Regulation of competence development in Bacillus subtilis

BIOCHEMICAL

Vol. 41, No. 4, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REGULATION OF COMPETENCE DEVELOPMENT IN BACILLUS Ira

C. Felknerl

and Orville

Department The University Austin,

Received

October

SUBTILIS

Wyss

of Microbiology, of Texas at Austin, Texas 78712

9, 1970

Summary: The number of cells in a population of Bacillus subtilis which become cornpetent can be either enhanced or reduced by several amino acids which includes arainine, histidine or lysine. Which effect is observed, depends upon the time of addition and the concentration. Inhibitors of protein synthesis block competence development at low concentrations during the early stages but become less effective when cells are committed to competence. These results indicate synthesis --de nwo protein occurs during competence development and that it is subject to regulation. Monofluoroacetate, which specifically inhibits aconitase activity also blocks competence development during these early stages. Since it prevents sporulation but has no effect some relationship between competence and sporulation through on vegetative grcwth, the tricarboxylic acid cycle seems to exist. It

has been reported

development

of competence

subtilis. first

Arginine,

in the

depend

on an intact

tive report

(21),

iments

suggest

fully

as regulators

acid

(TCA) cycle

sporulation

acid

27) are

(9,

11).

asporogenous

regulation,

were

mutants

and isocitric

occur

The dehydro-

growth

does not

without

established

on the

in Bacillus

among these.

are

the TCA cycle,

effect

7, 27)

Vegetative

cannot

,and competence

(2,

included

of aconitase

sporulation

transformable

how amino

(7,

whereas

have a positive

and transformation

act

TCA cycle,

acids

it.

Posi-

in an earlier

known

(20).

sporulation

Our exper-

and competence

related.

MATERIALS

AND METHODSi

W R. Romig

(University

and 569 ery-r method

were

of Marmur

used were 1

also

between but

transfection

and histidine

tricarboxylic

correlations

some of the amino

for

lysine

two and glutamate

genase

are

that

Present 79409.

Brain

Bacterial of California

described (15)

or that

Heart

Infusion

address:

strains.

Department

B. subtilis

at Los Angeles).

previously

(6).

of McDonald (BHI)

--et.

901

-B. subtilis

DNA isolation. al.

and Nutrient

of Biology,

168 - ind was obtained

Texas

(16). Broth.

569,

569 str-r

DNA was isolated Media. Spore

Tech University,

Commercial stocks

from

were

Lubbock,

by the media mainTexas

Vol. 41, No. 4, 1970

tained

on potato

2g (NH4>2S04,

BIOCHEMICAL

extract

medium

14g K2HP04,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(25).

Minimal lg sodium

6g KH2P04,

ter

of deionized,

distilled

M-l

medium was MM supplemented

water.

Minimal

with

tion

8-fold

All

solid

Two procedures

were

used

bation,

unless

ken for

16 hr at 37 C and 0.1

medium.

otherwise

After

resuspended

4 hr

an OD)boO of 0.22 and 0.1

amino

at ODj600

acid,

and ery-r

by first lecting

RESULTS AND DISCUSSION:

5 &g/ml

tryptophan

tence ways

was reached

was often

2

of the

assessed

higher

a for

were

than

it

Dihydrostreptomycin

after

added

was when they shorter

150 min incubation

was 1 mg/ml

test

in stage

amino

were than

and erythromycin

DNA at a concen-

with

curve). HA type)

plus

were

amino regimen

acid

are

regimen

3.

was lpglml.

27).

se-

scored. acids b,

only

present.

b, peak compe-

frequency (7,

the

with

2 of regimen

omitted

in

To insure

of individual Hmever,

the

in stage

902

of 0.22.

(Millipore,

The transformation

3.

and resuspended

hr transformants

plus

until

16 hr in M-2

them to BHI agar

growth.

MS and

and 200&g/ml

with

filters

the effects

by the mutant)

peak was considerably

reached

24-48

with

on a saturation

on membrane

determine

regimen

or histidine

incubated

transferring for

90 to 100 min in stage

several-fold

duration

using

always

washed

washed

maximum transformation

incubation

(required

lysine

were

sha-

10 ml of ~-1

was for

to an OD >600

incu.

in BHI were

tryptophan

4 hr,

a, incubated

and then

One cannot

development

When arginine,

were

for

All

was continued

growth

to MM, 5.0pg/ml

cells

gives

on BHI agar

b, initial

cultures.

were

shaking

was

Transforma-

to inoculate

cells

with

was shaken

regimen

transformants

After

shaking,

tryptophan

agar.

a, cells

used

Incubation

was added

like

In regimen

li-

hydrolysate

except

competent

per

0.5% glucose.

