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Applied and Environmental Microbiology, November 2001, p. 5247-5253, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5247-5253.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of the isiA Gene across
Cyanobacterial Strains: Potential for Probing Iron Deficiency
U.
Geiß,1,2
J.
Vinnemeier,2
A.
Kunert,2
I.
Lindner,2
B.
Gemmer,1
M.
Lorenz,3
M.
Hagemann,2 and
A.
Schoor1,*
Institut für Ökologie,
Botanisches Institut, Ernst-Moritz-Arndt-Universität Greifswald,
D-17487 Greifswald,1 FB
Biowissenschaften, Universität Rostock, D-18051
Rostock,2 and Experimentelle Phykologie
und Sammlung von Algenkulturen, Albrecht-von-Haller-Insitut
für Pflanzenwissenschaften, Universität
Göttingen, D-37073 Göttingen,3
Germany
Received 18 April 2001/Accepted 7 August 2001
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ABSTRACT |
The use of isiA expression to monitor the iron status
of cyanobacteria was investigated. Studies of laboratory cultures of the cyanobacterium Synechocystis sp. strain PCC 6803 showed
that isiA expression is dependent on the organism's
response to iron deficiency; isiA expression starts as soon
as a decline in the rate of growth begins. isiA expression
is switched on at concentrations of iron citrate of less than 0.7 µM.
A PCR method was developed for the specific amplification of the
iron-regulated isiA gene from a variety of cyanobacteria.
After we developed degenerate primers, 15 new internal isiA
fragments (840 bp) were amplified, cloned, and sequenced from strains
obtained from algal collections, from new isolates, and from enriched
field samples. Furthermore, isiA expression could be
detected by means of reverse transcription-PCR when enriched field
samples were exposed to restricted iron availability. These results
imply that determining the level of iron-regulated isiA
expression can serve to indicate iron deficiency in
cyanobacterial samples of differing origins from the field.
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INTRODUCTION |
Iron as an important micronutrient
of phytoplankton had been neglected for a long period before Martin and
coworkers (28-30) revived its consideration in their
approach to defining the basic pattern of plankton productivity in the
open oceans. Subsequent research dealt with iron as the crucial
nutrient in high-nitrate, low-chlorophyll regions of the open oceans
and its ecological importance (1, 12, 16, 22, 43). Some
evidence of iron-limited phytoplankton development also exists for
coastal upwelling regions (13). Owing to the complex
chemical behavior of iron, the bioavailable iron concentration
cannot easily be manipulated in oxygen-rich waters by iron
enrichment and iron depletion procedures. Trace-metal precipitation and
adsorption to particles as well as ion-exchange processes (for a
review, see reference 39) are only a few of the potential
effects of these procedures that can hamper the desirable effects of
fertilization and depletion experiments. Although these are methods
that provide support for the characterization of the concentration of
bioavailable iron in water (42), the great number and
variety of variously efficient mechanisms of organismic iron
sequestration (6, 14, 43) limit the interpretation of the
corresponding analyses. Thus, the use of molecular markers to detect
the physiological state of iron limitation in microalgae without the
use of artificial manipulations of water chemistry would represent an
important achievement in proving iron limitation in parts of phytoplankton.
Besides producing results of general interest, the use of molecular
approaches became necessary to go beyond the limits of conventional
methodology in aquatic ecology. Scanlan et al. (36) introduced the phosphate-binding protein PstS as a potential diagnostic marker for investigating phosphate stress in photosynthetic
picoplankton. For iron limitation, flavodoxin accumulation was used as
a biochemical marker and its detection in single cells provided some
insight into the physiological adaptation of natural phytoplankton on the molecular level (22). However, variability in levels
of flavodoxin expression has been found and strains of coastal origin, among them the only tested cyanobacterial representative, showed no
flavodoxin expression under iron-depleted conditions at all (8). In addition, the study of iron limitation in
cyanobacterial field populations using molecular markers is more
difficult than in eukaryotic algae (23).
