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Arch Microbiol (2006) 186:367–376
DOI 10.1007/s00203-006-0150-4
O RI G I NAL PAPE R
The rice Weld cyanobacteria Anabaena azotica and Anabaena
sp. CH1 express vanadium-dependent nitrogenase
Gudrun Boison · Caroline Steingen · Lucas J. Stal ·
Hermann Bothe
Received: 25 November 2005 / Revised: 29 May 2006 / Accepted: 10 July 2006 / Published online: 19 August 2006
© Springer-Verlag 2006
Abstract Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated
from Chinese and Taiwanese rice Welds, expressed
vanadium-containing nitrogenase when under molybdenum deWciency. This is the second direct observation of an alternative nitrogenase in cyanobacteria.
The vanadium nitrogenase-speciWc genes vnfDG are
fused and clustered in a phylogenetic tree next to the
corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and
of nifH in Mo-grown cells was shown for the Wrst time by
sequencing cDNA derived from cultures of A. azotica
and Anabaena sp. CH1. The vnfH sequences clustered
with that of Anabaena variabilis. The vnf genes were
strongly transcribed only in cultures grown either in
Mo-free medium, or in W-containing medium, but
also weakly in Mo-containing medium. NifH was
transcribed in all media. On-line measurements of
acetylene reduction by Mo-free A. azotica cultures
demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that
of ethylene. Acetylene reduction of cultures grown
either with or without Mo had a high temperature
optimum of 42.5°C. The uptake hydrogenase gene
G. Boison (&) · L. J. Stal
Department of Marine Microbiology,
Centre for Estuarine and Marine Ecology,
Netherlands Institute of Ecology (NIOO-KNAW),
P.O. Box 140, 4400 AC Yerseke, The Netherlands
e-mail: [email protected]
C. Steingen · H. Bothe
Institute of Botany, University of Cologne, Gyrhofstr. 15,
50931 Köln, Germany
hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1,
and A. variabilis.
Keywords Alternative nitrogenase ·
Vanadium nitrogenase · Cyanobacteria · Tungsten ·
vnfDG · vnfH · Uptake hydrogenase · Gene expression
Abbreviations
ARA Acetylene reduction assay
RT
Reverse transcription
Introduction
Most described nitrogenases contain an FeMo-cofactor
as a prosthetic group. As Wrst observed in Azotobacter,
some diazotrophic microorganisms Wx N2 using alternative nitrogenases, in addition to the Mo-containing
enzyme (Mo-nitrogenase). These organisms express a
V-dependent enzyme (V-nitrogenase) with an FeVcofactor under Mo-deWcient growth conditions. In
some strains, an Fe-only nitrogenase is active when
both Mo and V are unavailable (Loveless and Bishop
1999). The three types of nitrogenase are encoded by
separate gene clusters: nifHDK coding for the Mo-,
vnfH/vnfDGK for the V-, and anfHDGK for the
Fe-only nitrogenase (Bishop and Joerger 1990). The
alternative nitrogenases are distinguished from the Moenzyme by an additional subunit encoded by vnfG or
anfG, which may be involved in the insertion of the
FeV- or Fe-only cofactors into the apo-proteins (Chatterjee et al. 1997). Furthermore, alternative nitrogenases can be detected by their ability to reduce C2H2
partly beyond C2H4 to C2H6 (Dilworth et al. 1987).
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Alternative nitrogenases are generally repressed by
Mo, but expressed in the presence of tungsten even in
Mo-medium (Raina et al. 1992; Thiel et al. 2002b).
Remarkably, V-nitrogenases evolve three times more
molecular hydrogen and have a higher energy demand
than the Mo-enzyme (Eady 2003).
At present, the biological relevance of alternative
nitrogenases for the organism and its habitat is
unknown. Besides Azotobacter, vnf and anf genes have
been reported in only a few strains of taxonomically
unrelated bacteria: Rhodospirillum, Azomonas, Heliobacterium (Loveless and Bishop 1999), Clostridium
(Zinoni et al. 1993), Rhodobacter (Schuddekopf et al.
1993), Rhodopseudomonas (Larimer et al. 2004),
Methanosarcina (Chien et al. 2000), and Anabaena
variabilis (Thiel 1993). A. variabilis is the only cyanobacterium for which the presence of a V-nitrogenase
has unambiguously been shown (Kentemich et al.
1988; Thiel 1993). Circumstantial evidence for a
V-nitrogenase has been presented for Anabaena azollae (Ni et al. 1990; Thiel 1993). While vnfDGK genes
are clearly diVerent and separated phylogenetically
from nifDK, vnfH and nifH genes cannot be distinguished by phylogenetic analyses because of the limited number of known vnfH sequences obtained so far
(Zehr et al. 2003; Raymond et al. 2004).