0.05% casein

growing

contained

MgS04'7H20

had 1.5% Difco

suspension

For regimen

competent

antibiotic.2

on competence

of 0.10.

(o.lpg/ml

incubating

for

(22)

(MM) was MS plus

media

vigorous

The culture

of O.lOand

of lOpg/ml

Str-r

with

suspension

maximum transformation, tration

ml of this

incubation

0.2g

tryptophan,

was at 37 C.

was reached.

ml of this

of a single M2

stated,

in M-2 at ODhoo

citrate*2H20,

Mr2. medium was identical

and CH by 5-fold.

procedures.

(MS) of Spizizen

medium

40 pgfml

(CH) and 10W4M FeS04'(NH4)2S04.6H20. reduced

salts

was al-

However,

a and a second

the peak

Vol. 41, No. 4, 1970

Fig.

1.

Figure

strain

competence),

tion ences

with

Amino stage

acids

2 of regimen

formation

is

Although

used

competence

efficiency

pattern

for

instead

of competence

lysine

and histidine

were

in the number

gration

typical

569.

arginine

and ery-r

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Onset and loss of competence by J. subtilis strain 569 cells using lysine in the second stage of regimen b. Symbols: m , str-r marker; m , ery-r marker. Abscissa values represent the amount of time elapsed in the final stage of regimen b before the cells were challenged with transf rming DNA. Each sample plated contained about 2 x 10 8 recipient cells.

1 shows the

B. subtilis

str-r

BIOCHEMICAL

was the amino

give

markers

about

to insure

for

acid

the association in marker

these

markers

by regimen

added

the same pattern

of time variations

of transformants

development

during

stage

and maximum.

2 (preBoth

of peak transforma-

integration

could,

b for

(5).

however,

Differ-

reflect

inte-

(13). which b will

at 90 min.

can increase reduce

the level competence

The extent

of competence during

of reduction

903

stage

when present

during

3 when assessment

is dependent

upon

the

time

of transand con-

BIOCHEMICAL

Vol. 4 1, No. 4, 1970

centration

of amino

arginine

is added

formation

is

acid

at various

obviously

30 min no significant mitted

This

enzyme repression formants. sible tinued

(14,

Since

these

end product

is

is

are

observed.

the response

metabolic removed

3 relative

reduced

no longer

26) were

2 shows transformation

in stage

more severely

they is

Figure

times

effect

to competence

be blocked.

added.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

causing control

through

to the control

when addition This

if

is

indicates

affected,

expected

when ZOOpg/ml

but either

reduction

in

mechanisms

metabolism

those

system.

prior

that

once cells

in earlier

com-

stages

the number

of potential

can be reversed

After

are

inhibition

should

Trans-

to 4 min.

allosteric

competence

of L-

could

(12)

when

or

transthe respon-

develop

upon

con-

incubation.

Fig.

Cellular repressible. of B. subtilis

2.

Relative transforming activity of g. subtilis 168 @ grown through regimen a for competence and "pulsed" with arginine at various intervals of the final stage. All samples were assessed for transformation after 90 min growth in the final step. Str-r and cry-r frequencies were averaged wer several experiments in which the control transformations were between 2 and 4 x 10'4 transformants/ml.

systems Although

under

the above regulation

chloramphenical

competence

factor

(CFbs)

should

(CAP) has been by Akrigg 904

et.

be either shown al.

inducible

to inhibit (l),

precise

or de-

production sensitivity

Vol. 41, No. 4, 1970

at the onset the action

of competence

CAP will

inhibit

growth,

whereas

viability

of i.

as 0.13

ug/ml

regimen

b.

ing

when

(14),

(17,

Table

is

initiated.

will

stop

23).

Table

transformation

minutes

later,

results

synthesis,

1. The effect the final level

induction

total

when varying

without protein

50-fold

12.5 pg/ml

were

of protein

synthesis with

concentrations

of chloramphenicol of transformation 168 -'ind

only

total

of

protein

without

or

affecting of CAP as low

at 0 min in stage

a 5-fold

reduction.

is more sensitive 7-Azatryptophan, were

should

(1.4pglml)

concentrations

when added

caused

obtained

by wercoming inhibitors

affecting synthesis

1 shows that

about

occurs

Low concentrations (18)

if --de novo protein

Similar

If

low concentrations

enzyme synthesis

subtilis

can be explained

of protein

then

10 to 20 pg/ml

Thirty

established,

the process

inducible

reduced

initiation.