Cyanobacteria have a significantly higher demand for iron than
eukaryotic algae (4, 44). Investigations of cyanobacterial iron accumulation in the field are rare. Cyanobacterial responses to
iron stress resemble those of heterotrophic bacteria (for a review, see
reference 44), but some filamentous species express flavodoxin constitutively after N deprivation in heterocysts
(35; U. Geiß et al., unpublished data). In coccoid
cyanobacteria, the flavodoxin-encoding gene isiB is
cotranscribed in a dicistronic operon with isiA. Under
iron-limiting conditions, transcription is highly induced but the
dicistronic message is 10-fold less abundant than the monocistronic
isiA transcript (25, 40), which is iron
regulated as well. IsiA (also known as CP43') is a chlorophyll
a-binding protein similar to CP43 (PsbC) of photosystem II
and the Pcb protein of prochlorophytes (21). Its exact
function in iron-starved cells is still a matter of discussion. It has been hypothesized to serve as a chlorophyll a storage system
or an extra light-absorbing system to protect the photosystems against excess light under iron-depleted conditions (5, 9, 11, 32). Four isiA genes, which reveal a high degree of
homology, are known so far (24-26).
In the present study, the reliability of isiA expression
with respect to iron depletion was investigated by means of a model cyanobacterium. The relation of growth rate to isiA
expression as well as the induction of the isiAB promoter
was examined. Additionally, a PCR method was developed for amplifying
specifically isiA genes from diverse cyanobacterial sources.
Application of this method extended the sequence information on
isiA genes from miscellaneous cyanobacterial samples and
provided a tool for a first isiA expression study using
enriched cultures originating from brackish phytoplankton. Besides the
integrated flavodoxin approach (7, 8), detection of
isiA expression may support investigations into the iron
supply available to cyanobacterium-dominated coastal phytoplankton.
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MATERIALS AND METHODS |
Organisms and culture conditions.
The strains listed in
Table 1 were derived from the Sammlung
von Algenkulturen (SAG), Universität Göttingen,
Göttingen, Germany, and the Pasteur Culture Collection. The
cyanobacterial isolates were kindly provided by the Pharmaceutical
Institute, University of Greifswald. Field samples were taken on 30 July 2000 at a water depth of 0.3 m from the Darss-Zingst estuary
(Bodstedter Bodden, pier of Zingst, Germany), which is characterized by
salinities of 5 to 10
. Fifty-milliliter phytoplankton samples
were concentrated by centrifugation and plated on 1% Kobe agar (Roth)
in medium C (17). After growth on petri dishes in
daylight, the algal consortia were transferred into the synthetic
growth medium BG11 (33), leading to the growth of mixtures
of cyanobacteria, green algae, and bacteria.
Cyanobacterial strains were grown at 29°C under constant illumination
(40 µmol of photons m
2 s
1) for
physiological investigations and mRNA expression studies
using BG11
medium (
33). All other strains were grown in medium
as
recommended by the culture collections. For all iron depletion
experiments, media were prepared without an iron source. Only
significant sources of iron contamination (water, NaNO
3
stock
solution) were purified in a Chelex 100 (Bio-Rad) column in order
to minimize secondary iron contaminations of less abundant ultrapure
salts owing to extensive handling of solutions and glassware.
Acid-rinsed glassware was used throughout the experiments. At
the onset
of iron limitation experiments, cells were washed three
times with
iron-deficient medium. During incubation, cells were
transferred every
second day into fresh medium. Strains were harvested
when cultures
showed a blue shift of chlorophyll
a absorbance
from
approximately 680 to 673 nm, indicating iron deficiency
(
11).
Owing to the presence of other algae, this indicator
could not
be applied to enriched field samples that were harvested
after
about 12 days of cultivation, when slight chlorosis could be
observed.
For investigations on growth rate and induction of the
isiAB
promotor,
Synechocystis strain MpIGisi (
18) was
used. This
mutant is a derivative of
Synechocystis sp.
strain PCC 6803T with
a chromosomally integrated fusion product of the
isiAB promotor
with the
gfp gene
(
18). MpIGisi cells were cultivated semicontinuously
(daily medium exchange and adjustment of cell density) in the
presence
of kanamycin (50 µg ml
1) at 29°C under constant
illumination (175 µmol of photons m
2 s
1)
and continuous aeration (2% [vol/vol] CO
2) using a
nitrate-containing
mineral medium (
2).