This study addresses the questions whether the vnf
system is more widespread among cyanobacteria than
known hitherto and whether nifH and vnfH sequences
can be distinguished by phylogenetic analyses. Ecophysiological comparisons of V-nitrogenase-positive
cyanobacteria could shed light on the function of this
enzyme in nature. In this study, cyanobacteria maintained in culture collections were screened for the presence and expression of vnf genes.
Arch Microbiol (2006) 186:367–376
tively, vnf transcription was induced without vanadate
in Mo-medium supplemented with 10 M Na2WO4 £ 2
H2O (MoW-medium). The cultures were concentrated
by centrifugation (3,500g, 5 min at room temperature),
washed with the corresponding medium and diluted
three times (1:10) with fresh medium in 25 cm2 plastic
tissue culture Xasks (TPP, Switzerland) to minimize
Mo-contamination from glassware.
Isolation of genomic DNA, Southern blots, and dotblot hybridization
Genomic DNA was extracted from cyanobacterial cultures by vortexing cells with glass beads in phenol
(Tamagnini et al. 1997). For Southern blots, 2 g of
genomic DNA was restricted with EcoRI, separated on
an 0.7% agarose gel, and transferred to a positively
charged nylon membrane (Roche) by capillary transfer
with 0.4 N NaOH. For dot-blots, 0.5 g of genomic
DNA was denatured in 0.4 M NaOH/5 mM EDTA at
95°C for 5 min and placed on ice. The denatured DNA
(total volume 8.5 l) was spotted onto a positively
charged nylon membrane (Roche), which had previously been equilibrated with 0.4 M NaOH. Dot-blot
and Southern blot membranes were washed in 2£ SSC,
and the DNA was Wxed by UV cross-linking. Hybridization was performed at 60°C in 5£ SSC/0.5% blocking reagent (Roche)/0.1% N-lauroylsarcosine/0.02%
SDS. The blot was washed in 2£ SSC/0.1% SDS (2£,
5 min, 60°C) and stained using NBT/X-phosphate
(Roche) according to the “DIG application manual for
Wlter hybridization” (Roche). Hybridization probes
(vnfG from A. azotica, hupL and hoxH from A. variabilis) were labeled with digoxigenin-dUTP (Roche) by
PCR using the primers given in Table 1 together with
Taq polymerase (Fermentas) in 35 cycles as follows:
92°C for 40 s, 50°C for 40 s, and 72°C for 40 s.
Materials and methods
PCR and DNA sequencing
Strains and growth conditions
Anabaena azotica FACHB-118, formerly A. azotica
HB686 (X. Wu, Wuhan, China, personal communication), was obtained from the Freshwater Algae Culture
Collection of the Institute of Hydrobiology, Chinese
Academy of Sciences, Wuhan. Anabaena sp. CH1,
maintained as CCY 9910 in the Culture Collection
Yerseke, was kindly supplied by Prof. P. Böger, Konstanz, Germany. The cyanobacteria were cultivated in
BG11 medium (Rippka et al. 1979) without combined
nitrogen at 25°C (Mo-medium). To express V-nitrogenase activity, molybdate in the trace element solution
was substituted by 10 M V2O5 (V-medium). Alterna-
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Hot-start PCR was performed using genomic DNA or
cDNA as templates with HotStar Taq DNA polymerase (Qiagen), which required activation (15 min, 95°C)
prior to cycling. All programs were terminated with an
end-elongation for 7 min at 72°C. The primers used are
given in Table 1. The program for ampliWcation of the
16S rRNA was described elsewhere (Boison et al.
