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CURRENT MICROBIOLOGY Vol. 36 (1998), pp. 253–258
An International Journal
R Springer-Verlag New York Inc. 1998
Unusual Gene Arrangement of the Bidirectional Hydrogenase
and Functional Analysis of Its Diaphorase Subunit HoxU
in Respiration of the Unicellular Cyanobacterium Anacystis nidulans
Gudrun Boison, Oliver Schmitz, Barbara Schmitz, Hermann Bothe
Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, D-50923 Köln, Germany
Received: 11 August 1997 / Accepted: 23 September 1997
Abstract. The bidirectional, NAD1-dependent hydrogenase from cyanobacteria is encoded by the
structural genes hoxFUYH, which have been found to be clustered, though interspersed with different
open reading frames (ORFs), in the heterocystous, N2-fixing Anabaena variabilis and in the unicellular
Synechocystis PCC 6803. In another unicellular, non N2-fixing cyanobacterium, Anacystis nidulans, hoxF
has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH. An
ORF (termed hoxE gene) is located immediately upstream of hoxF in A. nidulans and in Synechocystis. Its
deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E.
coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino
acids of HoxF in A. nidulans and Synechocystis. The diversity in the arrangement of hydrogenase genes in
cyanobacteria is puzzling. The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional
hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria. Different hoxU mutants were obtained by inserting a lacZKmR cassette into the
gene both in A. nidulans and Anacystis PCC 7942. Such mutants showed reduced H2-evolution activities
catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O2-uptake. A common link
between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria
may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants
impaired in one of the diaphorase genes.
Cyanobacteria can express at least two different hydrogenases catalyzing the evolution and/or the consumption
of molecular hydrogen. The uptake hydrogenase encoded
by the hupLS genes [5, 19] performs only H2-uptake with
methylene blue or phenazine methosulfate as electron
acceptor. The enzyme is particularly active in the heterocysts of the N2-fixing Anabaena or Nostoc spp. [7, 21,
29]. Recent work with a defined mutant demonstrated its
occurrence also in the unicellular, non-N2-fixing Anacystis nidulans [4]. The hupLS genes are, however, not
present on the completely sequenced chromosome of
another cyanobacterium, Synechocystis PCC 6803 [12].
The reversible, bidirectional hydrogenase catalyzes
both H2-uptake and the Na2S2O4-dependent evolution of
the gas. This enzyme occurs both in unicellular cyanobacCorrespondence to: H. Bothe
teria and in heterocysts and vegetative cells of filamentous forms [11, 20], but not in Nostoc sp. strain PCC
73102 [29]. The molecular [2, 4, 26, 29] and biochemical
[11, 20, 24, 28] analyses showed that it is composed of
the H2-cleaving dimer HoxYH and the diaphorase moiety
HoxFU transferring electrons to NAD1 [24]. It is related
to the NAD1-reducing hydrogenase from Alcaligenes
eutrophus [8] and to the NADP1-dependent enzyme from
Desulfovibrio fructosovorans [17]. In the heterocystous
Anabaena variabilis, two ORFs are interspersed with the
hoxFUYH gene cluster [26]. The function of one of these,
ORF8, is completely unknown, whereas the gene product
of ORF3 has recently been found to show sequence
similarities to the nuclear-encoded chloroplast protein
CP12 in higher plants [22] and thus to a protein unrelated
to hydrogenases. The completely sequenced genome of
the unicellular Synechocystis PCC 6803 [12] contains
254
also a hoxFUYH cluster with three ORFs that, however,
share no sequence homology to those in A. variabilis. The
gene cluster of S. PCC 6803 contains a further ORF,
termed hoxE, which might code for another diaphorase
subunit [2, 12]. In another unicellular cyanobacterium,
Anacystis nidulans, hoxYUH but not hoxF could be
identified as yet [4]. The missing hoxF gene, as well as
hoxE, will be described in the present communication.
HoxE, F and U show significant sequence similarities to three subunits of the mitochondrial complex I
(NADH:Q oxidoreductase) and the corresponding subunits NuoE, F, and G in E. coli [2, 25, 26]. The genome of
S. PCC 6803 [12] does not contain other sequences with a
high degree of homology to these respiratory subunits,
which have also not been identified in any other cyanobacterium as yet. The bidirectional hydrogenase of A.
nidulans is associated with the cytoplasmic membrane
[13], which shows NADH oxidase activity when isolated
[23]. These findings could indicate that HoxE, F, and U
are shared both by the bidirectional hydrogenase and
respiratory complex I functioning as an electron input
device both from NADH and H2 at the cytoplasmic
membrane. This recently proposed model [25] has now
been tested with newly constructed hoxU mutants of
Anacystis.