Cells of
Escherichia coli strain TG1 were used for routine
DNA manipulations after cultivation in Luria broth at 37°C
(
34).
DNA and RNA techniques.
All DNA techniques such as plasmid
isolation, transformation of E. coli, and ligation were
performed according to standard procedures (34).
Chromosomal DNA was extracted from cyanobacterial cells by phenol and
chloroform treatment (41). Nucleotide sequences of known
isiA genes from the strains Synechocystis sp.
strain PCC 6803, Synechococcus sp. strain PCC 7942, Synechococcus sp. strain PCC 7002, and Anabaena
sp. strain PCC 7120 were aligned using the software package ALIGN
(Align Plus, version 2.0, copyright 1989, 1992; Scientific & Educational Software) to design the degenerate primers isiAfw
(5'-AAD TAY GAH TGG TGG GC-3' [bp 28 to 44 of the Synechocystis sp. strain PCC 6803 isiA gene])
and isiArev (5'-CGT TTC GGC AAA RTA RGG-3' [bp 849 to 866 of the Synechocystis sp. strain PCC 6803 isiA
gene]). A predicted 840-bp-long fragment was obtained in the PCR
performed with Taq PCR Master Mix (Qiagen). For the combined
amplification of psbC, pcb, and isiA fragments, the reverse primer HLWHA-1 (5'-GCG TGC CAS AGR TGA CC-3'
[bp 948 to 964 of the Synechocystis sp. strain PCC
6803 isiA gene]) was used together with the isiAfw primer
mentioned above. The PCR program consisted of an initial 94°C
denaturation step for 5 min and 1 min each of denaturation
(94°C), annealing (52°C), and polymerization (72°C) for 33 cycles
before the final elongation step (10 min). PCR fragments were evaluated
by Southern hybridization by applying digoxigenin-labeled
isiA probes from Synechocystis,
Synechococcus strain PCC 7002, and a Fischerella
sp., which were obtained using a PCR digoxigenin probe synthesis
kit (Roche Biochemicals). All PCR products were separated on 0.8%
agarose gels, and the bands were excised and eluted using a Qiaex kit
(Qiagen). The obtained DNA fragments were cloned into plasmid pGEM-T
(Promega). Both strands of these fragments were sequenced using the
dideoxy chain termination method by applying a Thermo Sequenase
fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP
(Amersham Life Science). Universal primers, which were fluorescently
labeled with an infrared dye, IRD 800 (MWG Biotech),
were used for sequencing.
Prior to RNA isolation, cells were broken by freezing them in liquid
nitrogen and crushing them with a pestle. After an additional
preextraction step with phenol (Aqua-Roti-Phenol; Roth) for 10
min (pH
4.5 to 5; 65°C), total RNA was isolated using a High Pure
RNA
isolation kit (Roche Biochemicals). The separation of the
RNA and the
transfer onto nylon membranes were carried out as
described previously
(
40). DNA probes for Northern blot experiments
were
synthesized by PCR using the above-mentioned degenerate
isiA primers. A 16S rRNA-specific probe was generated using the universal
primers for the eubacterial 16S rRNAs 27f and 1525r (
20).
The
PCR products were purified on agarose gels, and specific bands
were
excised and labeled with [
-
32P]dATP (Amersham) using a
random primer labeling kit (MBI-Fermentas).
Quantification of
the signals obtained in the Northern blot experiments
was done with a
phosphorimager (BAS-1000; Fuji). To avoid differences
caused by
improper gel loading, the quantitative data were calculated
on the
basis of signals obtained after rehybridization of the
same filters
with the 16S rRNA-specific probe, with slight variations
due to
potential growth rate-dependent variations of rRNA content
being
accepted. Reverse transcription (RT)-PCR was done by applying
SUPERSCRIPT II RNase H reverse transcriptase (Gibco BRL, Life
Technologies) with the degenerate isiArev primer before regular
PCR.
