Vol. 137, No. 1, 1986
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
May 29, 1986
Pages
CONJUGAL
108-113
TRANSFER OF HYDROGEN-OXIDIZING ABILITY OF ALCALIGENES HYDROGENOPHILUS TO PSEUDOMONAS OXALATICUS
F. Umeda,
H.
Department of Sciences,
M. Urushihara,
Min,
M. Okazaki,*
Biochemical Engineering, Osaka University, Suita,
Faculty Osaka
and Y. Miura of Pharmaceutical 565, Japan
Received March 27, 1986 Conjugal transfer of hydrogen-oxidizing ability (Hex) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined. Intraspecific cross of plasmid pHG21-a that encodes hvdrogenases that mediate hydrogen oxidation was most frequent at 25-C; the optimal temperature for growth was 30 C. The plasmid could be transferred from fl. hydrogenophilus to Pseudomonas oxalaticus OX1 and 0X4, and the resulting strains gained the capacity for autotrophic growth with H2 and CO . Plasmid pHG21-a was maintained in P. oxalaticus OX1 an 3 OX4 as stably as in 3. hydrogenophilus. 0 1986 Academic Press, Inc.
Alcaligenes
hydrogenophilus
chemolithoautotrophic energy
source
hydrogen other
(2).
Cells
ribulose key
from
is
as the
soil
oxidation.
the
(1).
It
One is
of
on
plasmid
transmissible bacteria
(2). (2).
In
*Present address: Faculty of Textile Ueda 386, Japan.
and
Hox plasmid study,
0 I986
Inc. reserved.
with
This
108
that
mediate and
hydrogenase and
CO2
fixation.
have a In
hydrogenases Hox
plasmid
can be exchanged we transferred
organism
EC 4.1.1.39)(2),
dioxide
this
as an
hydrogenase
H2
membrane-bound and
H2
blue-reducing
Department of Applied Science and Technology,
by Academic Press, in any form
of reproduction
with
hydrogenases
(RuBPCase,
0006-291X/86 $1.50 Copyright All r&h&
source.
grown
carbon
pHG21-a,
this
carbon
facultative
NAD+-reducing
carboxylase
soluble
grows
methylene
autotrophic
hydrogenophilus,
which
has two
soluble
membrane-bound
bisphosphate
encoded
sole
of A. hydrogenophilus
enzyme
a gram-negative,
bacterium, and CO2
was isolated
is
Hox plasmid
Biological Shinshu
are
is
between
A.
self-
hydrogen of
Science, University,
A.
Vol.
137,
No.
BIOCHEMICAL
1, 1986
hydrogenophilus bacteria
to
and
AND
C02-fixing
cultivated
BIOPHYSICAL
bacteria
these
as
bacteria
MATERIALS
RESEARCH
well
with
COMMUNICATIONS
as
to
hydrogen
H2 and CO2.
