1 1 2
Dinitrogen fixation in a unicellular chlorophyll d-containing
3
cyanobacterium
4 5
Ulrike Pfreundt1, Lucas J. Stal2,3, Björn Voß1, Wolfgang R. Hess1
6 7
1
8
Germany
9
2
University of Freiburg, Faculty of Biology, Schänzlestr. 1, D-79104 Freiburg,
Department of Marine Microbiology, Centre for Estuarine and Marine Ecology
10
(NIOO-KNAW), Yerseke, The Netherlands
11
3
12
Netherlands
Department of Aquatic Microbiology, IBED, University of Amsterdam, The
13 14 15
Running title: Nitrogenase in Acaryochloris spp. HICR111A
16 17
*Corresponding author: Wolfgang R. Hess; Phone: +49-761-203-2796
18
Fax: +49-761-203-2745; Email:
[email protected]
19 20
ISMEJ-11-00621OAR In press, NOV 27, 2011
2 1
ABSTRACT
2
Marine cyanobacteria of the genus Acaryochloris are the only known organisms that
3
use chlorophyll d as a photosynthetic pigment. However, based on chemical
4
sediment analyses, chlorophyll d has been recognized to be widespread in oceanic
5
and lacustrine environments. Therefore it is highly relevant to understand the genetic
6
basis for different physiologies and possible niche adaptation in this genus. Here we
7
show that unlike all other known isolates of Acaryochloris the strain HICR111A,
8
isolated from waters around Heron Island, Great Barrier Reef, possesses a unique
9
genomic region containing all the genes for the structural and enzymatically active
10
proteins of nitrogen fixation and cofactor biosynthesis. Their phylogenetic analysis
11
suggests a close relation to nitrogen fixation genes from certain other marine
12
cyanobacteria. We show that nitrogen fixation in Acaryochloris sp. HICR111A is
13
regulated in a light-dark dependent fashion. We conclude that nitrogen fixation, one
14
of the most complex physiological traits known in bacteria, might be transferred
15
among oceanic microbes by horizontal gene transfer more often than anticipated so
16
far. Our data show that the two powerful processes of oxygenic photosynthesis and
17
nitrogen fixation co-occur in one and the same cell also in this branch of marine
18
microbes and characterize Acaryochloris as a physiologically versatile inhabitant of
19
an ecological niche which is primarily driven by the absorption of far-red light.
20 21
Keywords: Acaryochloris / chlorophyll d / coral reef / cyanobacteria / dinitrogen
22
fixation / microbial diversity / nitrogenase
23
3 1
INTRODUCTION
2
The fixation of dinitrogen is a major and crucial biogeochemical process in the ocean
3
where it replaces the losses of bioavailable combined nitrogen due to denitrification
4
and anaerobic ammonium oxidation (ANAMMOX) and allows new primary production
5
(Zehr & Kudela 2011). The capacity of fixing dinitrogen is widely distributed among
6
Bacteria and a few Archaea, but not among Eukarya and it gives the organisms that
7
possess this capacity access to the largest pool of nitrogen, atmospheric N2.
8
Nitrogenase, the enzyme complex that is responsible for the fixation of dinitrogen is
9
extremely oxygen sensitive and therefore most of the N2-fixing microorganisms are
10
obligate or facultative anaerobes. A notable exception is the oxygenic phototrophic
11
cyanobacteria, many of which possess the capacity of fixing dinitrogen (Zehr 2011).
12
Therefore, it is generally assumed that cyanobacteria are the main group of
13
organisms that are responsible for the fixation of N2 in the ocean, although this
14
viewpoint has been challenged (Riemann et al. 2010). Obviously, cyanobacteria
15
need adaptations and follow certain strategies to provide nitrogenase with an
16
anaerobic environment (Bergman et al. 1997; Berman-Frank et al. 2003).
17
Heterocystous cyanobacteria confine nitrogenase to special differentiated anoxic
18
cells that lack photosystem II, thereby separating it spatially from oxygenic
19
photosynthesis. Many marine unicellular diazotrophic cyanobacteria, such as
20
Crocosphaera or Cyanothece follow another strategy and separate the two
21
incompatible processes temporally, confining the fixation of dinitrogen to the dark
22
period. A notable exception is the filamentous non-heterocystous cyanobacterium
23
Trichodesmium, which is abundant in the tropical oceans and one of the most
24
important diazotrophs (Karl et al. 2002). This organism may exhibit a combination of
25
spatial and temporal separation in which cells temporally switch off oxygenic
26
photosynthesis and fix N2 (Berman-Frank et al. 2001). Another exception is the
4 1
hitherto uncultivated putative unicellular diazotrophic cyanobacteria UCYN-A. The
2
analysis of the metagenome of flow cytometrically sorted cells has shown that the
3
genes for the oxygenic photosystem II and for CO2 fixation are absent (Tripp et al.
4
2010). The nitrogen fixation (nif) genes of this organism are expressed during the
5
daytime and N2 fixation also occurs during the day but further nothing is known about
6
the identity of this enigmatic cyanobacterium.
7 8
Acaryochloris is a unicellular marine cyanobacterium that is atypical in using
9
chlorophyll d for light harvesting (Miyashita et al. 1996). Based on chemical analyses,
10
chlorophyll d has only recently been recognized to be widespread in oceanic and
11
lacustrine environments (Kashiyama et al. 2008). Therefore it is highly relevant to
12
understand the genetic basis for different physiologies and possible niche adaptation
13
in this genus. At present, genome sequences are available of two Acaryochloris
14
strains. Acaryochloris marina MBIC 11017 (from here A. marina) was isolated from
15
waters around the Palau islands in the Pacific Ocean (Miyashita et al. 1996),
16
Acaryochloris sp. CCMEE 5410 from the Salton Sea lake in California (Miller et al.
