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Functional analysis of hydrogenases and their effects on cell growth and magnetosome synthesis inMagnetospirillum gryphiswaldense

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May 2010 Vol.55 No.13: 1271−1277
doi: 10.1007/s11434-009-0744-8



Functional analysis of hydrogenases and their effects on cell growth
and magnetosome synthesis in Magnetospirillum gryphiswaldense
BAN Jia1, JIANG Wei1, LI Ying1, ZHANG YaoPing2 & LI JiLun1*

State Key Laboratory for Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, China;
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA

Received April 24, 2009; accepted August 20, 2009

This study addressed the effect of hydrogen metabolism on cell growth and magnetosome synthesis in Magnetospirillum gryphiswaldense strain MSR-1. Two deletion mutants were generated: L206, with single deletion of the hupL gene encoding
H2-uptake [NiFe] hydrogenase; and B206, with double deletion of the hyaB gene encoding H2-producing [NiFe] hydrogenase and
the hupL gene. The wild-type and mutant strains were compared in terms of hydrogen uptake capability, hydrogen yield, growth
rate, and iron uptake, and observed by transmission electron microscopy. Results indicate that HupSL protein is a specific
H2-uptake hydrogenase while HyaAB protein is a specific H2-producing hydrogenase. In comparison to wild-type and B206, L206
released a greater quantity of H2 under conditions that induce magnetosomes synthesis, and showed higher rates of growth and
iron uptake. M. gryphiswaldense appears to regulate reducing power in vivo, via H2-uptake hydrogenase and H2-producing hydrogenase, to promote iron absorption and magnetosome synthesis.
Magnetospirillum gryphiswaldense, hydrogenase, hydrogen uptake, reducing power, magnetosome synthesis

Ban J, Jiang W, Li Y, et al. Functional analysis of hydrogenases and their effects on cell growth and magnetosome synthesis in Magnetospirillum gryphiswaldense. Chinese Sci Bull, 2010, 55: 1271−1277, doi: 10.1007/s11434-009-0744-8

Hydrogenases are essential enzymes in microbial hydrogen
metabolism. There are th; ree distinct classes with respect to
the metal atoms at their active site: [NiFe]-(some kinds
contain Se) [1], [FeFe]-, and metal-free-hydrogenases [2,3].
The most numerous and best-studied class is the [NiFe]hydrogenases. The core enzyme consists of a heterodimer
with a large subunit containing the bimetallic active site,
and a small subunit containing Fe-S clusters as an electron
donor [3]. Different hydrogenases have different catalytic
activity. Most of them can catalyze reversible reactions for
uptake or release of hydrogen, with efficiency varying with
species [4]. Some kinds of hydrogenase only show hydrogen uptake activity, whereas others only release hydrogen
[5]. Microorganisms may expend reducing power via hydrogen release [6], or, alternatively, absorb hydrogen and
recover the energy in the form of reducing power to produce
*Corresponding author (email: lijilun@cau.edu.cn)

© Science China Press and Springer-Verlag Berlin Heidelberg 2010

ATP via the electron transport system [7].
Magnetotactic bacteria are characterized by the ability to
synthesize membrane-enclosed, nano-sized, ferrimagnetic
iron oxide (magnetite, Fe3O4) or iron sulfide (greigite, Fe3S4)
crystals, termed magnetosomes. Considerable research in the
past decade has focused on the formation and application of
magnetosomes. During our studies on physiological and genetic characteristics of Magnetospirillum gryphiswaldense,
we discovered that strain MSR-1 absorbs high levels of H2
under shaking flask culture conditions. The thesis work of
Liu showed that excessive accumulation of reducing power in
vivo results in suppression of magnetosome synthesis and cell
growth [8]. Microbial hydrogen absorption and release are
closely related with the level of reducing power in vivo, but it
is unclear whether hydrogen metabolism is associated with
magnetosome synthesis.
The genome sequences of several magnetotactic bacteria
are now available [9]. These include Magnetococcus sp.



BAN Jia, et al.