AND METHODS
Bacterial strains used in this study are Bacterial strains. mutant strain was isolated as a listed in Table 1. An auxotrophic spontaneous mutant by the penicillin-cycloserine method (3). Antibiotic-resistant mutant strains were isolated as spontaneous strain of A. A plasmid pHG21-a cured Hoxmutants. isolated after treatment with acridine hydrogenophilus was orange. Media and culture conditions. The modified L-broth used as the nutrient broth consisted of 1 liter of water, 10 q of tryptone (Difco Laboratories, Detroit, Mich.), 5 g of yeast extract (Difco), 5 g of NaCl, and 1 g of fructose (pH 7.2). Autotrophic growth was in minimal medium under a gas mixture of H2, ? and (7:2:1, 2' was vol/vol) (1). When necessary, amino aci CD2 supplemented at a final concentration of 50 ug/ml. Solid media contained 1.5% (wt/vol) agar. All cultures were incubated at 30 C. Conjugation was performed by mating on membrane Conjugation. filters in a modification of Murooka et aA. (4). Equal volumes of exponentially growing donors and recipients were mated overnight on a membrane filter in a nutrientagar plate.Bacteria were then suspended in 2 ml of 0.9% saline, and 0.1 ml samples of suitable dilutions were spread on the selective medium. Bacterial growth for the mating period was estimated from the ODeGO,in 2 ml of bacterial suspension after conjugation. For estimation of conjugal transfer of Hox plasmid pHG21-a, donors and Hex+ transconjugants were incubated autotrophically under H2, 02, and CO2 for 7 to 10 days. When strains resistant to streptomycin (Sm) were used as recipients, Sm was added to the final concentration of 500 or 1,000 ug/ml. The transfer frequency of the plasmid
Table Strain Alcaligenes
hydrogenophilus
Source
0X1-SR OXI-Hl
HoxHex-, Hex+,
Smr
0x4 0X4-Hl
HoxHex+
0X4-H2
Hex+
OX6 OX23
HoxHoxHex-,
oxalaticus
0X23-SR Hex,
ability
strains
Relevant phenotype Smr Trp-
0x1
Abbreviations:
Bacterial
Hex+ Hex-, Hex+,
1970
CH30SR MT105 Pseudomonas
1.
to oxidize
Wild-type (1) Mutant of 1978, Mutant of 1978,
this this
study study
Wild-type, NCIB 8642 Mutant of 0X1, this study Conjugant, A. hydrogenophilus MT105 x -p. oxalaticus 0X1-SR, this study Wild-type, NCIB 8543 Conjugant,&. h>drogenophilus MT105 x _p. oxalaticus 0X4, this study Conjugant, A. hJdrogenophilus MT105 x 1. oxalat&us 0X4, this study Wild-type, NCIB 8544 Wild-type, ATCC 11884 Mutant of 0X23, this study
Smr
Sm'
hydrogen; 109
Smr, resistance
to streptomycin
Vol. 137, No. 1, 1986
transfer cell in
BIOCHEMICAL
was expressed mating mixture
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
as the number of transconjugants at the end of conjugation.
per donor
Isolation of plasmid. Bacterial cultures for plasmid isolation were harvested at the end of the logarithmic growing phase nutrient broth. Crude lysates of plasmid DNA were prepared described by Yano e_t &. (51, who ~1 ightly modified the method Hansen and Olsen. RESULTS Influence
of
temperature ability
was
the
used and
37
recipient.
C overnight.
transconjugants temperature. optimum Hex+
conjugal with
pHG21-a
temperature
the
hydrogenophilus
intraspecific
growth
the
are
and
growth
than
at
at of
for
(Cl
Growth (OD660)
(Smr) was
30 C, which but
the
25, of
Hex+
at
each
was the number
Hox plasmids
30 C (6,7).
The optimum
hydrogen
pHG21-a bacteria.
on conjugal ability
Number of Hex+ transconjugants (c.f.u.. per filter)
20
2.4
2.1
x 106
25
7.7
1.4
x
108
30
11.8
7.8
x
lo7
37
8.7
1.3
x
104
A. hydrogenophilus 1978 was used as the donor, and A. hydrogenophilus CH30SRwas used as the recipient. Three filter matings were performed at each temperature Hex+ transand mean values were estimated. conjugants were selected under H2, 0 minimal medium plates containing Sm ?i 110
30,
2).
Table 2. Influence of temperature transfer of hydrogen-oxidiziing Temperature
as
examined
Hox plasmid
other
was used
at 20,
25 C (Table
transferred
A.
formation
on hydrogen, at
of
CH30SR
were
was best
conjugation
was lower
type
strain
period
of
cross
wild
Hex-
effect
hydrogen-oxidizing
was performed
was largest
bacteria for
cured
growth
of
1978
mating
for
transconjugants
temperature
an
Conjugation
Bacterial
of hydrogen
transfer
Bacterial for
The
conjugation.
-A. hydrogenophilus
and plasmid
as the
on
investigated
hydrogenophilus. the donor
AND DISCUSSION
temperature on
in as of
of
of
&.