17
2005). Compared to other cyanobacteria, large and complex genomes were found for
18
both isolates. A. marina harbors a total of 8.3 million base pairs with more than 25%
19
of its 8462 genes being located on nine different plasmids, eight of which are larger
20
than 100 kb (Swingley et al. 2008). A similarly complex genome was also reported for
21
the Salton Sea strain (Miller et al. 2011). Despite this impressive coding capacity, no
22
genes for dinitrogen fixation were found in either of these two well-studied isolates.
23 24
We recently described Acaryochloris sp. HICR111A, a new member of the
25
Acaryochloris clade (Mohr et al. 2010). The strain was isolated from microbial mat-
26
like material on a dead coral skeleton in shallow water on the southern reef flat of
5 1
Heron Island, Great Barrier Reef (23° 26' 31.2'' S, 151° 54' 50.4'' E). The cells are rod
2
shaped, between 1 and 2 μm long with a diameter of 0.75–1 μm, which is somewhat
3
smaller than the 2–3 μm in length and the diameter of 1.5–2.0 μm reported for A.
4
marina (Mohr et al. 2010). The presence of Chl d was monitored spectro-
5
photometrically (Qy maxima of Chl d detected at 707 nm and a single fluorescence
6
maximum at 750 nm after Chl d excitation at 470 nm) and by high performance liquid
7
chromatography repeatedly from the early cultivation phase onwards. Purified
8
cultures use Chl d as main photopigment, as indicated by a 1:50 ratio of Chl a / Chl d,
9
which changed only little with increasing light intensity (from 5–10 µmol to 75–100
10
µmol photons m-2 s-1) (Mohr et al. 2010). Acaryochloris sp. HICR111A is, according
11
to 16S rRNA-based phylogeny, an early branching member of the genus
12
Acaryochloris (Mohr et al. 2010). Its 16S ribosomal RNA is 2% divergent from the two
13
previously described A. marina strains, which, together with several distinct genetic,
14
morphological and physiological differences, led us to conclude that cyanobacteria of
15
the genus Acaryochloris occur as distinct ecotypes. A preliminary sequence analysis
16
of its genome based on Illumina technology yielded a genome with an accumulated
17
length of 8,371,965 nt (Mohr et al. 2010). Here we report the existence of a complete
18
nitrogenase gene cluster in Acaryochloris sp. HICR111A, we demonstrate that the nif
19
genes are strongly induced upon removal of combined nitrogen, and we detected
20
nitrogenase activity.
21 22
MATERIALS AND METHODS
23
Culture conditions
24
Cultures of Acaryochloris sp. HICR111A (Mohr et al. 2010) were grown in ASN
25
medium (artificial seawater + BG-11 nutrients) (Rippka et al. 1979). For preliminary
26
analysis of dinitrogen fixation genes by qPCR, cultures were grown at room
6 1
temperature (summer: ~25 °C) under naturally changing light conditions (from here:
2
“summer cultures”) and later that year moved to an incubator at 26 °C under a
3
12h|12h light|dark cycle with warm white light (Sanyo fluorescent tubes, 40 watt) of
4
35 µmol quanta m-2 s-1. For measurement of nitrogenase activity two cultures of
5
Acaryochloris HICR111A were grown and used as biological replicates (the assays
6
were done on subsequent days). Both cultures were initially grown under a constant
7
12h|12h light|dark cycle at 26°C and 35 µmol quanta m-2 s-1. Nine days prior to the
8
nitrogenase assay, half of one culture was shifted to nitrogen depleted medium
9
(ASN0) by centrifugation in a sterilized tube at 6,000× g for 20 min, washed once with
10
ASN0 (a third of the original volume), and resuspended in the original volume of
11
ASN0. The other half of this culture served as control and was also centrifuged, but
12
resuspended in an equal volume of fresh ASN medium. Subsequently, both were
13
incubated under a light|dark regime of 12h|12h at 50-60 µmol quanta m-2 s-1. The
14
replicate culture had been incubated at 50-60 µmol quanta m-2 s-1 for 22 days and
15
half of it was subsequently shifted to ASN0 7 days prior to the assay as described
16
above. One day prior to the nitrogenase assay, all cultures were shifted to 150 µmol
17
quanta m-2 s-1 and 29 °C. For the nitrogenase assay six 15-ml samples of the ASN0
18
culture and one 15-ml sample of ASN culture were filtered onto GF-F filters and
19
placed on a perforated support in contact with ASN0 (or ASN in case of the control) in
20
a custom made, temperature controlled incubator. Each of the seven chambers was
21
closed by a glass window, leaving 3 ml headspace. The cultures were incubated
22
under a constant flow of N2 + 0.03 % CO2. The light/dark cycle, light intensity and
23
temperature were kept the same.
24 25
Determination of nitrogenase activity
7 1
Nitrogenase activity was measured in custom-made incubators with separate
2
chambers to hold filters of 45 mm diameter (one incubator containing 6 chambers,
3
the other containing a single chamber which served as the control). The chambers
4
were identical to the one described in (Staal et al. 2001). The incubator was
5
connected to a circulating cryostat water bath in order to thermostat the incubation
6
chambers. The single chamber was temperature controlled by a computer-controlled
7
Peltier element. The chambers were connected in series to a gas line of N2 with
8
0.03% CO2 premixed (Westfalen Gases, Münster, Germany) at 3 L h-1, controlled by
9
a mass flow controller (5850S) and control unit (0152, both Brooks Instrument, Ede,
10
The Netherlands). The incubator was covered with a hood containing the light source
11
(two cool white fluorescent tubes) that was turned on and off by a timer.