Chinese Sci Bull

MC-1, Magnetospirillum magnetotacticum MS-1, M. magneticum AMB-1, and M. gryphiswaldense MSR-1. Two
NiFe-hydrogenases have been identified in MSR-1 genome
sequences: H2-uptake hydrogenase gene hupSL and H2producing hydrogenase gene hyaAB. The present study was
designed to elucidate the functions of these two hydrogenases in H2 metabolism of M. gryphiswaldense, and to
analyze their effects on cell growth, iron uptake capability,
and magnetosome synthesis. For this purpose, we generated
two mutant strains, one with single deletion of the gene encoding the H2-uptake hydrogenase, the other with double
deletion of that gene and the gene encoding the H2producing hydrogenase.

1 Materials and methods

Bacterial strains, plasmids and growth conditions

Bacterial strains and plasmids used are listed in Table 1. M.
gryphiswaldense strain MSR-1 (DSM 6361) was purchased
from Deutsche Sammlung von Mikroorganismenund
Zellkulturen GmbH (Brunswick, Germany), and grown at
30°C in LAY medium [10], or, to eliminate magnetosome
synthesis, ferric citrate-free LAY medium. Solid culture
conditions were described previously [11]. For conjugation
experiments, MSR-1 was cultivated in LG medium [12].
Cloning host Escherichia coli DH5α and conjugation donor
E. coli S17-1 were grown at 37°C in Luria-Bertani (LB)
medium. For M. gryphiswaldense, antibiotics were used at
the following concentrations (mg/mL): nalidixic acid (Nx) 5,
gentamicin (Gm) 5, kanamycin (Km) 5. For E. coli, antibiotic concentrations used were: ampicillin (Ap) 100, chloromycetin (Cm) 25, gentamicin 10, kanamycin 50, and tetracycline (Tc) 12.5.
1.2 Construction of H2-uptake hydrogenase and H2producing hydrogenase deletion mutants
Ligation, transformation, PCR, sequencing, and DNA extraction were performed by standard methods [13]. For con-

May (2010) Vol.55 No.13

struction of a hupL deletion mutant, two pairs of primers
were designed to amplify the 3′ region and 5′ region of the
A gentamicin resistance cassette of the plasmid pUCGm
was excised with Sac I and purified. The three fragments
were ligated into pSUP202, which was excised with BamH I
and Hind III, yielding suicide plasmid pSUPL. pSUPL was
introduced into E. coli S17-1 by transformation, and then
into M. gryphiswaldense MSR-1 via conjugation. Colonies
which were sensitive to chloromycetin and resistant to both
nalidixic acid and gentamicin were selected and confirmed
by PCR. The hupL mutant of MSR-1 was named L206
For construction of a hyaB deletion mutant on the basis of
L206, two pairs of primers were designed to amplify the 3′
region and 5′ region of the gene: pB1, 5′-CGGATCCGGCC
A kanamycin resistance cassette of the plasmid pUC4k
was excised with Pst I and purified. The three fragments
were ligated into pSUP202 which was excised with BamH I
and Hind III, yielding suicide plasmid pSUPB. pSUPB was
introduced into E. coli S17-1 by transformation and then
into L206 via conjugation. Colonies which were sensitive to
chloromycetin and resistant to both nalidixic acid and
kanamycin were selected and confirmed by PCR. This
hupL/hyaB double mutant of MSR-1 was named B206

In vivo hydrogen production assay

Hydrogen production assay of bacterial strains was performed in shaking flasks. The three strains were grown in 2
L gas-tight vials, filled with 1.5 L LAY medium or ferric

Table 1 Strains and plasmids used in this work
Strains and plasmids
E. coli DH5α
E. coli S17-1
M. gryphiswaldense MSR-1
M. gryphiswaldense hupL206
M. gryphiswaldense hyaB206


Source or reference

Cloning strain
Mobilizing strain
Wild type
Δ hupL::aaaC1, NxrGmr
Δ hyaB::KanΔ hupL::aaaC1, NxrGmrKmr

Our lab collection
Our lab collection
Our lab collection
This work
This work

TcrAmprCmr, ColE1ori, Mob+
Containing a 0.85-kb Gm resistance cassette, AprGmr
Containing a 0.81-kb Km resistance cassette, AprKmr
pSUP202 containing hupL 5′ and 3′ flank fragment and Gm fragment, AprCmrGmr
pSUP202 containing hyaB 5′ and 3′ flank fragments and Km fragment, ApmrKmr

Our lab collection
Our lab collection
Our lab collection
This work
This work

BAN Jia, et al.