Materials and Methods
Culture, growth, preparation of extracts, and activity measurements. Anacystis nidulans (5 Synechococcus leopoliensis 5 Synechococcus sp. PCC 6301) was purchased from the Algensammlung des
Pflanzenphysiologischen Instituts der Universität Göttingen, Germany
(SAUG 1402-1). Anacystis PCC 7942 (5 A. R2 5 Synechococcus PCC
7942) came from the Pasteur Collection. The wild-type and the mutants
were grown in exactly the same medium as described previously [4].
Likewise, the preparation of hydrogenase extracts and the activity
measurements were performed as before [4]. The respiratory O2-uptake
was determined by a conventional Clark-type electrode. Cells of the late
logarithmic growth phase (200 ml, O.D. ,1.0 at 540 nm) were
centrifuged (4000 g, 2 min at room temperature) and suspended in 10 ml
20 mM Tris-HCl-buffer pH 7.6. Cells were then adapted to darkness for
2 h, and the O2-uptake activity was determined with 5–20 mg protein in
the electrode chamber.
Construction and isolation of the mutants. Construction of the
mutants was performed by using natural transformation (not electroporation). The method has been described exactly in the previous publication [4]. The protocols for isolation of DNA, hybridizations, and
labeling are also given in the previous paper [4]. Screening for the hoxF
gene was done with the l-gene bank described in [4]. Sequencing was
performed with the ABI PRISM Ready Reaction Dye Terminator Cycle
Sequencing Kit and the ABI PRISM 310 Genetic Analyzer (Perkin
Elmer). The DNA sequences of all identified genes coding for the
bidirectional hydrogenase (hoxEFUYH) and for the accessory proteins
(hoxW, hypA, and hypB) from A. nidulans have been deposited to the
EMBL/GenBank/DDBJ databases (accession no. Y13471 for hoxEF,
no. X97797 for hoxUYHWhypAB).
CURRENT MICROBIOLOGY Vol. 36 (1998)
Results
Characterization of the hoxEF genes of Anacystis
nidulans. Screening of a l-GEM-11 gene bank from
Anacystis nidulans [4] with a dig-labeled 400-bp EcoRV/
HindIII probe out of the N-terminus of hoxF from A.
variabilis [26] allowed identification of four positive
clones giving the same restriction pattern. An 1.5-kb
HindIII/SstI fragment hybridizing with the hoxF probe
and the adjacent 1.6-kb BglII/XhoI and 1.4-kb XhoI/XhoI
fragments partly overlapping with the HindIII/SstIfragment (Fig. 1) were subcloned into pUC18 and
pBluescript. Altogether, a 3.0-kb segment with the hoxEF
genes was sequenced on both strands (Fig. 1).
The putative hoxE gene product (165 amino acids)
has 53% identical amino acids with the same protein from
Synechocystis PCC 6803 [12], (only 46% identity to this
gene product of the same organism follows from the
sequence given in a different publication [2]). The
identity to the corresponding gene products HndA of the
Desulfovibrio fructosovorans hnd operon [17] and NuoE
of the NADH:ubiquinone oxidoreductase of E. coli [30]
is 35% and 28%, respectively. HoxE carries the consensus motif CX4CX35CXGXC probably harboring a [2Fe2S] cluster, like the homologous gene products from
other organisms [2, 12, 17, 30]. This motif is also present
in hoxF, though in a slightly modified way.
HoxF (534 amino acids, 61% or 59% amino acid
identities to the A. variabilis [26] or Synechocystis [12]
hoxF gene product, respectively) follows downstream of
hoxE. The hoxF gene product carries the four characteristic binding sites for the [2Fe-2S]-cluster just mentioned,
for NAD1 and FMN and for a [4Fe-4S]-cluster as in A.
variabilis [26] and Synechocystis [12]. Transcription is
probably terminated downstream of hoxF because of the
presence of ORF1 encoded on the opposite strand (Fig.
1). This ORF1 shows homologies to the unidentified gene
slr0427 from Synechocystis [12], which is obviously not
related to any of the hydrogenase genes or to the other
ORFs found in the bidirectional hydrogenase gene clusters of A. variabilis and S. PCC 6803 [12, 26].