12 13
Nitrogenase activity was measured using the acetylene reduction assay (ARA)
14
(Steward et al. 1967). Nitrogenase activity was measured at six time points: (1) -2 – 0
15
h, (2) 0 – 2 h, (3) 3 – 5 h, (4) 6 – 8 h, (5) 10 – 12 h, and (6) 12 – 14 h (where the
16
beginning of the dark phase is taken as 0 h). The ASN culture (nitrate-supplemented)
17
was taken as the control and measured along with the second and the fifth time
18
points in the two replicate experiments, respectively. The samples had been
19
incubated under anaerobic conditions for 7-8 h before the acetylene reduction assay
20
was started to prevent a false negative result due to oxygen inactivation.
21 22
For each time point one chamber was disconnected from the gas flow and 1 ml of
23
acetylene was injected into the chamber (~30 % of gas phase) while the gas was
24
displaced into a second syringe connected to the gas outlet of the incubator. The gas
25
in the headspace was mixed by ‘pumping’ using the two syringes. After 2 h of
26
incubation the gas was mixed again, two 0.5 ml gas samples (technical duplicates)
8 1
were withdrawn from the headspace, and the filter with the culture snap-frozen in
2
liquid nitrogen for later RNA extraction. Acetylene and ethylene in the gas samples
3
were determined gas chromatographically (Chrompack CP9001, Middelburg, The
4
Netherlands). The gas chromatograph was equipped with a flame ionization detector
5
(FID) and a wide-bore silica fused (0.53 mm internal diameter) Porapak U column
6
(Chrompack). The carrier gas was N2 at 10 ml min-1 and the flows of H2 and air for
7
the FID were 30 and 300 ml min-1, respectively. The temperatures for injector,
8
detector and oven were 90, 120, and 55 °C, respectively. The total amount of
9
ethylene produced by the culture was calculated using acetylene as an internal
10
standard (Stal 1988) and normalized to chlorophyll d (measured in a separate 15-ml-
11
sample of the original culture).
12 13
Determination of chlorophyll d
14
Fifteen ml of culture were transferred to a 50-ml Falcon tube and centrifuged for 15
15
min at 5,000 g. Five ml of 96 % ethanol was added to the cell pellet, vortexed, and
16
the chlorophyll was extracted overnight in the dark at room temperature. The extract
17
was centrifuged for 20 min at 6,000 g and the absorption was read
18
spectrophotometrically at 665 and 696 nm. The concentration of chlorophyll d was
19
calculated using the formula Chl d [µg/ml] = A665nm (-0.2006) + A696nm (12.0995)
20
(Ritchie 2006).
21 22
RNA extraction
23
Acaryochloris cells were collected by rapid filtration on hydrophilic polyethersulfone
24
filters (Supor 800 Filter, 0.8 µm, Pall, NewYork, USA) and transferred to 15-ml
25
Sarstedt tubes containing 2 ml of PGTX RNA extraction solution (Pinto et al. 2009).
26
Alternatively, the frozen GF-F filters with the samples from the acetylene reduction
9 1
assays were quickly thawed, the cells removed with forceps and immediately placed
2
into a 15-ml Sarstedt plastic tube containing 2 ml of PGTX extraction solution.
3
Samples in Sarstedt tubes were vortexed together with the PGTX, frozen in liquid N2
4
and stored at -80 °C until extraction. Frozen Acaryochloris cells in PGTX were
5
transferred from -80 °C immediately to a 95 °C water bath for 5 min with occasional
6
vortexing and subsequently put on ice for 5 min. For phase extraction, 700 µl of
7
chloroform/isoamylalcohol (IAA) (v/v 24:1) per 1 ml PGTX was added, the mixture
8
shaken vigorously and left at 24°C for 10 min with occasional shaking and vortexing.
9
The phases were separated by centrifugation for 15 min (20 min for GF-F filter
10
samples) at 3,750 rpm (Allegra 6 KR, Beckman Coulter, Krefeld, Germany). The
11
aqueous phase containing the RNA was transferred to a new reaction tube and 1
12
volume chloroform/IAA (v/v 24:1) was added, followed by a second centrifugation
13
step (for GF-F filter samples, the aqueous phase was first re-extracted with phenol
14
(pH 4.5-5)/chloroform/IAA (v/v/v 25:24:1)). The aqueous phase was transferred to a
15
2-ml reaction tube (1 ml per tube) and the RNA precipitated overnight with 1 volume
16
of isopropanol at -20 °C. Precipitated RNA was pelleted by centrifugation for 25 min
17
at 13,000 g at 4 °C, the pellet washed with 1 ml 70 % ethanol and centrifuged for 10
18
min at 13,000 g at 4 °C. The pellet was subsequently air-dried and dissolved in 100
19
µl (30 µl for GF-F filter samples) nuclease-free Milli-Q H2O. RNA concentration and
20
grade of contamination of a sample were measured with a spectrophotometer
21
(NanoDrop ND-1000 or ND-2000, Peqlab, Erlangen, Germany). Quality of RNA was
22
verified on 1.5% formaldehyde-agarose gels.