Computer analysis.
We searched for sequence similarities in
databases with the assistance of the BLAST software program
(3). Sequence alignments were performed with the software
Align Plus (version 2.0; Scientific & Educational Software). The
phylogram was constructed by means of a multiple sequence alignment of
protein sequences using the software program PAUP (phylogenetic
analysis using parsimony, beta version 4.0; David Swofford, Laboratory
of Molecular Systematics, Smithsonian Institution).
Nucleotide sequence accession numbers.
The nucleotide
sequences of the isiA/psbC gene fragments of
Synechococcus sp. strain PCC 7942 (EBI accession no.
P15347), Synechococcus sp. strain PCC 7002 (EBI accession
no. P31157), Synechocystis sp. strain PCC 6803 (EBI
accession no. P73884), and Anabaena sp. strain PCC 7120 (isiA, EBI accession no. S42648; psbC, EBI
accession no. S42647) were extracted from databases. Complete and
partial sequences of cyanobacterial isiA genes from Fischerella muscicola PCC 73103 (EBI accession no. AJ296146; for a previous study, see reference 10),
Stanieria sp. strain SAG 27.84 (EBI accession no. AJ311682),
Oscillatoriales sp. strain 99-2/6.2.2 (EBI accession no.
AJ311683), Oscillatoriales sp. strain 99-3/5.3.1 (EBI
accession no. AJ311684), Lyngbya lagerheimii SAG 24.99 (EBI
accession no. AJ311685). Anabaena torulosa (EBI accession
no. AJ311686), Anabaenopsis elenkinii SAG 252.80 (EBI
accession no. AJ311687), BB 1 clone 1 (EBI accession no. AJ311688), BB
1 clone 2 (EBI accession no. AJ311698), BB 1 clone 5 (EBI accession no.
AJ311690), BB 2 clone 4 (EBI accession no. AJ311691), BB 3 clone 4 (EBI
accession no. AJ311692), BB 7 clone 3 (EBI accession no. AJ311693), R3
(EBI accession no. AJ311694), and R10 (EBI accession no. AJ311695) were obtained during this study and were submitted to the database.
| |
RESULTS |
Iron-dependent alterations of growth and isiA
expression in Synechocystis sp. strain PCC 6803.
To
document isiA induction, we focused on an appropriate model
strain to investigate the iron dependency of the growth rate and its
relation to isiA expression. During an experiment using aerated Synechocystis cultures, the growth rate and
isiA mRNA synthesis were monitored. Slight effects on the
growth rate were detected 3 h after the onset of iron-depleted
conditions. After 3 days of iron depletion, a >10-fold reduction in
the growth rate was observed (Fig. 1A).
Additionally, RNA samples were taken each day to measure the content of
isiA mRNA in Northern blot experiments with an
isiA-specific probe (Fig. 1B to D). When the iron
requirements of the cells were met, no transcription of isiA
was detectable. However, as soon as 3 h after transfer into
iron-depleted medium, isiA transcription was completely
induced and the transcript level remained almost constant as long as
these conditions were maintained. When cells were incubated under
iron-replete conditions again, after as little as 3 h of cultivation,
most of the isiA mRNA had disappeared. The monocistronic
isiA-specific transcript predominated, whereas the
dicistronic isiAB-specific mRNA gave only faint signals in
iron-starved cells (not shown).
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FIG. 1.
Growth rate and isiA expression of
Synechocystis strain MpIGisi and dependence on the starting
and stopping of iron-depleted growth in batch cultures with
CO2-enriched aeration. +Fe, 19.2 µM Fe(NH4)
citrate;  Fe, 0 µM Fe(NH4) citrate. (A) Specific growth
rate; (B) Northern blot hybridization signals after application of an
isiA-specific probe; (C) Northern blot hybridization signals
after application of a 16S rRNA-specific probe; (D) quantitative
estimation of isiA mRNA normalized to the 16S rRNA content
(highest content corresponds to 100%). d, days.
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Induction of the isiAB promoter as a function of the
Fe(NH4) citrate concentration.