The region upstream of hoxE (160 bp sequenced)
does not carry motifs with apparent sequence homologies
to any known gene. The hoxEF gene cluster is separated
by at least 16 kb from the residual structural genes
hoxUYH of the bidirectional hydrogenase from A. nidulans (Fig. 1), which is inferred from the comparison of
the restriction pattern of the two regions analyzed and the
localization of the genes on them hybridizing with either
the hoxF or the hoxU probe from A. nidulans.
Generation of hoxU mutants by insertional mutagenesis and their physiological activities. In the preceding
publication [4], physiological experiments with a hoxH
G. Boison et al.: HoxU in Respiration of Anacystis nidulans
255
Fig. 1. Gene clusters of the bidirectional hydrogenase of Anacystis nidulans, Synechocystis PCC
6803, and Anabaena variabilis. The same gene
names were used as in [12] for Synechocystis
PCC 6803 and in [26] for Anabaena variabilis.
Abbreviations for the restriction enzymes: Bg 5
BglII, H 5 HindIII, S 5 SstI, X 5 XhoI.
mutant of A. nidulans demonstrated the presence of an
uptake hydrogenase in addition to the bidirectional
enzyme in this cyanobacterium. Such mutant was completely unable to perform Na2S2O4- and methyl viologendependent H2-evolution (see Table 1), but still consumed
H2 in the presence of phenazine methosulfate to approximately 50% of the rate of the wild type [4]. In a similar
approach, two mutants of A. nidulans (EH-01 and EH-02)
have now been generated within hoxU by inserting a
lacZKmR-cassette (construct placKmEH in Fig. 2) to
demonstrate any link between the bidirectional hydrogenase and respiratory complex I. The insertion of the
cassette in the hoxU gene in the mutants was verified by
Southern blot analysis with a hoxU probe and a probe of
the lacZKmR-cassette in comparison with the wild type
(Fig. 3). In the mutants, no wild-type specific hybridization bands were detected even when the amount of DNA
from the mutants was increased tenfold for the blots and
when the length of the DNA-staining reaction was
increased ten times. Thus, the segregation was apparently
complete in the mutants. In contrast to hoxH mutants,
both EH-01 and EH-02 of hoxU consistently showed
,10% of the H2-evolution activity of the wild type (Table
1). Since the genes hoxU and hoxY overlap and hoxY and
hoxH are separated from each other by only 10 bp, these
genes may form a transcriptional unit. This appears to be
transcribed at a reduced level despite the insertion of the
lacZKmR cassette, which carries a termination signal
within (Fig. 2). Alternatively, it could be argued that
solely hoxY and hoxH are transcribed from an independent weak promoter, and Na2S2O4- and methyl viologendependent H2-evolution requires these two hydrogenase
structural genes only.
In an attempt to find a hoxU mutant absolutely
negative in Na2S2O4- and methyl viologen-dependent
H2-evolution, the plasmids pKmlacEB and pKmlacEH in
addition to placKmEH (Fig. 2) were inserted into the
genome of Anacystis PCC 7942 (5 A. R2). This latter
strain can be transformed with high efficiency, is the
cyanobacterium most frequently used for molecular analyses, and is genetically related to A. nidulans SAUG
1402-1 [9]. Therefore, the same constructs can be used
for generating mutants in both strains. The position of the
inserts in the mutants of A. PCC 7942 was verified by
Southern analysis. A. PCC 7942 mutants of hoxU carrying lacZ of the cassette in the sense direction (EHlacKm-4,
EHlacKm-10) have a high residual Na2S2O4- and viologen-dependent H2-evolution activity (Table 1). In con-
256
CURRENT MICROBIOLOGY Vol. 36 (1998)
Table 1. Na2S2O4- and methyl viologen-dependent H2-evolution
activities of the A. nidulans wild type and its bidirectional hydrogenase
mutants
Isolate
wild type
hoxH mutants
hoxU mutants
A. nidulans
A. nidulans
SAUG 1402-1
PCC 7942 (5R2)
(activity in %)
B16
lacKmB4-1
lacKmB4-2
EH-O1
EH-O2
100a
0
0
0
10
7
100a
R2lacKmB4
R2KmlacB2
EHlacKm-4
EHlacKm-10
EHKmlac-2
EBKmlac-42
0
0
58
86
4.5
1.7
100% refers to the specific activity of 348 nmol H2 3 h21 3 mg
protein21 for A. nidulans SAUG 1402-1 and 53 nmol H2 3 h21 3 mg
protein21 for A. PCC 7942. H2-evolutions were determined in crude
extracts. The data represent average values from five independent
experiments. The mutants EH-O1, EH-O2, EHlacKm-4, and
EHlacKm-10 were obtained with the plasmid construct placKmEH,
whereas EHKmlac-2 and EBKmlac-42 were generated by transformation with pKmlacEH and pKmlacEB, respectively. The generation of
mutant B16 is described in [4]. LacKmB4-1, LacKmB4-2, and
R2LacKmB4 were obtained by transformation with the plasmid construct placKmB4, the mutant R2KmLacB2 by using the construct
pKmlacB2, both inserting the lacZKmR-cassette at the same site in
hoxH as the KmR-cassette in mutant B16 [4]. For plasmid constructs,
see Fig. 2.