23 24
Northern blot analysis
25
Blots for the detection of nifD mRNA were prepared from the separation of 1 µg of
26
total RNA on 1.5 % denaturing formaldehyde-agarose gels. Separated RNA was
10 1
transferred to Amersham Hybond-N nylon membranes (GE Healthcare, Munich,
2
Germany) by capillary blotting with 20x SSC buffer (3 M NaCl, 0.3 M C6H5Na3O7, pH
3
7.0). The membranes were hybridized with [α-32P]UTP-labeled transcript probes
4
overnight at 62oC in 50 % deionized formamide, 7 % SDS, 250 mM NaCl, 120 mM
5
phosphate buffer (sodium salts), pH 7.2 in screw-cap hybridization tubes. The
6
membranes were washed in 2× SSC, 1 % SDS for 10 min; 1× SSC, 0.5 % SDS for
7
10 min; and in 0.1× SSC, 0.1 % SDS for 10 min at 57 °C. Signals were detected and
8
analyzed on a Personal Molecular Imager FX system with Quantity One software
9
(Bio-Rad, Munich, Germany).
10 11
Quantitative real-time reverse transcription PCR (RT-qPCR)
12
For elimination of contamination with genomic DNA, 2 µg or 8 µg (for first and second
13
qPCR experiment, respectively) of each RNA sample were incubated twice with 2
14
units of TURBO DNase (TURBO DNA-freeTM Kit, Ambion, Austin, TX, USA) for 20
15
min at 37 °C. The DNase was inactivated following the manufacturer’s instructions.
16
RNA was re-extracted by phenol (pH 4.5 - 5)/chloroform and precipitated with 3
17
volumes of NaOAc/ethanol. RNA samples were tested for residual contamination with
18
DNA by PCR (30 amplification cycles) using the nifD primers of the qPCR reactions.
19
All tests were negative. cDNA synthesis was done with 350 ng RNA of each sample
20
using the Quantitect kit (Qiagen, Hilden, Germany) with a mix of 9 primers (the
21
reverse primer for each gene to be tested), each at a final concentration of 0.5 µM,
22
and following manufacturer’s instructions. A 7500 Fast system (Applied Biosystems,
23
Foster City, CA, USA) was used for qPCR. The modified “Fast Mode” (10 min at 95
24
°C, 40 x (3 s at 95 °C, 30 s at 60 °C)) was used for the preliminary analysis of the
25
“summer culture” samples. For analysis of the samples from the acetylene reduction
26
assays, the longer “Standard Mode” (2 min at 50 °C, 10 min at 95 °C, 40 x (15 s at 95
11 1
°C, 60 s at 60 °C)) was used. Each 15-µl reaction contained 7.5 µl of Power SYBR
2
Green Mix (Applied Biosystems), 4.5 µl of diluted cDNA (1:1280 for nifB, nifD, fdxH,
3
psbA, psaA and coxA; 1:80 for pdhB and 1: 5,242,880 for 16S rRNA), 1.5 µl each of
4
forward and reverse primer. Primers were designed manually to contain similar GC
5
content and have an equal length of 24 nt. The “PrimersList” tool on
6
http://primerdigital.com/tools/ was used for all calculations; for melting temperature
7
calculation the option “using Allawi’s thermodynamics parameters” was chosen
8
(Kalendar et al. 2011). Prior to the final experiment, a primer-matrix was tested
9
(amplifying genomic DNA) to determine the most efficient primer concentration for
10
each primer pair and to confirm primer efficiencies to be similar. Primer sequences,
11
concentrations, and efficiencies are shown in Table 1. Primers were synthesized by
12
Sigma-Aldrich, Hamburg, Germany. All reactions were done in triplicate and for each
13
reaction a ‘no RT’ control was included. 16S rRNA was amplified as reference. Melt
14
curves for all amplifications showed only one product (no product for “no RT” control).
15
Data were analyzed with the 7500 Fast System SDS software (Applied Biosystems),
16
with manual Ct and automatic baseline setting (followed by manual control). Relative
17
transcript quantities were calculated with the ΔΔCt method (Livak & Schmittgen
18
2001). As calibrator conditions, samples of the nitrate-supplemented culture were
19
used, namely the one harvested at 12:00 and 22:00 for the 1st and 2nd experiment
20
(replicate), respectively.
21 22
Software
23
The here described contig was fully annotated and submitted to Genbank (accession
24
JN585763). Artemis Release 12 (Rutherford et al. 2000) was used for visualization
25
and annotation of genome information stored in Genbank or text files. For multiple
26
sequence alignments and BLAST searches in local databases, BioEdit version
12 1
7.0.5.3 (Hall 1999) was used. Phylogenetic analyses were done with MEGA version 5
2
(Tamura et al. 2011) and with Geneious Pro version 5.4.6 (Drummond et al. 2011)
3
using the Mr. Bayes PlugIn (Huelsenbeck & Ronquist 2001). The BLAST algorithm
4
(Altschul et al. 1990) was used with the BLOSUM62 matrix to find putative homologs
5
of genes (BLASTN) or proteins (BLASTP) in the NCBI database and to determine
6
conservation between homologous sequences.
7 8 9
Results and Discussion
10
A cassette for nitrogen fixation genes in Acaryochloris sp. HICR111A
11
During annotation of the Acaryochloris sp. HICR111A genome we encountered a
12
possible cassette for nitrogen fixation (nif) genes on a 63005 bp long contig
13
(GenBank accession JN585763). This contig harbours 60 protein-coding genes, from
14
which 22 physically clustered genes are related to dinitrogen fixation. With a few
15
exceptions like cysteine synthase A and serine-O-acetyltransferase, these have no
16
homologs in the genomes of the two other sequenced Acaryochloris (Swingley et al.
17
2008; Miller et al. 2011) but several flanking genes on both sides of the nif gene
18
cluster have homologs in A. marina (Figure 1), albeit at different positions in the
19
genome. It should be noted that the genome was assembled from DNA of a non-
20
axenic culture containing one strain of an α-proteobacterium and possibly another
21
bacterium. However, these were the only nif genes found in the whole metagenome.