Vol.
137,
No.
BIOCHEMICAL
1, 1986
Conjugal
transfer
hydrogenophilus transfer
to
of
OX1
metabolizes
of
pHG21-a
is
an
was
of
not
C02,
to
OX23
(8).
OX6 and
hydrogen P.
by
Calvin
the
of
to
OX1
cycle
donor
and
recipient.
(9). the
donor
donor
Hox plasmid
Smr
in
transferred
per
A.
Plasmid
per
and
P.
oxalaticue
10m2
10e7
and
conjugal
bacteria.
(8).
as the as
harbourinq
soluble
the
3.
A. hydroqenophilus
hydroqenophilus, of
from
the to
(Table pHGZ?-a,
membrane-bound
P. 3).
grown
hydrogenases
shown).
grow
other
-A.
OX1
Microorganisms often
used
a frequency
a frequency
had
to
used
was
at
P. oxalaticus and
as
was
OX1
of
at
from
CO2 fixation
(Trp-)
transferred
OX1
H2
the
COMMUNICATIONS
examined
oxalate-utilizer
via
cross
We
well
RESEARCH
ability
ability
P. oxalaticus
oxalaticus
(data
oxalaticus.
MT105
intraspecific
with
-P.
as
formate
hydrogenophilus
Cells
hydrogen-oxidizing
bacteria
oxalaticus
BIOPHYSICAL
of
hydrogen-oxidizing
C02-fixing
strain
AND
able
on oxalate
to
(9).
grow So,
oxalate-utilizers Plasmid OX23
we examined
such
pHG21-a
except
autotrophically
at
not
was
less
as
than
on the
conjugal
P. oxalaticx
per
0X6,
to
-P. oxalatjcus
donor,
but
Table 3. Conjugal transfer of hydrogen-oxidizing ability from 5. hydroqenophilus to P_. oxalaticus Recipient
Transfer frequency (Hex+ transconjuqants per donor)
-A. hydrogenophilus
CH30SR
1.7
x
10-2
p. oxalaticus
OXI-SR
3.8
x
1O-7
p. oxalaticus
OX4
3.3
x
10-7
_P. oxalaticus
OX6
<
10-10
_P. oxalaticus
0X23-SR
< 10-10
A. hydrogenophilus MT105 was used as the donor. Conjugation was performed at 25 C. Donor and Hex+ transconjuqants were selected for minimal medium ;l-;;;~pb~;~r ( Iis, ,4A,fnL~2. For donor wo;;Er; supplemented. Sm-resistant recipients were used, Sm was added at 500 ug/ml. 111
can
transfer
0X4,
transferred IO-lo
formate
it
and
was
Vol. 137, No. 1. 1986
BIOCHEMICAL
12
AND BIOPHYSICAL
3
4
5
6
7
RESEARCH COMMUNICATIONS
8
Figure 1. Agarose gel electrophoresis of plasmid DNAs from Hex+ and Hoxstrains of 4. hydrogenophilus and p. oxalaticus. Agarose gel electrophoresis was carried out on 0.7% (wt/vol) agarose in Tris-borate buffer (89 mM Tris, 89 mMboric acid, 2.5 mMEDTA, pH 8.5) at 100 V for 8 h. lane 1. A. hydrogenophilus 1978; lane 2. A. hydrogenophilus CH30SR; 0X1; lane 4. p. oxalaticus lane 3. E. oxalaticus 0X1-Hl; lane 5. A. hydrogenophilus 1978; lane 6. p. oxalaticus 0X4; lane 7. 2. oxalaticus 0X4-Hl; lane 8. p. oxalaticus 0X4-H2.
transferred (Table
to P. oxalaticus
of
10m7
per donor
3).
Plasmid from
Agarose
analysis. Hex+
oxalaticus
and is
two large
shown in
plasmids
plasmid (lane
had only
3),
plasmid
transconjugants plasmid plasmid
pHG21-a
(lane pHG21-a
pHG21-a
were
(lane
of
(lane
the.