Chinese Sci Bull

citrate-free LAY medium, for 24 h. A 100 μL sample of gas
in each vial was measured using a Beifen SE-206 gas chromatograph (2 m 5 Å molecular sieve column, carrier gas
argon, column temperature 60°C, heater temperature 150°C,
detector type TCD). Under these conditions, the area value of
100 μL hydrogen was 448546. Hydrogen yield of each strain
was calculated as milliliter per liter broth, A600 = 1.0.

In vivo hydrogen uptake capability assay

The medium 100 mL was incubated in a 250-mL vial
plugged by a rubber stopper, with inoculum concentration
10%. Hydrogen was injected into the vial to a concentration
of 55% of gas phase in the headspace (v/v). Samples of gas
in the vial were taken regularly, to assay relative hydrogen
concentration of the gas phase, and record the reduced value
of hydrogen.

Determination of Fe3+ concentration

The method was described previously [14]. Samples of the
broth were taken regularly, and centrifuged to assay Fe3+
concentration of the supernatant. Iron uptake capability was
determined by the reduced value of Fe3+ in the supernatant.

Transmission electron microscopy

Cells were centrifuged, and washed twice with phosphatebuffered saline, and the bacterial suspension was added to
an electron microscopy copper grid. Water was soaked up
by filter paper, the grid was washed twice with water,
air-dried again, and then observed by transmission electron
microscopy (Hitachi H-7500).

2 Results
2.1 Identification and sequence analysis of the hup
gene cluster and hya gene cluster
Inspection of the working draft sequence of M. gryphiswaldense led to identification of two gene clusters with
similarity to hydrogenase-related genes (Figure 1). One is

Figure 1

May (2010) Vol.55 No.13


the hup gene cluster (nt1809278−1822888), which contains
14 ORFs. This cluster starts upstream of the hydrogenase
structural genes hupSL, belonging to the group of membrane-bound respiratory hydrogenases which perform respiratory hydrogen oxidation linked to quinone reduction.
These enzymes link oxidation of H2 to reduction of electron
acceptors, with recovery of energy in the form of a proton-motive force. The hypothetical HupS protein displays
extensive (77%) sequence similarity to M. magnetotacticum
MS-1 and M. magneticum AMB-1 HupS over its whole
length. The sequence similarity of HupL protein to M.
magnetotacticum MS-1 and M. magneticum AMB-1 HupL
is 84%. An Fe-S-cluster-containing hydrogenase component
gene hybA, showing 59% and 57% sequence similarity to M.
magnetotacticum MS-1 and M. magneticum AMB-1 uptake
NiFeSe-hydrogenase, and a cytochrome component of uptake NiFeSe-hydrogenase gene hybB showing 59% and
51% sequence similarity to M. magneticum AMB-1 and
Dechloromonas aromatica RCB uptake NiFeSe-hydrogenase cytochrome component, are between hupSL. Adjacent to the structural genes, one hydrogenase activity regulatory factor (hoxX), 2 hydrogenase accessory genes (hypA,
hypB), 2 hydrogenase maturation genes (hypF, hyaD), and 5
hydrogenase expression/formation genes (hypC1, hypC2,
hypD, hypE, hypK) were found downstream from hupL.
The other is the hya gene cluster (nt3760338–3765023),
which contains 6 genes, as follows. hycA corresponds to
4Fe-4S ferredoxin, the physiological electron acceptor of
the class I hydrogenases. crpA belongs to the CAP family of
transcription factors, which bind cAMP to activate transcription of hya gene cluster. fnrA was identified as cytochrome-c3 hydrogenase subunit gamma. The next two,
hyaA and hyaB, are soluble H2 producing NiFe-hydrogenase structural genes. The hypothetical HyaA protein
shows 66% and 64% sequence similarity to Azotobacter
vinelandii AvOP and Nitrococcus mobilis Nb-231 NiFehydrogenase. HyaB protein shows 54% sequence similarity
to Cyanothece sp. PCC 7425 and Thioalkalivibrio sp. HLEbGR7 NiFe-hydrogenase. The last gene, iclR, belongs to
the IclR family, suggested to repress transcription of the
Hya gene cluster.

Putative hydorgenase-related genes in M. gryphiswaldense MSR-1. (a) Putative uptaken hydrogenase genes; (b) putative hydrogenase genes.


BAN Jia, et al.

Chinese Sci Bull

May (2010) Vol.55 No.13

2.2 Molecular characterization of the hupL deletion
mutant, and hupL& hyaB double deletion mutant

2.3 In vivo hydrogen uptake capability assay and hydrogen production assay

Screened NxrGmrCms colonies were selected and confirmed
by PCR (Figure 2). Two pairs of primers were designed to
verify the double crossover mutant: PA (5′-CCAATGCCG
GGTGGCGTGCATCTC-3′). PA was located outside the
hupL 5′ flank, and PB was located outside the hupL 3′ flank.
A 2.8-kb fragment was amplified from genomic DNA of
wild-type (WT) which had the whole hupL gene with PA
and PB, but a 1.5-kb from the hupL deletion mutant whose
hupL gene was replaced by a Gm resistance cassette. By
contrast, PC lay in the Gm resistance cassette, and PD was
located outside the hupL 3′ flank. A 1.3-kb fragment was
amplified from genomic DNA of the mutant with PC and PD,
but nothing from WT. This result confirmed that the Gm
resistance cassette really exchanged with the hupL gene.
The verification steps of B206 were similar to those of
L206. The verified primers were PE (5′-TCTGCAACAG
-3′), PG (5′-CTCTCTACAAAGTTGGGC-3′), and PH (5′-C
TTACGAAAAAGCCGGCC-3′). Results indicated that a
3.1-kb fragment was amplified from genomic DNA of WT
with PE and PF, but none with PG and PH. A 1.3-kb fragment
was amplified from genomic DNA of B206 with PE and PF,
and a 1.1-kb fragment with PG and PH.

The above analysis indicated that hupSL are the structural
genes of the H2 uptake NiFe-hydrogenase, and hyaA/hyaB are
the H2 producing NiFe-hydrogenase structural genes. By
constructing L206 and B206, we determined the hydrogen
uptake capability and hydrogen yield among the three strains,
to verify the function of the two Ni, Fe-hydrogenases
(Figure 3).
MSR-1 (WT) showed rapid and constant absorption of
hydrogen in the growth process, during which hydrogen
concentration dropped from 55% to 15%. Thus, WT has
strong hydrogen uptake capability. By contrast, L206 showed
almost no drop in hydrogen concentration, and did not have
the capability to absorb hydrogen. These findings indicate
that hupL is the structural subunit of the uptake hydrogenase.
In the deletion mutation of hupL, the uptake hydrogenase
was inactivated in vivo. B206 showed properties similar to
those of L206. These findings indicate that the hydrogenase
composed from hyaA and hyaB has no hydrogen absorption
activity, and no significant influence on cellular hydrogen
uptake capability. It may be a specific H2 producing hydrogenase.
Hydrogen yields of 3 strains cultured in LAY medium or
ferric citrate-free LAY medium are shown in Table 2. WT
and B206 showed no hydrogen release under two culture
conditions, whereas L206 produced hydrogen. MSR-1 presumably released hydrogen, but this could not be detected
as a result of activity of uptake hydrogenase in vivo. The
results for B206 indicate that the hyaAB hydrogenase is a
specific H2 producing hydrogenase. L206 released a greater
quantity of H2 under conditions that induce magnetosome
synthesis, suggesting a relationship between these processes,
i.e., an accumulation of reducing power could occur in the
course of magnetosome synthesis, and an excessive amount
of reducing power in vivo could result in suppression of
magnetosome synthesis. Assuming that the purpose of H2
release in WT is to generate excessive reducing power to
promote magnetosome synthesis, one would expect to observe

Figure 2 Schematic to verify the double crossover mutants of L206 and
B206 (a), verification of L206(b) and Verification of L206(c). (b), lanes 1
and 2 show products of PA PB and PC PD using M. gryphiswaldense WT as
template; lanes 3 and 4 show products of PA PB and PC PD using L206 as
template. M, standard molecular weight. (c), lanes 1 and 2 show products
of PE PF and PG PH using M. gryphiswaldense WT as template; lanes 3 and
4 products of PE PF and PG PH using B206 as template. M, standard molecular weight.