22
Besides the nitrogen fixation cassette, the genome of Acaryochloris sp. HICR111A
23
contains genes for several other nitrogen assimilation pathways, including
24
nitrate/nitrite-, ammonia-, urea-, and cyanate assimilation. It is missing the
25
nitrate/nitrite transporters encoded by nrtABCD and focA, but contains the nrtP gene
13 1
which also codes for a nitrate/nitrite transporter. A similar situation can be found in
2
the reference strain A. marina.
3 4
Genome assemblies solely based on Illumina technology may be erroneous in
5
particular in case of the Acaryochloris genomes which are among the biggest
6
(Swingley et al. 2008; Miller et al. 2011) and most complex genomes in the phylum
7
cyanobacteria (Hess 2011). Therefore, the sequence assembly linking this
8
nitrogenase gene cluster to the neighbouring genes with homologs in A. marina was
9
verified by PCR yielding three amplicons of 365, 2802 and 2182 nt (Figure 2). Among
10
the 22 N2 fixation-related genes are the nifHDK genes encoding the three different
11
nitrogenase subunits, genes related to molybdenum cofactor biosynthesis or
12
transport (hesAB, nifBENX, modABC), Fe-S cluster assembly (nifSU), nif-specific
13
ferredoxins (fdxHN) and a flavodoxin. On the left side, this cluster is flanked by a glgP
14
gene for a putative glycogen/starch/alpha-glucan phosphorylase, closely related to A.
15
marina gene AM1_2114, on the right side by several genes of different phylogenetic
16
matches, including four restriction/modification (RM) genes, followed by 12 homologs
17
of A. marina genes AM1_4104 to AM1_4121 (Figure 1). Very surprisingly, the RM
18
genes are interspersed by a gene encoding a protein of the XisH superfamily. The
19
only known function of these proteins is the cell-type specific excision of a 55 kb
20
element interrupting the fdxN gene during heterocyst differentiation in nitrogen-fixing
21
filamentous cyanobacteria of the Nostocales group (Ramaswamy et al. 1997). The
22
function of this XisH homolog in a unicellular non-heterocystous cyanobacterium
23
appears enigmatic: the fdxN gene in Acaryochloris sp. HICR111A is not interrupted
24
by a genetic element, moreover, the sequence comparison of XisH yielded mainly
25
matches in the Nostocales, but also in some unicellular dinitrogen fixers such as
14 1
Cyanothece and Crocosphaera, as well as in some cyanobacteria lacking this ability,
2
such as Microcystis aeruginosa and, most interestingly, A. marina (gene AM1_4785).
3 4
When the 22 core nif genes were compared against sequence databases, we noticed
5
a remarkable co-linearity with a similar gene cluster in Synechococcus PCC 7335
6
including the same peculiar drop in GC content upstream of nifB (Figure 1). The
7
average blastP amino acid identity of all nif genes is 80%, showing the highest
8
similarity between the homologs of the structural genes nifHDK (90% average
9
identity) and the lowest similarity between the homologs of the nif related genes from
10
cysE to nifT (73% average identity, compare Figure 1). Of all proteins in the NCBI
11
database, Synechococcus PCC 7335 homologs had the highest similarity to those of
12
Acaryochloris sp. HICR111A in all cases (Table 2). Phylogenetic analyses of
13
Acaryochloris sp. HICR111A Nif proteins confirmed a very high sequence similarity,
14
locating the respective orthologs from both strains in the same clade. For these
15
analyses, we concatenated the NifHDK sequences from 19 different cyanobacteria
16
and used the homologs from Rhodospirillum rubrum as outgroup (Figure 3b).
17
Remarkable is not only the tight clustering of Synechococcus PCC 7335 and
18
Acaryochloris sp. HICR111A Nif proteins in a single clade but also the totally
19
unexpected closer relatedness to the respective orthologs in Trichodesmium
20
erythraeum (Figure 3b). Furthermore, a discrepancy to the general phylogeny of
21
these species is evident when comparing NifHDK phylogeny to the 16S rRNA-based
22
phylogeny (Figure 3a). This pattern of presence/absence of nif genes throughout
23
different clades can only be explained by events of selective gene gains or losses
24
and horizontal gene transfer must have played an important role in these processes.
25
Of special interest was also the fact that Acaryochloris sp. HICR111A expresses a
26
fused version of the genes nifE and nifN (Figure 1). Of all fully sequenced
15 1
diazotrophic cyanobacteria, only four strains have such a fused version of nifEN
2
(Figure 3c). Remarkably, besides Acaryochloris sp. HICR111A, these are
3
Synechococcus PCC 7335 (GenBank accession: EDX83984), Trichodesmium
4
erythraeum (NCBI reference sequence: YP_723620.1), and Anabaena variabilis
5
(nifEN2, GenBank accession: AAA93024) which has one fused and one separate
6
version in the nif2 and nif1 clusters, respectively. The expression of nifEN was
7
verified experimentally for Trichodesmium (Dominic et al. 2000) and is evident at the
8
DNA sequence level in the other three organisms. The phylogenetic analysis (Figure
9
3c) of the NifEN protein shows that the occurrence of fused nifEN coincides with
10
higher sequence similarity, clearly separating the Acaryochloris nif genes from those
11
of typical unicellular diazotrophic cyanobacteria. This strengthens the hypothesis that
12
the Acaryochloris nif cluster had originally been acquired by horizontal gene transfer.