a
trast, mutants with the inverse insertion of the cassette
into hoxU (EHKmlac-2 and EBKmlac-42) have ,2% of
the activity of the wild-type (Table 1). Thus, insertion of
the cassette in dependence of its orientation has polar
effects on the transcription of the hoxYH genes located
downstream of hoxU. Since incomplete segregation is
unlikely, it is concluded that hoxU is inactivated. The
residual hoxYH expression is unlikely to play a role when
the mutants are assayed for respiratory activity.
Determination of the respiratory activity of the mutants. Intact cells of all hoxU and hoxH mutants of A.
nidulans (Table 2) and A. PCC 7942 (not documented)
showed nonimpaired O2-uptake activities. No differences
in the respiratory rates between mutants and wild type
were observed in cells incubated aerobically, anaerobically (under argon), or anaerobically with a mixture of
argon/H2 5 80/20 (vol/vol) for at least 2 h. Growth rates
of all these cultures were also not affected by the
mutagenesis (not documented).
Discussion
The genes coding for the structural hydrogenase proteins
are contiguous on the genomes of microorganisms [31].
The only exception to this rule has now been reported for
Fig. 2. The plasmids used for insertional mutagenesis of hoxU and hoxH
in Anacystis nidulans SAUG 1402-1 (5 Synechococcus PCC 6301) and
A. nidulans R2 (5 S. PCC 7942). The constructs contain the cassette
with the kanamycin resistance-conferring gene and the lacZ gene from
pKOK6 [14] at the position indicated by arrows in the given orientation.
Each construct bears one cassette only. Restriction enzymes: E 5
EcoRI, P 5 PstI, H 5 HindIII, B 5 BamHI.
the stable hydrogenase of the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina, where an
approximately 2-kb insert resides between the structural
genes hydS and hydL [15]. Cyanobacteria apparently
show a puzzling richness with respect to the organization
of the hydrogenase structural genes. In Synechocystis
PCC 6803 [12] and in Anabaena variabilis [26], the
structural genes of the bidirectional hydrogenase are
clustered, though interspersed with different ORFs at
different positions. In A. nidulans, hoxUYH are clustered,
and hoxF has now been found after a longer search by
heterologous hybridization under lower stringency with a
probe from A. variabilis. This gene, together with an ORF
of unknown function (hoxE), is separated by at least 16
kb from the residual structural genes hoxUYH in an
unusual way. With respect to the accessory genes, hoxW,
hypA, B, and F are contiguous downstream of hoxH in A.
nidulans [4], whereas they are scattered throughout the
genome of Synechocystis [12]. The three cyanobacteria
A. variabilis, A. nidulans, and Synechocystis PCC 6803
are obligate photoautotrophs, presumably with rather
similar cell metabolism. Furthermore, the similarities in
the amino acid level of their homologous hydrogenase
proteins range between 60% and 75%. Despite this, the
organization of the hydrogenase genes is strikingly
dissimilar with no obvious explanation. Variations in the
257
G. Boison et al.: HoxU in Respiration of Anacystis nidulans
Table 2. In vivo O2-uptake activity in the A. nidulans SAUG 1402-1
wild type and its bidirectional hydrogenase mutants
Isolate
O2-uptake specific
activity
[nmol · h21 · mg
protein21]
A.
hoxU mutants
hoxH mutants
nidulans
WT EH-O1 EH-O2 lacKmB4-1 lacKmB4-2
213
231
219
206
199
646
673
664
637
646
O2-uptake determined polarographically represents average values and
standard deviations from different experiments (n 5 3–5). For the
generation of the mutants, see legend in Table 1.