Figure 3 Concentration of hydrogen uptaken by M. gryphiswaldens WT,
L206 and B206 by shake-flask culture.

BAN Jia, et al.

Table 2

Chinese Sci Bull


May (2010) Vol.55 No.13

Hydrogen yield of M. gryphiswaldense MSR-1 (WT) and deletion mutants with or without addition of Fe3+a)

Hydrogen yieldb) (mL)
LAY medium


Fe3+-free LAY medium

LAY medium

Fe3+-free LAY medium














a) The data are mean values of three independent experiments results; b) hydrogen yield (mL) H2 per liter broth; c) ND, not detectable. A600 = 1.0.

higher H2 production under the magnetosome synthesis
2.4 Shaking-flask culture, and iron uptake capability
Results from shaking-flask culture (Figure 4) indicate significant differences among the 3 strains under conditions
without vs. with addition of hydrogen. Gas volume ratios
under these 2 conditions were, respectively, oxygen/nitrogen 1:4, and hydrogen/oxygen/nitrogen 1:1:3. Under
no-hydrogen-added condition, L206 had the highest growth
rate, and went into logarithmic phase at 6 h, whereas WT
and B206 went into logarithmic phase at 8 h (Figure 4(a)).
Similar results were obtained under the hydrogen-added
condition (Figure 4(b)). The presence of hydrogen appeared
to have no effect on growth rate of the two deletion mutants,
but slowed down growth of WT. Therefore, growth of WT
under the two conditions was compared independently.

WT showed higher growth rate without hydrogen addition,
i.e., A600 after 24 h culture was 0.8173, compared to a value
of 0.6650 with hydrogen addition. These findings indicate
that hydrogen release and absorption in WT had a significant effect on cell growth rate.
To explore the influence of hydrogen metabolism on iron
uptake capability, we performed time course comparison
among the three strains under culture conditions without vs.
with hydrogen addition (Figure 5). Under both conditions,
L206 had the highest iron absorption rate, with peak absorption dose values being 19.59 μm without hydrogen, and
13.75 μm with hydrogen. Rates and dose values for WT and
B206 were similar to each other, and less than those of
L206. WT showed peak dose values of 14.79 μm without
hydrogen and 12.47 μm with hydrogen. These findings
suggest that iron absorption (iron uptake capability) and cell
growth are both suppressed by hydrogen absorption, and
promoted by hydrogen release.

Figure 4 Growth curves of M. gryphiswaldens WT, L206&B206 by shake-flask culture under no hydrogen added (a), under hydrogen added (b), growth
curves of M. gryphiswaldens WT under hydrogen added and no hydrogen added (c).

Figure 5 Time course comparison the iron uptake capabilities between M. gryphiswaldens WT, L206&B206 under no hydrogen added (a) and under hydrogen added (b).


BAN Jia, et al.

Chinese Sci Bull

2.5 Transmission electron microscopy (TEM) of magnetosomes
Cells were sampled over time to observe progression of
magnetosome synthesis (Figure 6). For L206, scattered

May (2010) Vol.55 No.13

magnetosomes appeared after 8 h culture, and orderly chains
of magnetosomes appeared after 12 h. By contrast, for WT
and B206, few magnetosomes were visible after 8 h; after
12 h, the number of magnetosomes was higher but no
chains could be observed. The faster rate of magnetosome
synthesis in L206 than in WT and
B206 suggests that deletion of uptake
hydrogenase promoted magnetosome
synthesis, which is consistent with
the difference in iron absorption for
the three strains.