13 14
Expression of Acaryochloris sp. HICR111A nif genes is controlled by the light/dark
15
cycle
16
Horizontally transferred genes are not necessarily actively expressed genes. They
17
might just have been acquired and now be in the process of decline. The
18
incompatibility between photosynthetic oxygen production and the well-known
19
oxygen sensitivity of the nitrogenase enzyme should also be considered. Therefore,
20
we set out to measure the expression of these genes in two different ways.
21 22
First, we measured expression of nifD in N-deplete (-N) versus N-replete (+N)
23
conditions in cultures growing under natural light/dark cycles (German summer,
24
varying light intensities and length of day/night). Using quantitative RT-PCR we
25
observed a several thousand fold induction of gene expression in the –N cultures
26
during the dark, starting already towards the end of the light phase (Figure 4a) and
16 1
detected the appearance of a strongly accumulating transcript of about 2.3 kb in
2
Northern blot hybridization in -N cultures only (Figure 4b). The large size of the nifD
3
transcript characterizes it as a likely polycistronic mRNA. Similar results were
4
obtained for other probed nif genes (not shown). The fact that nif genes were already
5
induced before the onset of the dark phase indicates that the circadian clock
6
probably played a role in the timing of the expression, the key components of which
7
are encoded by the kai genes present in this genome. Follow-up experiments
8
including hoaxing experiments with shifted light and dark phases should give further
9
insight.
10
To prove that the induction of gene expression detected at the mRNA level actually
11
results in enzymatically active nitrogenase, we measured nitrogenase activity during
12
six 2h-periods and in parallel analyzed the expression of nif genes in each sample.
13
This experiment was done twice with two different cultures. Figure 5a shows that
14
both cultures, despite different periods of adaptation to the final light and temperature
15
conditions (9 and 22 days, respectively) and differing time points of their shift to N-
16
deplete medium (9 and 7 days, respectively), were performing dinitrogen fixation in
17
the second half of the 12h dark period. In contrast, the expression of three different
18
genes, nifB, nifD and fdxH, measured at the RNA level, started to increase with the
19
begin of the dark period, whereas a gene for a typical photosynthetic protein, psbA,
20
chosen for control, showed an inverse behavior with high expression during the light
21
period and a minimum at night (Figure 5b,c). Thus, compared to the induction of nifB,
22
nifD, and fdxH expression at the RNA level, the peaks of dinitrogen fixation are offset
23
by about 6 hours. The maximum observed nitrogenase activity rate was 485 nmol
24
C2H4 / mg Chl d /2h-1. This relatively low N2 fixation rate corresponds to the slow
25
growth of Acaryochloris sp. HICR111A observed in culture. Our measurements were
17 1
done under micro-aerobic conditions. In an aerobic environment the small diameter
2
of the cells would make it impossible for Acaryochloris sp. HICR111A to maintain an
3
anaerobic intracellular environment for nitrogenase as the diffusion of oxygen would
4
be higher than the scavenging of O2 by respiration. However, Acaryochloris sp.
5
HICR111A forms aggregates in culture (Mohr et al. 2010). Such aggregates may
6
enhance anoxic conditions in the dark and hence support the formation of active
7
nitrogenase. The energy required for N2 fixation in Acaryochloris sp. HICR111A is
8
most likely obtained from the aerobic respiration of the intracellular glycogen reserve,
9
possibly involving the enzymatic activity of the putative glycogen phosphorylase
10
encoded by the glgP gene next to the nif gene cluster. However, we can also not
11
exclude the possibility of anaerobic fermentation, when aggregates of biofilms of this
12
organism turn anoxic at night as observed for other cases (Stal & Moezelaar 1997).
13 14
Conclusions
15
Acaryochloris sp. HICR111A differs from other chlorophyll d-containing Acaryochloris
16
strains in that it possesses a complete and fully functional cluster of nitrogenase
17
genes that seems to be acquired through horizontal gene transfer from other
18
unicellular
19
nitrogenase activity was detected in the second half of the dark phase. Nitrogenase
20
activity was comparatively low but in line with the slow growth rate of this organism.
21
We conclude that Acaryochloris sp. HICR111A may thrive diazotrophically in its
22
natural habitat by forming aggregates or biofilms at or close to the intertidal zone of
23
tropical reefs which are characterized by low oxygen levels during the night.
24
marine
cyanobacteria.
Nitrogenase
genes
were
expressed
and
18 1
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22 1
FIGURE LEGENDS
2 3
Figure 1
Comparison of the Acaryochloris HICR111A and Synechococcus PCC
4
7335 nif clusters. (a) GC content aligned with the scaled map of the Acaryochloris nif
5
cluster and surrounding genes. Numbers in the top right corner are %GC. Two
6
intergenic regions exhibiting a characteristic drop in GC content are boxed in red. (b)
7
Map and GC content of the Synechococcus PCC 7335 nif cluster. Orange block
8
arrows indicate genes associated with dinitrogen fixation, grey block arrows indicate
9
genes with known products, but not known to be associated with dinitrogen fixation,
10
white block arrows indicate genes encoding hypothetical proteins. Grey triangles
11
indicate stretches of sequence containing multiple genes not explicitly associated
12
with dinitrogen fixation.
13 14
Figure 2 Three linking PCRs were performed to validate the sequence assembly of
15
contig 62 of the Acaryochloris sp. HICR111A sequencing project containing the nif
16
gene cluster (for a detailed map of the cluster see Figure 1). The positions of forward
17
and reverse primers are indicated by red arrows. Genes with the closest orthologs
18
found in Acaryochloris marina are colored green; genes with the closest orthologs
19
found in the Synechococcus sp. PCC 7335 nif gene cluster in the same order are
20
colored orange; other genes in grey. Genes are clustered into boxes with only those
21
important for the PCRs shown as single arrows. Arrows and boxes are not drawn to
22
scale. The inset shows a 0.8% agarose gel with the three PCR products compared to
23
a λ (PstI) ladder.