bitor

was not

of a repressor

be more effective

result

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

during

and 7-azatryptophan of Bacillus subtilis

This

to CAP duralso

added

3 of

an inhistage

3.

on

1

I-

I

CBLOl tMPHENICOL* Concentration

0 min

(udml

*

7-AZATRYPTOPHAN* 30 min

Concentration

% of Control

mM

0 min

15 min

30 min

% of Control

50

0,015

0.25

2.0

0.04

0.15

0.43

12.5

0.012

0.20

1.0

0.045

0.68

0.67

5.0

0.015

0.25

0.5

0.05

0.60

0.73

1.3

0.020

0.40

0.2

0.15

0.82

0.83

0.13

0.020

0.50

0.1

0.40

0.97

0.93

None

1.00

1.00

None

1.00

1.00

1.00

These inhibitors of protein synthesis were added,.to cells up to 30 min during stage 3 of regimen b. Since all values after 30 min were around lOO%, they were not included in able 1. Control frequencies for ery-r and str-r were between 2 and 4 x 10' 6 , 905

Vol. 41, No. 4,197O

Transformation

BIOCHEMICAL

was reduced

to above 40% by 30 min. 0 min.

This

indicates

sential

for

competence

Inhibition tion

of cells

puromycin cule lished

would

of both

support must

11).

aconitase

TCA cycle.

We have

Table

(CFdp)

function

evidence

(Ribble,

should

(Ribble,

is

C. Felkner,

Min

of Monofluoroacetate

% of Control

Transforming

other 10,

linked

and low

with

poor

upon

the data)

on

(mM) 0.1

Activity

0.8

0.6

1.5

3.0

20

1.1

2.0

4.5

10.2

30

4.3

10.0

8.6

35.0

40

16.7

60.0

33.0

100.0

50

43.8

81.0

*100.0

93.8

906

and

unpublished

of magnitude

CFbs.

ll),

dependent

0.5

transformation was in the same order population but was slightly higher.

unpubthan

10

The percent the control

and

of a mole-

concentrations strain 168

1.0

interac-

to fluorocitrate

processes

2. Time-effect of varying Monofluoroacetate development of competence by Bacillus subtilis in M-2 broth. -ind growing

5

(9,

es-

3, 8).

C. Felkner,

molecule

converted

R. J. and I.

Concentration

and I.

have been

inhibit

the

induction

can occur

dehydrogenase

it

for

R. J.

is

of competence

protein

monofluoroacetate (4),

needed

at

be CFbs (1, during

acquisition

sporulation

and isocitric

activity

blocks

(24)

and

significant synthesis

could

pneumoniae

of a critical

before

was only

protein

synthesis

experiments

the idea

Since

Growth in M-2 Broth

*

CAP in ;.

factor

to 15% at 15 min,

upon new protein

One possible

with

Recent

aconitase

(10,

inhibits

synthesis

at 0 min,

inhibition

dependent

development.

CFdp.

The TCA cycle

sporulation

a process

some --de novo protein

than

data)

levels

When 0.2mM was added,

and competence

inhibits

other

to 4% when 2mM was added

that

of protein

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as

Vol. 41, No. 4, 1970

that

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

l.OmM MFA prevents

out affecting during

the appearance

vegetative

stage

growth.

3 of regimen

Since

3.

Inhibition

preincubation

mainstream ing

citrate

blocking

for

aconitase

in 2.

show that es is

with

for

subtilis.

TCA cycle

Experiments

sporulation

has this

even more significant processes.

tein

is not

established,

are

affected.

This

Although

the data relationship

link

reported is

currently

2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15.

3.

This

link

Completely

being

Since

development in the

literature

between

these

for

normal

is unnecessary the TCA cycle

defines

Withhold-

of com-

processvegeta-

and --de novo pro-

the period

investigated

MFA is

at which for

both

both sporu-

and transformation.

LITERATURE

1.

for

and others

between here

the metabolic

growth.

must be necessary

the TCA cycle

of O.lmM to 5.OmM.

MFA to enter

vegetative

same requirement.

the precise

a range

on transformation.

affect

to MFA

to 30 min incubation

10 min of stage

in progress

since

prior

allows

effect

induction

with-

2.

within

the first

MFA does not

an intact

tive

lation

of citrate

up to 20 min had little

specific,

petence

dependent

was withheld

transformation

in Table

of transformation

is concentration

citrate

of competence

reported

inhibitor

in the absence

(4),

enzymes and sporulation

The sensitivity

a and b is

MFA is a more effective in stage

of proteolytic

Akrigg,

CITED

A.,

S. R. