Conjugal transfer of hydrogen-oxidizing ability of Alcaligeneshydrogenophilus to Pseudomonasoxalaticus

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