6).
(2). strain
and harboured OX1
of
size
was plasmidOX1
OX4 harboured
an
between
of
that
Hex+ transconjugants Nine
both
plasmid
pHG21-a
other
two
transconjugants
analysis
a
P. oxalaticu2
types.
112
harbours
A_. hydrogenophilus
two
8). Plasmid
-P.
(270 Md) and pHG21-b
4). P. oxalaticus
(lane
DNAs and
1978
P. oxalaticus
a intermediate
contained 7) and
Hex-
transconjugant
Hex+
with
hydrogenophilus
et -- al.
2).
of plasmid
hydrogenophilus
cured
(lane
pHG21-a
OX4
-A.
-.A
1.
pHG21-a and pHG21-b
oxalaticus
of
by Friedrich
and the
plasmid
electrophoresis
1 and 5),
pHG21-b
plasmid
indigenous
Fig.
(lane
CH30SR was a plasmid cryptic
gel
strains
Hox-
(230 Md) as reported
free
OX4 at a frequency
out
of
of 11
P.
Hex+
and indigenous
showed that
had only all
HOX+
BIOCHEMICAL
Vol. 137, No. 1, 1986
transconjugants
AND BIOPHYSICAL
of P. oxalaticu-s
RESEARCH COMMUNICATIONS
and OX4 contained
OX1
plasmid
pHG21-a. Stability
of
oxalaticus
plasmid
in
each
plated
cultivated
on nutrient
nutrient
agar
Plasmid
plates.
plates
pHG21-a
hydrogenophilus ---..__--__ pHG21-a
in
were
and in
or
maintained
without in
-P.
broth
examined
97% of
for in
those
oxalaticus --I____
of
overnight
96% of
with
the
OX1 and
OX4 as
strain
on
H2 and CC2.
0X1.
of
A.
Plasmid
of P. oxalaticus
Hox plasmid
plasmid.
OX1
colonies
of -P. oxalaticus
A.
and were
of each
in 100% of the colonies indigenous
cultures
growth
-P.
plasmid
of P. oxalaficus
100 colonies
was maintained
was maintained
OX4 with
Then,
of
grown
nutrient
and in
stability
transconjugants
and Hex+
and OX4 were
the
Autotrophically
host.
hydrogenophilus
hydrogenophilus
in -A.
We examined
and 0X4.
OX1
pHG21-a
pHG21-a
stably
pHG21-a was as
in
A.
hydrogenophilus. CONCLUSIONS Hox plasmid to
2.
oxalaticus
expressed
pHG21-a was transferred OX1
and coupled
P. oxalaticus
OX1
and with
0X4.
from
The
originally
A. hydrogenophilus
hydrogenase existing
enzyme
genes
were
systems
in
and 0X4. REFERENCES
1.
Ohi,
K.,
Takada, N., Komemushi, S., Okazaki, M., and Miura, Y. J. Gen. Microbial. 2_5, 53-58 Friedrich, B., Friedrich, C. G., Meyer, M., and Schlegel, H. G. (1984) J. Bacterial. 12, 331-333 Ornston, L. N., Ornston, M. K., and Chou, G. (1969) Biochem. Biophys. Res. Commun. 36, 179-184 Murooka, Y., Takazawa, N., and Harada, T.(1981) J. Bacterial. 145, 358-368 Yano, K., and Nishi, T. (1980) J. Bacterial. 43, 552-560 Reh, M., and Schlegel, H. G. (1981) J. Gen.-3icrobiol. 126, 327-336 Behki, R. M., Selvaraj, G., and Lyer, V. N. (1983) Can. J. Microbial. 29, 767-774 Khambata, S<- R., and Bhat, J. V. (1953) J. Bacterial. 66, 505507 Friedrich, C. G., Bowien, B., and Friedrich, B. (1979) J. Gen. Microbial. II, 185-192 (1979)
2. 3. 4. 5. 6. 7. 8. 9.
113