2004). The thermal cycle for ampliWcation of nifH
using degenerated primers was as follows: 94°C for
40 s, 52°C for 40 s, and 72°C for 80 s for 35 cycles. A
gradient PCR with annealing temperatures between 50
and 65°C was used to determine the highest possible
annealing temperature for ampliWcation of nifH and
Arch Microbiol (2006) 186:367–376
Table 1 Oligonucleotide
primers used in this study
The name of the target gene is
followed by the position of the
primers in nucleotide numbers referring to the GenBank
accession number given
Primer
369
Sequence in 5⬘!3⬘ orientation
Source
16S rRNA (N359–N1404 of E. coli K12)
CYA359
GGGGAATYTTCCGCAATGGG
Nübel et al. (1997)
1387R
GGGCGGWGTGTACAAGGC
Marchesi et al. (1998)
nifH (N276–N634 of V01215)
nif-f
TGYGAYCCNAARGCNGA
Zehr and McReynolds (1989)
nif-r
ADNGCCATCATYTCNCC
Zehr and McReynolds (1989)
nifH (N296–N425 of V01215), sequence speciWc for A. azotica
AaznifH-f
CACCCGTTTGATGCTCCATGCC
This work
AaznifH-r
TTCTACGCAACGAACACCACGG
This work
nifH (N297–N411 of V01215), sequence speciWc for A. variabilis and Anabaena sp. CH1
Av99niH-f
ACCCGTTTGATGCTCCACGC
This work
Av99nifH-r
CGCCACGGAAACCGGTCAACAT
This work
vnfH (N296–N409 of V01215), sequence speciWc for A. azotica
AazvnfH-f
TACTCGTTTGATCCTCCACTGT
This work
AazvnfH-r
TCACGAAAACCATTGATAACG
This work
vnfH (N297–N416 of V01215), sequence speciWc for A. variabilis and Anabaena sp. CH1
Av99vnfH-f
ACCCGCTTGATTCTCCACAC
This work
Av99vnfH-r
TTTGATATCGCGGAAGCCTT
This work
vnfDG (N1481–N2137 of L20472)
D6fm
GAAGACTTYGARAAGGTCAT
This work, shortened D6f,
Loveless and Bishop (1999)
Ga2r
TGGTKCARTTCRCSGTT
This work
vnfG (N346–N475 of AY422692)
GAaf1
TTCAAGAGCGTTGTTTGT
This work
GAar1
TTGCCAGTCAGTGTTTCC
This work
hupL (N18605–N18912 of AF368526)
LF1
GAYCCYTGGTATATYAARCC
This work
LR1
TCRCCAGTYTTMGCATCATG
This work
hoxH (N2385–N2608 of X97797)
HAnv3
GTAAGTGAGGCACCTMGTGGK
Boison et al. (2000)
HAn2
CAGGAAAGGCAGGGRTCAAAR
Boison et al. (2000)
vnfH using sequence and strain-speciWc primers: 94°C
for 30 s, 50–65°C for 30 s, and 72°C for 1 min for 35
cycles. The annealing temperatures used in further
PCR reactions with this program were 65°C for the
nifH-speciWc primers, 53°C for the vnfH-speciWc primers of A. azotica, and 59°C for the vnfH-speciWc primers of Anabaena sp. CH1 and A. variabilis. For the
latter primers, the program was changed to 38 cycles
and denaturing and annealing times of 1 min each. A
40-cycle program was used to amplify vnfDG: 94°C for
60 s, 46°C for 60 s, and 72°C for 80 s. AmpliWcation of
vnfG was performed by nested PCR using a 1-l product of a vnfDG-PCR as template according to the following protocol: 40 cycles of 94°C for 60 s, 48°C for
60 s, and 72°C for 30 s. The thermal cycle for ampliWcation of hupL and hoxH was as follows: 94°C for 40 s,
52°C for 40 s, and 72°C for 40 s for 35 cycles.
PCR products of the right size were cloned with the
pGEM-T Easy vector system (Promega) in Escherichia
coli XL1-blue (Stratagene). White colonies were controlled by PCR using some cells as template and the
general primers directed against the Sp6- and T7-promoter sites from the cloning vector. This PCR was run
with 35 cycles as follows: 94°C for 45 s, 55°C for 45 s,
and 72°C for 45 s. For every product, three clones of
the right size were sequenced on both strands in an
ABI 3130 Genetic analyzer (Applied Biosystems).
Nucleotide sequence accession numbers
New sequences were deposited in GenBank under the
accession numbers: A. azotica (vnfDG = AY422692,
nifH = DQ294218, vnfH = DQ294219, 16S rRNA =
AY422691), Anabaena sp. CH1 (vnfDG = DQ294215,
nifH = DQ294216, vnfH = DQ294217, 16S rRNA =
DQ294214).
Computing programs for sequence analysis
Sequence data were compared with the NCBI data
bank entries using the BLAST program (Altschul et al.
1997). Sequences were aligned with the ClustalX software (Thompson et al. 1997), and corrected manually.
The aligned sequences, editing out the primers, were
used for phylogenetic analysis using the MEGA3 software package (Kumar et al. 2004). Neighbor-joining
trees were calculated with Poisson correction and bootstrap values from 1,000 replicate trees.
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Arch Microbiol (2006) 186:367–376
Isolation of total RNA, and reverse transcription
Total RNA was extracted from cultures with warm
acidic phenol followed by puriWcation with the RNeasy
Kit (Qiagen) and reversely transcribed with random
primers and Superscript II (Invitrogen) as described
(Boison et al. 2000). PCR was performed with cDNA,
a sample where Superscript has been omitted (negative
control) and genomic DNA (positive control) as
described above.