24 25
Figure 3 Bayesian trees showing the phylogeny of 16S ribosomal RNA sequences
26
and of concatenated amino acid sequences of nitrogenase structural proteins NifHDK
23 1
and NifEN. (a) Phylogenetic analysis of 40 sequences of 16S ribosomal RNA from N2
2
fixing and other cyanobacteria. Rhodospirillum rubrum ATCC 11170 served as
3
outgroup;
4
(Prochlorococcus and Synechococcus) was collapsed for better overview. (b)
5
Phylogenetic analysis of 21 amino acid sequences of concatenated NifHDK proteins
6
from 19 different cyanobacteria and Rhodospirillum rubrum ATCC 11170 (outgroup).
7
For Anabaena variabilis, NifHDK protein sequences from nif clusters 1 (expressed in
8
the heterocysts) and 2 (expressed in the vegetative cells under anaerobic conditions)
9
were included. Variable C- and N-termini were cut out of the alignment (with a
10
maximum of 55 and 33 amino acids deleted at the NifH-NifD and NifD-NifK junctions,
11
respectively). (c) Phylogenetic analysis of the fused NifEN protein in N2 fixing
12
cyanobacteria and Rhodospirillum rubrum (outgroup). For all strains without the fused
13
version of these genes, amino acid sequences were concatenated for this analysis.
14
Strains which express the fused nifEN are indicated by a grey arrow. In all trees,
15
Acaryochloris strains are highlighted in orange, members of the Nostocales are
16
highlighted in grey. Synechococcus sp. 7335 is framed in orange for comparison with
17
Acaryochloris. All strains from (b) and (c) are included in (a).
one
subtree
showing
11
species
of
marine
picocyanobacteria
18 19
Figure 4 Transcript levels of nifD by quantitative real-time reverse transcription PCR
20
(RT-qPCR) and Northern blot analysis in two different experiments. a) Relative
21
transcript levels of nifD obtained by qPCR in Acaryochloris HICR111A grown with
22
(+N) and without (-N) combined nitrogen (nitrate) in summer under natural light
23
conditions. The shaded grey area shows the measured light intensity during the
24
sampling period. The data were normalized to 16S rRNA and calibrated to the 12:00
25
nitrate sample. (b) Northern blot analysis of nifD transcripts during the dark phase in
26
Acaryochloris HICR111A grown under controlled conditions with a defined 12h/12h
24 1
light/dark cycle. –N: grown without combined nitrogen; NO: grown with nitrate (NO3-).
2
5S rRNA was hybridized as internal standard.
3 4
Figure 5 Nitrogenase activity and real-time reverse transcription PCR (RT-qPCR)
5
analysis of relative transcript levels from genes involved in dinitrogen fixation and
6
photosynthesis. Shown are two different experiments with Acaryochloris HICR111A
7
cultures grown under a 12h/12h light-dark cycle. Nitrogenase activity (acetylene
8
reduction) and transcript levels were measured at six points in time during the
9
12h/12h light-dark cycle. (a) Nitrogenase activity. The culture used for measurements
10
on 02.06.2011 had adapted to the final temperature and light conditions for 9 days
11
and was shifted to nitrogen-deplete medium 9 days prior to the assay, the culture
12
used for measurements on 03.06.2011 had adapted to the final temperature and light
13
conditions for 22 days and was shifted to nitrogen-deplete medium 7 days prior to the
14
assay. Data points are set in the middle of the acetylene incubation time (total
15
incubation 2h). (b) and (c) relative transcript levels of nifB, nifD, and fdxH (nif cluster
16
genes) as well as psbA in nitrogen deplete culture samples. All transcript data were
17
normalized to 16S rRNA and calibrated to their respective mRNA levels in the one
18
sample of nitrate-supplemented culture harvested at 12:00 and 22:00 for the 1st (a)
19
and 2nd (b) experiment, respectively (relative expression for these samples is 1). The
20
black and white bars above each graph indicate light- and dark-periods.