3 Discussion

Figure 6 TEM observation cells of M. gryphiswaldens WT, L206 and B206. 4-h culture: A1, MSR-1; B1,
L206; C1, B206. 8-h culture: A2, MSR-1; B2, L206; C2, B206. 12-h culture: A3, MSR-1; B3, L206; C3,
B206. 24-h culture: A4, MSR-1; B4, L206; C4, B206. The bars show 500 nm.

In order to clarify relationships between hydrogen metabolism, cell
growth, and magnetosome synthesis
in Magnetospirillum gryphiswaldense
strain MSR-1, we successfully constructed hupL deletion mutant L206
and hupL/hyaB double deletion mutant B206. By comparing hydrogen
uptake capability and hydrogen
yield among the 3 strains, we concluded that HupSL protein is a specific uptake hydrogenase, HyaAB
protein is a specific H2 producing
hydrogenase, and they have correlated effects on the balance of hydrogen metabolism in MSR-1.
Experiments on the relationship
between hydrogen metabolism and
magnetosome synthesis in the 3
strains showed that deletion of the
uptake hydrogenase accelerated
rates of cell growth and iron absorption, increased the iron absorption
dose, and promoted formation of
magnetosomes into orderly chains.
By contrast, deletion of the H2 producing hydrogenase resulted in
suppression of cellular hydrogen
release, cell growth, iron absorption,
and magnetosome synthesis.
Hydrogen metabolism in microorganisms is closely related to intracellular energy metabolism and
degree of “reducing power”. The
absence of the cytochrome system
and the oxidative phosphorylation
mechanism in anaerobic Clostridium spp made the formation of
NADH faster than its oxidation, so
that cells spent the excessive reduc-

BAN Jia, et al.

Chinese Sci Bull

ing power through hydrogen release by hydrogenases
[15,16]. Rhizobium and Rhodospirillum rubrum gave off
considerable hydrogen gas during the nitrogen fixation
process, and recycled the gas via uptake hydrogenases to
supply energy and reducing power to nitrogenases [7,17].
Our previous studies demonstrated that MSR-1 is a typical microaerobic bacterium, and needs to control oxygen
partial pressure in the process of magnetosome synthesis.
Reducing power is significantly increased during magnetosome synthesis; in cells cultured in medium with iron concentration 30 μmol/L, reducing power was 20% higher than
in medium containing only trace iron. Cells spent the excessive reducing power via polyhydroxybutyrate (PHB) synthesis and hydrogen release [8]. In the present study, we
hypothesize that: (i) L206 cells, which lack hydrogen absorption capacity, can consume redundant reducing power
quickly to promote magnetosome synthesis; (ii) WT cells
recycle hydrogen released in the growth process via uptake
hydrogenases, while B206 cells lack the ability to consume
reducing power because of deletion of H2 producing hydrogenase, such that both of them are unable to release reducing power over time, resulting in suppression of magnetosome synthesis.
In addition to genes directly involved in magnetosome
synthesis, other intracellular enzymes clearly affect levels of
reducing power and iron absorption in vivo. H2-uptake hydrogenase and H2-producing hydrogenase both appear to be
involved in the regulation process.
Recent in-depth studies on the magnetosome function
have shown that these important cell organelles may be involved in energy storage, oxidation-reduction cycle, and
iron storage, in addition to their role as “magnetic micro-needles” that give magnetotactic bacteria their capacity
for magnetotaxis and aerotaxis. Spring et al. [18] suggested
the existence of a second respiratory chain located in the
magnetosome membrane, such that magnetosome synthesis
is coupled with simultaneous Fe(II) oxidation and transfer
of electrons to oxygen to generate ATP via proton concentration gradient. Our present results confirm the significant
effect of hydrogen metabolism on cell growth and iron uptake capability. Microbial cells may regulate the release rate
of reducing power in vivo through hydrogen absorption and
production to control magnetosome synthesis; i.e., magnetosome synthesis represents an accumulation of reducing
power from the perspective of hydrogen metabolism. This
concept is consistent with the connection between magnetosome synthesis and oxidation-reduction cycle found in
previous studies.
This work waks supported by the National Natural Science Foundation of

May (2010) Vol.55 No.13


China (Grant Nos. 30570023 and 30870043) and National High Technology Research and Development Program of China (Grant No.














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