Acetylene reduction assay
Nitrogenase activity was determined by acetylene
reduction assay (ARA) with an on-line system (Staal
et al. 2001). The cells were concentrated on a glass
Wber Wlter (47 mm, Whatman GF/F) and incubated in
a temperature-controlled chamber under continuous
gassing with 70% N2, 20% O2 (each containing 0.4%
CO2), and 10% C2H2. For anoxic measurements, the
gas Xow controller was adjusted to 90% N2 and 0%
O2. Injections were made automatically every 5 min.
C2H4 and C2H6 were separated and quantiWed on a
gas chromatograph (Chrompack CP 9001, Varian,
Netherlands) equipped with a capillary column
(CP-PoraPLOT U, 27.5 m, 0.53 mm, 20 m, Varian,
Netherlands) and a Xame ionization detector. The
temperatures were 90, 60, and 120°C for the injector,
oven, and detector, respectively. The reaction chamber was illuminated with a slide projector. The light
intensity was changed using a set of neutral Wlters as
slides in the projector to give an exponentially
increasing light intensity. The incident light in the
reaction chamber was measured using a Licor Li 250
light meter. The assay temperature was changed for
temperature-dependent measurements at a random
order from 15 to 47°C. Three to Wve injections were
made at each temperature until the values were stable. All ARA experiments were repeated three times
with cultures pre-incubated at 25°C in a 12 h/12 h
light/dark regime.
Chlorophyll a determination
Chlorophyll a was extracted from the Wlters used for
the ARA measurements by 90% acetone and soniWcation for 1 h. After removing the debris by centrifugation, absorption of the supernatant was measured
at 664 nm and the chlorophyll a concentration was
calculated using an absorption coeYcient of 87.7 ml
mg¡1 cm¡1.
Results and discussion
Characterization of vnfDG genes in A. azotica
and Anabaena sp. CH1
Heterocystous cyanobacteria from the culture collections in Köln (Germany) and Yerseke (CCY; The
Netherlands) were screened for the presence of the
V-nitrogenase-speciWc gene vnfDG by PCR. Sequences
from PCR products obtained from A. azotica FACHB118 and Anabaena sp. CH1 were identiWed as cyanobacterial vnfDG by BLAST comparison. The sequences
from A. azotica and Anabaena variabilis shared identities of 93% on DNA and 98% on amino acid levels,
whereas identity values between the Anabaena sp. CH1
and A. variabilis sequences amounted to only 77% for
the nucleotides and 73% for the amino acids. VnfD and
vnfG, which are separate genes in other bacteria, are
fused into a single entity in A. azotica and Anabaena sp.
CH1, as was Wrst observed in A. variabilis (Thiel 1993).
Surprisingly, this sequence in Anabaena sp. CH1 is 21
nucleotides longer than those from the other Anabaena
strains. This additional stretch is located at the 3⬘ end of
the vnfD part of the fused gene (Fig. 1). It is neither
known whether the functional and regulatory properties
An. azotica
AIYSPLMQLAAFDVRDDAPKAP------AKTKEIEH-LNEKVTNITTYIQERCLW
An. variabilis
AIYSPLMQLAAIDVRDDAPKAP------AKTKEIEH-LNEKVTNITTYIQERCLW
An. CH1
AIYSPLMQLAGIDVRDDEPKKDNSESLKQQSEEVTAYIQERTEEITKFIQERCLW
M. acetivorans AIYSPMWKLAGKDPRETDSPMWSLT-EKDSGVVQESM-NEKIEEITALIQERCLW
M. barkeri
GIYSPMWSLAGKDPR----------------VVQELM--KKLEEVTALIQKQCLW
R. palustris
AVHNPLLKLAATDIRGETSTR---------LLEAAEMSAEQIDQLYNYCQERYLW
Az. chroococcum AVHNPLRHLAAVDIRDSSQTTP-------VIVRGAAMSQSHLDDLFDYTEERCLW
Az. paspali
AVHNPLRHLAAVDIRDKSQTTP-------VIVRGAAMSQSHLDDLFAYVEERCLW
Az. salinestris
AVHNPLRHLAAVDIRDKSQTTP-------IIVRGAAMSQSRLDDLFAYVEERCLW
Az. vinelandii
AVHNPLRHLAAVDIRDKSQTTP-------VIVRGAAMSQSHLDDLFAYVEERCLW
*
** * *
**
Fig. 1 Alignment of the C-terminal end of VnfD and N-terminal
end of VnfG sequences. The arrow indicates the start methionine
of VnfG. In Anabaena spp., VnfD and VnfG are fused into one
123
entity. Identical amino acids are marked by an asterisk. An
Anabaena, M Methanosarcina, R Rhodopseudomonas, Az Azotobacter
Arch Microbiol (2006) 186:367–376
371
of the VnfDG products are altered by this gene fusion,
nor whether the unique addition of 21 nucleotides in the
vnfDG fusion area of Anabaena sp. CH1 is of functional
signiWcance. In a neighbor-joining analysis of all available deduced VnfDG sequences, those from cyanobacteria clustered next to those from Methanosarcina spp.
and were clearly separated from AnfDAnfG (Fig. 2).