A reporter strain of
Synechocystis named MpIGisi (18), carrying a
fusion of the isiAB promoter with a promoterless
gfp reporter gene, was used to investigate the iron
concentration-dependent expression of isiA. The iron
requirements of this strain were monitored in BG11 medium by observing
the appearance of green fluorescent protein (GFP) fluorescence. In our
first attempt, fluorescence-induced cells pregrown under iron-depleted
conditions were transferred into medium with Fe(NH4)
citrate concentrations ranging from 0 to 19.2 µM. The results shown
in Fig. 2 reveal an increase in GFP
fluorescence of cells grown with less than 0.77 µM, whereas faint to
nondetectable fluorescence was observed at 0.96 to 19.2 µM
Fe(NH4) citrate. The highest level of GFP fluorescence was
obtained from cells in iron-free medium. These results were corroborated by a second experimental series in which cells with repressed GFP fluorescence after cultivation in iron-replete medium were transferred into media with increasing levels of iron depletion. As found before, iron concentrations above 0.77 to 0.96 µM failed to
induce GFP fluorescence in the cells, while increases in fluorescence were induced by further decreases in iron concentrations (data not
shown). The kinetics of fluorescence change as well as the level of
fluorescence in the different samples reveal a strict dependence of
isiAB promoter activity on the extracellular concentration of Fe(NH4) citrate.
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FIG. 2.
GFP fluorescence in cells of Synechocystis
strain MpIGisi after 9 days of cultivation in media containing
different concentrations of Fe(NH4) citrate. The
fluorescence level of precultivated cells [0.7 µM
Fe(NH4) citrate] at the beginning of the experiment
corresponds to 100%. Error bars denote standard deviations of
triplicate samples.
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Identification of the isiA gene in various
cyanobacterial strains.
In order to use iron-dependent expression
of isiA as a molecular marker to monitor iron supply in
different cyanobacterial species, further investigations into whether
the isiA gene is widespread among cyanobacteria are
necessary. The search for this gene was carried out via a PCR approach
with degenerate primers. For the generation of degenerate PCR primers,
the amino acid sequences deduced from the four known isiA
genes were compared to those of the structurally and functionally
similar proteins PsbC and Pcb. Several regions of high similarity were
detected. A sequence region at the N terminus contains the peptide
sequence WWAGNAR, which is completely conserved in all known
IsiA sequences and occurs similarily in PsbC. It was chosen to generate
the primer isiAfw. After optimization experiments, a degenerate primer
that includes the WWA motif and a few bases upstream was derived. In order to create a degenerate primer for isiA-specific
amplifications, the second primer (isiArev) was deduced from the
peptide sequence (PYFADT) inside the shortened lumenal loop E' of IsiA,
which represents the major difference between IsiA and PsbC
(26). The nucleotide sequences encoding these peptides in
the different cyanobacterial strains were aligned and used for the
primer design. These isiA-specific degenerate primers were
tested with DNAs from various cyanobacterial strains of all the basic
taxonomic sections (Table 1).
From many of the investigated strains, we amplified an expected 840-bp
fragment (Fig.
3) which showed strong
hybridization
signals with
isiA-specific probes in Southern
blot experiments
(data not shown). Cloning and sequencing revealed that
the 840-bp-sized
fragments which appeared indeed represented
isiA genes. The high
specificities of the degenerate
isiA primers were further supported
by control experiments
using the
Prochlorococcus marinus strain
MED4. Its
pcb gene, which possesses very high sequence similarity
to
isiA, was not amplified, whereas it could be amplified in a
PCR using a further degenerate primer, HLWHA-1, which is homologous
to
a highly conserved region at the N termini of the genes
isiA, psbC, and
pcb (data not shown).
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FIG. 3.