a
Acaryochloris sp. HICR111A ~ 20000 bp
orthologs to A. marina genes AM1_4121 to AM1_4104
1 kb
b ~ 10000 bp
~ 38800 bp
63.0
Synechococcus PCC 7335
31.0
1st amplicon365 bp
2nd amplicon2802 bp
3rd amplicon2182 bp
forward primer
modA - nifB
cysE – nifT 1 t 1st
reverse primer 4507 bp 2838 bp 2140 bp
805 bp 448 bp 339 bp
2 d 2nd
3d 3rd
genes of mixed origin
A. marina orthologs
a
Nostoc punctiforme PCC 73102 Anabaena variabilis ATCC 29413
100
100
Anabaena sp. PCC 7120 'Nostoc azollae' 0708
81
65
Raphidiopsis brookii D9
90 100
53
Cylindrospermopsis raciborskii CS-505
Microcoleus chthonoplastes PCC 7420 Trichodesmium erythraeum IMS101 100
Cyanothece sp. PCC 8802 Cyanothece sp. PCC 8801
77 100
Cyanothece sp. WH 8904 Gloeothece sp. KO68DGA
16S ribosomal RNA
98
Cyanothece sp. ATCC 51142
61
100
100
Crocosphaera watsonii WH 8501
UCYN-A Cyanothece sp. PCC 7822
100
100
b
Cyanothece sp. PCC 7424 70
Synechocystis sp. PCC 6803 99
concatenated nifHDK
Synechococcus sp. PCC 7002
Cyanothece sp. PCC 7425 73
Acaryochloris sp. HICR111A
97
100
Acaryochloris marina MBIC11017
Synechococcus sp. PCC 7335
Anabaena variabilis ATCC 29413 (nif1) 100
98
Anabaena variabilis ATCC 29413 (nif2) Anabaena sp. PCC 7120
Synechococcus elongatus PCC 7942
99
97
Cyanothece sp. PCC 7425
100
100
A Acaryochloris hl i sp. CCMEE5410
62
100
Picocyanobacteria (11 species)
Synechococcus sp. OS-B 100
Nostoc punctiforme PCC 73102
100
'Nostoc azollae' 0708
100
Synechococcus sp. OS-A
100
Cylindrospermopsis raciborskii CS-505
68
Gloeobacter violaceus PCC 7421
Gloeothece sp. KO68DGA
100
Cyanothece sp. ATCC 51142
100
Rhodospirillum rubrum ATCC 11170 100
UCYN-A
0.05
Crocosphaera watsonii WH 8501
Nostoc punctiforme PCC 73102
c
100 100
Cyanothece sp. PCC 7424
100
Anabaena variabilis ATCC 29413 nif1 100
100
98
Anabaena sp. PCC 7120
100
'Nostoc azollae' 0708 100
68
100
Cylindrospermopsis raciborskii CS-505 100
Trichodesmium erythraeum IMS101 51
Synechococcus sp. OS-B Acaryochloris sp. HICR111A 100
C Cyanothece th sp. PCC 7425 98
Synechococcus PCC 7335 100
Acaryochloris sp. HICR111A Synechococcus sp. OS-A
0.05
100
Synechococcus sp. OS-B Cyanothece sp. PCC 7822 Cyanothece sp. PCC 8801 100
Cyanothece sp. PCC 8802 UCYN-A 100 100
concatenated/fused nifEN
Gloeothece sp. KO68DGA Cyanothece sp. ATCC 51142 100
Crocosphaera watsonii WH 8501 Rhodospirillum rubrum ATCC 11170
0.05
Synechococcus sp sp. PCC 7335 Trichodesmium erythraeum IMS101
Rhodospirillum rubrum ATCC 11170
100
100
Cyanothece sp. PCC 8802
Synechococcus sp. OS-A
Anabaena variabilis ATCC 29413 nif2
100
Cyanothece sp. PCC 7822
Cyanothece sp. PCC 8801
a Nitrogena ase activity [nmol C2H4 / m mg Chl d /2h-1]
500.00 450 00 450.00 400.00 350.00 300.00 250.00
02.06.2011
200.00
03.06.2011
150.00 100.00 50.00 0 00 0.00 8:00
10:00
12:00
14:00
16:00
18:00
20:00
22:00
0:00
time of the day
b
c
relative e gene expressio on (log2 scale)
16384 0 16384.0 4096.0 4096.0 1024.0
1024.0
256.0
nifB
64.0
nifD fdxH
16.0
psbA
256.0 64.0 16.0
4.0
4.0
1.0
1.0
0.3
0.3 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 0:00 2:00
time of the day
8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 0:00 2:00
time of the day
Table 1 Oligonucleotides used in this work
primer name
sequence in 5'3' direction
coxAfw coxArev fdxHfw fdxHrev nifBfw nifBrev nifDfw nifDrev pdhBfw pdhBrev psaAfw psaArev psbAfw psbArev 16S_fw 16S_rev
aagcacaccatctagacgaagtgg gaatcccaatcaccttatggtcgg atatcacgattcccgtggatgagg cttgccaacacagctagaacaagc aaacgaaggtcaagaagtctggtg gtagcagggatgcttttcaatccg agaaggcaccctgatttatgacga ggcaaagccattttctggaagaca agtactgttatttaacgccctccg tttgtaggagccaccatagtgtcc aacagctgaagttgacatcaaccc tgcaggttccaaatccaagttgtg tgacaacagtattgcaaagacgcg ccaaaccaaccgatgtataaacgg ttggaaacgactgctaataccgga actgatcgaagtcttggtaagcca
concentration in assay 0.6 µM 0.6 µM 0.6 µM 0.6 µM 0.6 µM 0.6 µM 0.6 µM 0.6 µM 1 µM 1 µM 1 µM 1 µM 0.6 µM 0.6 µM 0.6 µM 0.6 µM
primer PCR efficiency (Kalendar et al. 2011) 69 % 88 % 92 % 90 % 85 % 87 % 83 % 88 % 86 % 89 % 88 % 83 % 90 % 79 % 87 % 88 %
Table 2 Similarities between Acaryochloris HICR111A Nif proteins and their homologs in Synechococcus PCC 7335 (percentages of identical or similar residues measured by blastP). For Synechococcus PCC 7335, the sequence overlap was over 97% for 16 of the shown 22 proteins, and over 90% for the rest of the proteins except for NifB (80%) that is likely annotated too short in Synechococcus PCC 7335. An asterisk next to a number indicates that this is the top hit found in the NCBI database.
Gene or Gene Product
Synechococcus PCC 7335 blastP POSITIVES (%)
blastP IDENTITY (%)
modBC
78
68 *
cysteine synthase flavodoxin fdxH hesB hesA nifW ORF2 nifX nifEN nifZ nifK nifD nifH nifU nifS nifB cysE ferritin dps family nifV nifT/fixT parB
92 93 92 88 93 85 87 86 91 90 93 95 93 90 94 90 81 86 89 86 66
83 * 87 * 80 * 79 * 85 * 74 * 72 * 75 * 82 * 78 * 90 * 91 * 89 * 83 * 87 * 82 * 72 * 76 * 78 * 66 * 53 *