The close similarity of the cyanobacterial vnfDG to
vnfDvnfG of Methanosarcina spp. might strengthen the
idea of an archaeal origin of conventional and alternative nitrogenases (Raymond et al. 2004). Since alternative nitrogenases occur in bacteria of totally unrelated
taxonomic aYnities and because the composition of the
structural genes nifHDK and vnfHDGK is largely diVerent, a selection pressure must have existed for the acquisition of the V-nitrogenase genes by the organisms by
lateral gene transfer. The evolution of the cyanobacterial V-nitrogenase gene vnfDG may have occurred in a
common ancestor, since these genes are contiguous in
the three Anabaena spp. but not in other bacteria.
PCR experiments with vnfDG-primers and nested
PCR with vnfG-primers were negative for Fischerella
sp. SAG 1427-1, Chlorogloeopsis sp. ATCC 27193, Calothrix spp. (ATCC 27905, CCY 0013, and CCY 0202),
and Anabaena spp. (CH2 and CCY 0015). A dot-blot
hybridization using a vnfDG probe of A. azotica gave
unambiguous positive signals only with A. variabilis,
A. azotica, and Anabaena sp. CH1 (not documented).
IdentiWcation of nifH and vnfH sequences
An investigation of nitrogenase and hydrogenase genes
in A. azotica carried out by Southern blotting yielded
two signals with a probe for nifH, possibly due to the
presence of nifH and vnfH, and one signal each using a
probe for the uptake hydrogenase gene hupL and the
bidirectional hydrogenase gene hoxH. These results
were corroborated by PCR using the degenerate primers LF1/LR1 for hupL and HAnv3/HAn2 for hoxH.
In order to identify nifH and vnfH sequences in
A. azotica and Anabaena sp. CH1, reverse transcription
(RT)-PCR products obtained with the universal nifHprimers from cultures of these strains grown in Mo- and
V-medium were cloned and three clones were
sequenced from both media and strains. Two clearly
diVerent genes were expressed in the cultures grown in
Mo- or V-medium and the three clones from each
medium were identical in both cases. In a phylogenetic
analysis of deduced NifH sequences, the genes
expressed in Mo-medium clustered with the majority of
heterocystous NifH sequences next to NifH1 from
A. variabilis (Fig. 3), which is known to be active in Monitrogenase. The other gene, expressed in V-medium,
occupied a separate cluster comprising the sequence
from A. variabilis tentatively identiWed as vnfH (Thiel
et al. 2002a). Thus, the diVerent genes from A. azotica
and Anabaena sp. CH1 were termed nifH and vnfH.
X15077 Azotobacter chroococcum
95
71
AF152913 uncultured bacterium
AF152914 uncultured bacterium
100
AF058782 Azotobacter salinestris
84
88
98
100
99
100
99
100
84
100
100
68
AF058781 Azotobacter paspali
M32371 Azotobacter vinelandii
AF152909 uncultured bacterium
AF152910 uncultured bacterium
VnfDVnfG
AF152908 uncultured bacterium
AF152912 uncultured bacterium
AF152911 uncultured bacterium
NC005296 Rhodopseudomonas palustris
NC003552 Methanosarcina acetivorans C2A
AF254784 Methanosarcina barkeri
Anabaena sp. CH1
AY422692 Anabaena azotica
AY422707 uncultured bacterium BSC
VnfDG
CAA52044 Anabaena variabilis
M23528 Azotobacter vinelandii
AnfDAnfG
10 % substitutions
Fig. 2 Neighbor-joining tree of deduced amino acid sequences of
part of V-nitrogenase subunits VnfDG (corresponding to amino
acid residues 373–578 of A. variabilis, accession no. CAA52044),
rooted to AnfDAnfG of Fe-nitrogenase of A. vinelandii. The sequences of the separate subunits VnfD/AnfD and VnfG/AnfG
from bacteria other than cyanobacteria were concatenated (VnfDVnfG/AnfDAnfG) for the analysis. VnfDG is one entity in cyanobacteria. Numbers at the branches indicate the percentage of
occurrence of the respective node in a bootstrap analysis of 1,000
re-samplings; only values above 50% are shown
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Arch Microbiol (2006) 186:367–376
Anabaena sp. CH1 VnfH *
99
DQ315787 Anabaena variabilis VnfH
AAC36070 unidentified marine eubacterial clone BT1118
47
U49515 Fischerella sp. UTEX1931
Anabaena azotica VnfH *
74
AAQ64036 uncultured bacterium cluster M
73
ZP00111243 Nostoc punctiforme (extra copy)
U73129 Calothrix sp. ATCC27901
69
52
AJ716389 uncultured bacterial clone MING-50A
V01482 Nostoc sp. PCC7120 NifH1
AAS22069 Anabaena aphanizomenoides
A49831 Anabaena oscillarioides
49
AAA22011 Anabaena oscillarioides
AAT48543 Cylindrospermopsis raciborskii
AAC64640 Fischerella UTEX1903
44
AAB37315 Chlorogloepsis sp. ATCC27193
AAA22014 Anabaena sp. L-31
Anabaena azotica NifH1 **
43
43
40
62
AAA87251 Anabaena azollae
U89346 Anabaena variabilis NifH1
Anabaena sp. CH1 NifH1 **
42
58
69
AAB37316 Chlorogloeopsis sp. CCAP1411/1
AAP48972 Tolypothrix sp.