Separation of PCR fragments obtained with DNAs from
various organisms (Table 1) using degenerate isiA-specific
primers. In all cases, the 840-bp fragment could be verified as an
isiA gene fragment by sequencing. Lanes: 1, marker ( DNA
EcoRI/HindIII digested); 2, Synechococcus sp. strain SAG 14.02-1; 3, Synechocystis sp. strain PCC 6803; 4, Synechococcus sp. strain PCC 7942; 5, Synechococcus sp. strain PCC 7002; 6, Anabaena
sp. strain PCC 7120; 7, Fischerella muscicola PCC 73103; 8, Anabaena torulosa SAG 26.79; 9, Lyngbya
lagerheimii SAG 24.99; 10, Microcystis aeruginosa BM
Mi/5; 11, Oscillatoriales sp. strain 99-2/6.2.2 (isolate
from Baltic Sea shoreline, Zingst); 12 to 15, BB 1, BB 2, BB 3, and BB
7 (enrichment cultures of estuarine field samples); 16, Oscillatoriales sp. strain 99-3/5.3.1 (isolate from Lake
Kummerow); 17, Chlorogloeopsis fritschii SAG 1411-1; 18, Anabaenopsis elenkinii SAG 252.80.
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Fragments of
isiA genes from representatives of all five
taxonomic sections of cyanobacteria (
33) could be
amplified, indicating
a widespread distribution of this gene. In
further experiments,
these degenerate primers were applied to DNAs from
new isolates
and mixed phytoplankton cultures of different waters. In
isolates
from brackish water (shoreline, Zingst, Baltic Sea; salinity,
10
) and a freshwater lake (Lake Kummerow) as well as in mixed
samples from the brackish Bodstedter Bodden (Fig.
3, lanes 12
to 15),
isiA fragments could be amplified. After being cloned
and
sequenced, three different
isiA sequences appeared from the
mixed phytoplankton cultures (Bodstedter Bodden). The newly obtained
sequences of
isiA fragments from additional strains and the
field
samples fit very well into a joint phylogram with already known
isiA sequences (Fig.
4). The
clustering of
isiA sequences from
field samples basically
reflects expectations with respect to
their taxonomic relationships.
The partial
isiA sequences obtained
from a
Nodularia bloom fit into the cluster of filamentous
cyanobacteria,
whereas the
isiA sequences obtained from
enriched phytoplankton
from a eutrophic estuary in which unicellular
and colony-forming
cyanobacteria dominate (
38) fit in the
coccoid strains (Fig.
4).
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FIG. 4.
Rooted cladogram (TreeView version 1.5; R. D. M. Page, 1998) after phylogenetic analysis of translated IsiA protein
sequences (PAUP software package, neighbor-joining method). The
isiA sequences (830-bp internal fragment) come from
databases (marked by  ) and from
the present study of different cyanobacterial strains, clones from
field samples, and enrichment cultures. Bootstrap values were
calculated after 1,000 replications; only branchings with values above
50% are shown.
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Expression of isiA mRNA in enrichment cultures of field
samples.
Although the presence of isiA in field samples
is an important precondition for its use as a marker gene, its proper
function cannot be assumed in advance. The expression of
isiA in enriched cultures originating from brackish
phytoplankton was tested first in experiments by means of RT-PCR. The
four cultures BB 1, BB 2, BB 3, and BB 7 containing three different
isiA genes (Fig. 4) were cultivated in iron-depleted medium.
In order to ensure that isiA-bearing organisms were still
present after the iron starvation period in spite of potential
competition with other algae and/or bacteria, DNAs were extracted
separately at the sampling times and used in PCRs with
isiA-specific primers. At the beginning of and after 12 days
of iron-deficient cultivation, RNA was extracted from harvested cells.
RT-PCR led to the 840-bp internal isiA fragments from the
iron-starved enrichment cultures BB 2 and BB 7 (Fig. 5). A control PCR excluded DNA
contamination and confirmed isiA expression. For comparison,
we analyzed a sample from an iron-starved laboratory culture of the
cyanobacterial model strain Synechocystis sp. strain PCC
6803 that indicated synthesis of a corresponding target mRNA (Fig. 5).
These RT-PCR experiments resulted in the first proof of iron-dependent
isiA expression in noncharacterized phytoplankton species
from eutrophic coastal waters. This indicates similar responses to
reduced iron availability in at least parts of the coastal population
of cyanoplankton and in frequently investigated cyanobacterial
model strains.
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FIG. 5.
