dormancy of cells and organisms

1
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
2
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 KgtHLgtP

3
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)
4
Red fluorescence is lost in mature
akinetes Akinetes induction in P K medium
5
Fluorescence emission spectra of Aphanizomenon
cultures and akinetes
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Variations in fluorescence response of vegetative
cells and akinetes of A. ovalisporum Spectral
Laser Confocal Scanning Microscopy (LCSM) study
620 nm 655 nm 670 nm 690 nm 720 nm
Exponentially grown trichomes
Akinete-induced trichomes
Isolated Akinetes
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.
7
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.
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Principal component analysis (PCA) of
fluorescence emission spectra data of vegetative
cells and akinetes of A. ovalisporum
9
Is the loss of red fluorescence in mature
akinetes related to growth conditions Akinetes
induction in P K medium vs K P medium
10
Growth and akinete formation under different K
concentrations
Growth conditions BG11 (control) 0.23 mM K BG11 0.12 mM K BG11 0.06 mM K BG11 0.02 mM K BG11 K depleted
Doubling time (d) 5.2 5.4 7.4 14.8 No growth
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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)
P mM 0 100 200 400
germination (STD) 0 19 (6) 14 (3) 34 (10)
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Task Development of molecular tools to study the
development and germination of akinetes -
Progress, obstacles and plans

• Genomic Library construction
• Two cultures of Aphanizomenon ovalisporum were
  provided to Richard Reinhardt MPI Molecular
  Genetics Berlin-Dahlem, to create genomic
  libraries
• KLL strain grown in rich medium (BG11)
• HUJI strain grown in rich medium (BG11)
• The genomic libraries were cloned into pCC1Fos.
• The number of clones per culture
• 13,056 fosmids
• 15,360 fosmids
• A total of 3481 sequencing reads were made from
  both library fosmids, using
• primers T7 and M13(-28)

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Contig assembly of Aphanizomenon ovalisporum
genome
Anabaena variabillis ATCC 29413 Genome size 6.4
Mbp
Aphanizomenon ovalisporum 3500 sequencing
fragments from the genomic libraries
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/
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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.
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
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 akinetes
development/germination processes. Selected
fosmids were provided by M. Kube, from MPI
Molecular Genetics Berlin-Dahlem
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Working Plan

• 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.