ZP_00162494 Anabaena variabilis (extra copy)
42
42
AF012326 Nostoc sp. PCC7120 (extra copy)
AAS22068 Anabaenopsis sp. NRE1
86 ZP_00112319 Nostoc punctiforme NifH1
L23514 Nostoc commune UTEX584
AAS75595 Nodularia sphaerocarpa
53
AAB37308 Scytonema sp.
54
77
51
ZP_00109382 Nostoc punctiforme (extra copy)
U04054 Nostoc muscorum
NifH2
Trichodesmium, Symploca
Gloeothece, Synechococcus, Crocosphaera
X07866 Rhodobacter capsulatus
5 % substitutions
Fig. 3 Neighbor-joining tree of deduced amino acid sequences of
part of nitrogenase reductase NifH (corresponding to amino acid
residues 48–152 of Nostoc sp. PCC7120, accession no.
AAA22008) rooted to the sequence of Rhodobacter capsulatus.
Bootstrap analysis is as in Fig. 2; only values above 40% are given.
Gray boxes—sequences of the genomes of A. variabilis, Nostoc
sp. PCC7120, and N. punctiforme PCC73102, which are described
as VnfH or NifH1 of Mo-nitrogenase, and cDNA sequences of
A. azotica and Anabaena sp. CH1 expressed in V-medium (asterisk)
and Mo-medium (double asterisks). Copies of nifH known from
the genome sequences as not belonging to a nifHDK cluster are
marked as extra copies. The condensed NifH2 cluster comprises
the same sequences as in Boison et al. (2004). The other condensed clusters contain—L15554 Gloeothece sp., U22146 Synechococcus sp. RF-1, AAP48976 Crocosphaera watsonii,
AAB81940 Symploca atlantica PCC8002, AAP48970 Oscillatoria
sancta, M29709 Trichodesmium thiebautii, AAB70120 Trichodesmium erythraeum IMS101
Since only a few vnfH sequences have been deposited in the databanks, a clear separation between a
vnfH and nifH cluster in phylogenetic studies has not
emerged (Zehr et al. 2003; Raymond et al. 2004). The
situation is further complicated by the occurrence of
multiple copies of nifH in several heterocystous cyanobacteria. Hence, it is not known whether the sequences
from Fischerella sp. UTEX 1931 and Calothrix sp.
ATCC 27901 and a few environmental sequences that
cluster with the vnfH sequence of A. variabilis (Fig. 3)
belong to a V-nitrogenase system. No vnfDG genes
were detected in other strains of Fischerella or Calothrix in the present study, but it cannot be ruled out
that such genes exist but are too divergent to be
detected. However, it is known that the nifH copy from
Nostoc punctiforme clustering with the vnfH sequence
does not belong to a vnf system (Thiel et al. 2002a). By
the sequencing of cDNA of V- and Mo-grown cultures
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Arch Microbiol (2006) 186:367–376
in the present study, it appears to be possible for the
Wrst time to distinguish between vnfH and nifH. This
might give a new start for re-investigating the still unresolved nifH/vnfH phylogeny.
373
M 1 2 3
4 5 6
7 8
9 10 11 12 13 14 +
-
M
A
Transcription of vnf genes and hupL in Anabaena spp.
Primers were designed to distinguish between nifH and
vnfH sequences of the Anabaena strains and tested for
their speciWcity on cloned nifH and vnfH products.
These primers are sequence- and strain-speciWc and
ampliWed under high stringency only the desired target.