Separation of internal isiA gene fragments
obtained by RT-PCR using RNAs isolated from different cyanobacterial
samples cultivated for 12 days under restricted iron availability
(Fe) in contrast to conditions for control cells (+Fe). Lanes BB,
enrichment cultures of estuarine field samples (numbers signify the
different cultures); lanes 6803, Synechocystis sp. strain
PCC 6803; lanes 1, marker ( DNA EcoRI and
HindIII digested).
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DISCUSSION |
Reliability of isiA expression as a molecular marker of
cellular iron supply.
The coincidence of isiA
expression and iron-dependent growth limitation in semicontinuous
cultures of the model strain Synechocystis sp. strain PCC
6803 suggests a close relationship of the two processes. However, the
almost complete activation of transcription as well as the high level
of isiA transcription after the onset of iron depletion,
when the growth rate was reduced by only a few percentage points,
characterizes the system as a very sensitive one with regard to reduced
iron availability in the model strain Synechocystis. In
addition, the fast disappearance of isiA mRNA under
iron-replete growth conditions is a clear indication of a high rate of
turnover of isiA mRNA and cannot be explained by mRNA
dilution due to cell division. Under optimum growth conditions for
Synechocystis, expression of isiA is a fast and
sensitive means to detect even early stages of iron deficiency. This
efficacy is similar to that of the molecular marker flavodoxin in
diatoms (31), which is applied to oceanic phytoplankton.
In the cyanobacterial model strain, the changing
isiA
expression in response to fluctuating extracellular concentrations of
Fe(NH
4) citrate further indicates a well-balanced system of
iron
supply. With regard to its intracellular iron demand,
Synechocystis possesses a sensitive regulating system. Iron
concentrations above
and below the critical limit for the
Synechocystis strain MpIGisi
caused gradually changing
steady-state levels of GFP fluorescence.
The regulation of
intracellular iron metabolism seems to be adapted
(without "on-off"
limits) to the extracellular iron supply continuously.
Since a specific
ferric-citrate receptor is known in
Synechocystis (
19), the potential variability of iron uptake had been
reduced
in our experiments in advance. Thus, the estimated threshold
for
concentration-dependent
isiA expression depends on
ferric citrate
as the iron source. However, our demonstration of the
basic regulatory
scheme opens perspectives to the probing of the
sophisticated
relations of iron availability and iron sequestration in
the future,
when the extracellular iron speciation is more under the
control
of the model organism or competing organisms themselves. The
independence
of
isiA transcript decline and organism growth
rate after the
cessation of iron depletion is an important
feature in distinguishing
critical nutrients in the environment, since
the cessation of
iron limitation does not necessarily result in growth
promotion
when a second nutrient has an immediate limiting effect
(colimitation).
Distribution of isiA in extant cyanobacteria.
Provisional objections to isiA being able to serve as a
molecular marker of iron limitation (23) with regard to
the distribution of the gene among cyanobacteria can now be rejected.
The screening of different cyanobacterial strains for isiA
served mainly to give us the initial impression that we could amplify
gene fragments with one set of degenerate PCR primers. Using optimized
isiA-specific degenerate primers, internal isiA
fragments of cyanobacterial strains could be obtained without
amplification of any other gene coding for a chlorophyll-binding
protein like PsbC or Pcb. The isiA-bearing strains belong to
all five cyanobacterial sections (33), which indicates a
widespread distribution of the gene. The available sequences cover at
least one taxon in each of the five sections of cyanobacterial taxonomy
as well as heterocystous strains, indicating that isiA is
common in extant cyanobacteria.
Though the strains can also be separated into three different groups of
16S rRNA clustering (
27), most of the strains cannot
be
designated correspondingly, since 16S rRNA sequences are not
available
and cannot be easily obtained from nonaxenic strains.
Although the
obtained fragments show a sequence homology of 65
to 73% and reveal
different clusters in the phylogram (Fig.