Fig. 3. Demonstration of the insertion of the lacZKmR -cassette into the
diaphorase gene hoxU and of the complete segregation in the mutants
obtained. Genomic DNA of A. nidulans SAUG 1402-1 (lane 1), of the
mutants EH-01 (lane 2) and EH-02 (lane 3), and of A. PCC 7942 (lane
4) was restricted with HindIII/EcoRI. Hybridization [4] with the hoxU
probe from A. nidulans at 68°C resulted in two bands of the expected
size (4.6 and 1.1 kb) in the mutants after 2 h and 20 h of color
development, and no signal for the wild-type band (5.0 kb) even after 20
h of incubation time. The 1.0-kb band in the mutants refers to the
internal fragment of the cassette hybridizing unspecifically. S 5 1-kb
ladder, 1.6-kb band marked.
gene order on the cyanobacterial genome have also been
described for some other gene clusters unrelated to
hydrogenase [6]. Hydrogenases, though expressed, are
unlikely to be essential for cyanobacterial growth, and no
hydrogenase phenotype has been described for these
organisms as yet. These enzymes might have been
essential for the growth of cyanobacteria in earlier earth
periods.
An unusual ORF (termed hoxE) resides upstream of
hoxF in A. nidulans and Synechocystis 6803. The hoxE
gene product has a predicted molecular mass of 18 kDa;
bands of about that size are seen on SDS-gels of the
purified enzyme [26]. HoxE and the 58-terminus of hoxF
(the first 104 amino acids in A. nidulans) may have arisen
by gene duplication, since both sequences show 28%
identity on the amino acid level. Apparently, hoxE is not
present immediately upstream of hoxF in the A. variabilis
cluster [26] and can also not be detected on the genomes
of A. variabilis and A. 7120 by heterologous hybridization with an hoxE probe from A. nidulans even at low
stringency (G. Boison, unpublished data). In unicellular
cyanobacteria (Anacystis, Synechocystis), HoxE, if expressed at all, may be a special requirement of an
NAD1-dependent hydrogenase of the cytoplasmic membrane. The hoxE gene and the special [2Fe-2S] cluster
binding site at the N-terminus of hoxF are missing in the
sequences coding for the soluble cytoplasmic NAD1dependent hydrogenase of Alcaligenes eutrophus [8]. In
the heterocystous A. variabilis, the bidirectional hydrogenase possesses the special [2Fe-2S] cluster in HoxF. The
enzyme is present in both heterocysts and vegetative cells
[11], and immunological data indicated its cytoplasmic
location (however, with a higher specific labeling associated with the thylakoids) in this cyanobacterium [27].
The definitive role of HoxE and of the special [2Fe-2S]
cluster in HoxF remains to be elucidated.
The data of the present investigation indicated that
mutants in hoxU have unaltered respiratory O2-uptake
activities. The hoxU gene product has been discussed to
serve as the bridging unit in the link between respiration
and hydrogenase [25]; therefore, insertion mutants of this
gene were chosen on purpose. The construction and
analysis of mutants in other parts of the hydrogenase
genes are unlikely to provide other information. The
results (unimpaired respiratory O2-uptake, but virtually
inactive bidirectional hydrogenase) could indicate that a
link between hydrogenase and respiratory complex I does
not exist. Other interpretations of the results are favored
by us. Like many other microorganisms, cyanobacteria
might have further respiratory chains of different composition that could reside either on the cytoplasmic membrane or on the thylakoids. Any defect in respiration
involving the hoxU gene product may be compensated by
alternative respiratory electron transport chains. Indications for multiple respiratory chains in cyanobacteria are
manifold (e.g., NADH oxidation by a dehydrogenase
homologous to respiratory complex I [16] or by a
rotenone-insensitive NADH dehydrogenase II [1], NADH
oxidase activity in the thylakoids of Plectonema boryanum [18], NADH or NADPH as primary respiratory
electron donor in dependence of the culture condition [3,
10], NADPH, possibly via NADPH:ferredoxin oxidoreductase, can stimulate respiratory O2-uptake [3]). Disappointingly enough, the attractive hypothesis of the common link between respiration and hydrogenase cannot be
258
CURRENT MICROBIOLOGY Vol. 36 (1998)
verified simply by determining the activities in a defined
hydrogenase mutant.
ACKNOWLEDGMENT
16.
This work was kindly supported by a grant from the Deutsche
Forschungsgemeinschaft.
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