The expression of nitrogenase and hydrogenase genes
was then investigated in A. azotica, Anabaena sp. CH1,
and A. variabilis cultures grown in either Mo-, MoW-,
or V-medium by RT-PCR (Fig. 4). The diVerent products of each strain were ampliWed from the same
cDNA pool. The universal nifH-primers, which do not
distinguish between nifH and vnfH, were used as positive control, and products were obtained with these
primers from the cDNA of all three strains grown in all
media (Fig. 4). The nifH-speciWc primers revealed that
nifH was transcribed in all three strains in Mo-grown
cultures and also in MoW- and V-grown cultures. This
eVect was described earlier for A. variabilis (Thiel et al.
1997). However, vnfH and vnfDG was strongly
expressed in the Anabaena strains only in MoW- and
V-medium (Fig. 4). Expression in the presence of Mo
and W might be due to the fact that the high aYnity
Mo-transporter is competitively inhibited by W (Thiel
et al. 2002b; Zahalak et al. 2004). Surprisingly, the vnf
genes were weakly transcribed also in the Mo-medium.
Expression of vnf genes in the presence of Mo is a new
Wnding and might be explained by the higher sensitivity
of RT-PCR compared to -galactosidase assays and
Northern blots, which were used in earlier investigations (Thiel et al. 1997). Whether this basic transcription also reXects expression and activity of vanadium
nitrogenase cannot be concluded from these results.
The uptake hydrogenase gene hupL, which is
expressed under nitrogen-starvation only, was transcribed equally in Mo-grown and Mo-deWcient cultures
(not documented), suggesting that this enzyme recycles
H2 evolved by Mo- or by V-nitrogenase.
Activity of the V-nitrogenase from A. azotica
measured by ARA
V-grown cultures of both A. azotica and Anabaena sp.
CH1 reduced C2H2 partly beyond C2H4 to C2H6, which
is a characteristic of alternative nitrogenases (Dilworth
et al. 1987). Under all conditions tested, the C2H6
formed amounted to 2.1 § 0.5% of the measured C2H4,
B
C
D
Fig. 4 Transcription of nifH and vnf genes in Anabaena spp. Total RNA, isolated from A. azotica (Aa), Anabaena sp. CH1
(CH1), and A. variabilis (Av) cultures grown in Mo-, MoW-, or
V-medium was reverse-transcribed with hexanucleotides. PCR
from the cDNA pools and from negative controls to which no reverse transcriptase was added (nc) was performed with degenerated primers nif-f/-r directed against nifH/vnfH (A), with speciWc
primers AaznifH-f/-r and Av99nifH-f/-r directed against nifH
(B), with speciWc primers AazvnfH-f/-r and Av99vnfH-f/-r directed against vnfH (C), and with degenerated primers D6fm/Gar2
directed against vnfDG (D). Lane 1/2 cDNA/nc of Aa-Mo; lane
3/4 cDNA/nc of Aa-MoW; lane 5/6 cDNA/nc of Aa-V; lane 7/8
cDNA/nc of CH1-Mo; lane 9/10 cDNA/nc of CH1-V; lane 11/12
cDNA/nc of Av-Mo; lane 13/14 cDNA/nc of Av-V; (+) positive
control with genomic DNA in PCR; (¡) negative control with no
template added in PCR; M DNA ladder, low range (Fermentas).
A fragment size of 500 bp is indicated by a line
which is as high as in V-grown cultures of A. variabilis
(Kentemich et al. 1988; Zahalak et al. 2004) or Azotobacter spp. (Dilworth et al. 1987). Cultures grown in
Mo-medium did not form detectable amounts of C2H6,
except above 40°C when some C2H6 was formed, as
observed earlier with Azotobacter chroococcum (Dilworth et al. 1993).
Further studies were pursued only with A. azotica,
because the geographical origin and the characteristics
of this strain are well known (Li 1981). Thus, physiological experiments with this cyanobacterium were carried out under conditions reXecting the growth
123
Conclusions
In this paper, the occurrence of a functional V-nitrogenase in A. azotica and Anabaena sp. CH1, both isolates
from rice Welds (Ley et al. 1959; Chen 1984), is conclusively presented from physiological and genetic data.
This is the second unambiguous example of V-nitrogenase in cyanobacteria, previously shown to be in
A. variabilis (Kentemich et al. 1988; Thiel 1993).
123
50
2.5
40
2.0
30
1.5
20
1.0
10
0.5
0
C2H6 [µmol h-1 mg Chla-1]
demands experienced in its natural habitat. A. azotica
was isolated from rice Welds in the Chinese province
Hubei (Ley et al. 1959), where it is used as a bio-fertilizer in the late-rice crop (Li 1981). The area is characterized by hot summers with average temperatures of
about 30°C (Domroes and Peng 1988), and temperatures in the open water body of the rice Welds of up to
40°C (Halwart and Gupta 2004). One of the aspects of
this study was, therefore, to test whether high temperatures may favor V-nitrogenase activity in this special
cyanobacterium.