4),
they could be amplified
easily from miscellaneous and distantly
related cyanobacterial species
by a simple PCR protocol. However,
in some cyanobacteria no
isiA fragments could be amplified. These
strains do not
necessarily lack the
isiA gene, as no efforts have
so far
been made to vary the DNA extraction method. Although the
successful
DNA extraction method was tested with 16S rRNA-specific
primers, in
cases of nonaxenic cultures the results can reflect
the presence of
contaminating DNAs of heterotrophic bacteria.
Furthermore, the
degenerate primers might have restrictions for
a simultaneous
amplification of
isiA genes from the different
strains. In
two cases,
isiA fragments were found only after we
applied
the universal primers for the genes
psbC, pcb, and
isiA to DNAs (
Stanieria sp., Baltic
Nodularia bloom), but the reproducible
amplification of
isiA fragments from field samples verifies that
the utility
of our degenerate primers is not restricted to the
few culture strains
which served as sources for the primers. The
degenerate primers might
already be very useful for monitoring
isiA expression in
cyanobacterial blooms dominated by one or a
few species by means of
semiquantitative RT-PCR. Clear proof of
the existence of a
cyanobacterial strain lacking an
isiA gene
has not yet been
found, but the ongoing complete-genome sequencing
projects of several
cyanobacterial strains will provide definitive
information in the near
future.
The successful demonstration of
isiA mRNA accumulation under
iron-restricted conditions in enriched cultures from the estuary
indicates a relevant supplemental tool for gaining greater knowledge
of
estuarine ecosystems with respect to the interaction of iron
level and
cyanobacterial development. Though the total iron supply
in ecosystems
can be very high, this possibility does not prevent
cyanobacteria from
showing significant sensitivity to the micronutrient
(
15), and corresponding investigations would be supported
by
noninvasive
methodologies.
Any conclusions regarding
isiA expression in the field would
require balanced interpretations. Scanlan and Wilson (
37)
have
given an overview of the categorizeation of indicators of nutrient
deficiency in phytoplankton as sensitive or nonsensitive. In addition,
the coherence of signal intensity and the extent of iron limitation
have yet to be characterized. According to our study,
isiA
can
be placed into the category of indicators which are sensitive
to
the onset of limitation. Thus, signals can potentially be obtained
before field measurements of growth and primary production indicate
statistically significant cyanoplankton responses to iron deficiency.
However, such situations can also be expected when flavodoxin
is used
as a corresponding marker in eukaryotic algae (
31).
Our
study is one contribution to overcoming the provisional reservations
concerning
isiA's suitability as a molecular indicator of
iron
stress, owing to methodological problems (
23), since
specific
isiA signals can now be obtained in spite of their
high similarity
to those of other genes coding for chlorophyll-binding
proteins.
As the flavodoxin approach (
7) has been further
characterized
to perfect its practice (
8,
31), further
investigations with
respect to
isiA expression in
cyanobacteria will likewise be directed
to the detection of signals in
experimental phytoplankton
systems.
| |
ACKNOWLEDGMENTS |
This study was supported by a grant from the Deutsche
Forschungsgemeinschaft (DFG).
The excellent technical support of B. Brzezinka and K. Sommerey is
acknowledged. We are grateful for the provision of strains and isolates
by B. Kolpers, S. Mundt, (Pharmaceutical Institute, University of
Griefswald) and K. Ribbeck (FB Biowissenschaften, University of
Rostock). Cells of Prochlorococcus strain MED4 were kindly
provided by W. Hess (Department of Genetics, Humboldt University, Berlin, Germany), and Microcystis aeruginosa was kindly
provided by B. Meyer (Max-Planck-Institute for Limnology, Plön,
Germany). We thank M. Blank (FB Biowissenschaften, University of
Rostock) for introducing the PAUP software package. Samples of the
Nodularia bloom were kindly provided by H. Schubert
(Plant Ecology, University of Greifswald).
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Ernst-Moritz-Arndt-Universität Greifswald, Institut für
Ökologie, Botanisches Institut, Grimmer Str. 88, D-17487
Greifswald, Germany. Phone: 49 3834 864147. Fax: 49 3834 864114. E-mail: schoor{at}uni-greifswald.de.
| |
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0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5247-5253.2001
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Abstract
Introduction