Measurements of light response curves, at 25 and
35°C in a range of 0–980 mol photons m¡2 s¡1, were
Wrst performed under a Xow of 20% O2 and then
repeated without O2 using the same cells. The ratio of
the amounts of C2H4 and C2H6 formed in V-grown cultures was constant over the whole light range both
under aerobic and anaerobic conditions (not documented). Light saturation of C2H2-reduction was
reached at around 200 mol photons m¡2 s¡1 with both
Mo- and V-grown cultures under all conditions. Saturating light intensities of 245 mol photons m¡2 s¡1
were then chosen for assaying the temperature dependence of C2H2-reduction. Over the whole temperature
range, C2H4-production was 1.5-fold higher in Mothan in V-grown cultures and the ratio of the amounts
of C2H6 and C2H4 in V-grown cells was constant over
the measured temperature range (Fig. 5). The maximum of C2H4-formation for both media was at 42.5°C.
This rather high temperature optimum reXects the natural habitat of A. azotica. However, neither the light
response curve nor the temperature dependence
revealed diVerences in the optimal conditions for C2H2reduction of Mo- and V-grown cultures of A. azotica.
Since Mo-nitrogenases are more eYcient, the use of
this enzyme might be preferred in A. azotica in rice
Welds and will be independent of the temperature,
unless its expression is hampered by the presence of W.
Tungsten is an abundant mineral in the neighboring
province of Hunan (Tanelli 1982) and it is known to
interfere strongly with the activity of Mo-nitrogenases
(Kumar and Kumar 1980).
Arch Microbiol (2006) 186:367–376
C2H4 [µmol h-1 mg Chla-1]
374
0
0
15
25
35
Temp. [°C]
45
55
Fig. 5 C2H2-reduction and C2H4- and C2H6-formation in Moand V-grown cultures of A. azotica. Temperature-dependent
curve under saturating light conditions of 245 mol photons
m¡2 s¡1. C2H4 (Mo-culture)—solid triangle; C2H4 (V-culture)—
solid square; C2H6 (V-culture)—open square. One representative
measurement from three independent experiments is given.
Internal errors of integration were not signiWcant. C2H6 values
below 0.2 mol h¡1 mg Chla¡1 are at the detection limit of the
method and may be overestimated by up to 20%
Circumstantial evidence for the occurrence of V-nitrogenase in another rice Weld strain, A. azollae (Ni et al. 1990),
is not supported by published sequence information.
Hardly anything is known about the function, distribution, and expression of alternative nitrogenases in
nature. Bacteria containing alternative nitrogenase
genes have been isolated from diverse environments
(Loveless et al. 1999). However, to our knowledge, the
expression of anf genes in gut symbionts of termites
(Noda et al. 1999) is the only example where transcription of alternative nitrogenase genes in environmental
samples has been documented. Expression of alternative nitrogenases in Mo-deWcient soils or micro-zones
of microbial colonies (Maynard et al. 1994), at unusually high tungsten concentrations, high alkalinity (pH
10) (Tsygankov et al. 1997) or low temperatures
(Walmsley and Kennedy 1991) has been suggested in
several studies.
So far, all but one of the Anabaena strains able to
express V-nitrogenase have been isolated from rice
Welds of diVerent geographic origin. 16S rRNA analysis
suggests that Anabaena sp. CH1 is closely related to
A. variabilis, but that A. azotica is more closely related
to Trichormus azollae and Anabaenopsis circularis (not
documented). Thus, conditions that favor the use of
alternative nitrogenases might exist in the anoxic lower
soil levels of Xooded rice Welds . It was speculated that
these enzymes may have been advantageous in ancient
anoxic oceans, when Mo may have been scarce due to
the formation of insoluble sulWdes under anoxic conditions (Raymond et al. 2004). Tungsten sulWde, by contrast, is more soluble (Hille 2002). This might increase
Arch Microbiol (2006) 186:367–376
Mo-deWciency in anoxic environments by inhibiting
Mo-uptake. Indigenous cyanobacteria like A. azotica
are important biological nitrogen fertilizers in rice
Welds, and the distribution and possible contribution of
V-nitrogenases to total N2-Wxation in these habitats
need further investigation.
Acknowledgments We are indebted to Prof. M.G. Yates, Brighton, United Kingdom, for helpful comments on the English and
to Prof. H. Dai, Wuhan, Peoples Republic of China, for supplying
the A. azotica culture. We warmly thank M. Doeleman for the
excellent technical assistance, Dr M. Staal for generous help with
the on-line GC system, and Dr X. Zhai for kind help with the Chinese translations. This is NIOO publication number 3897.
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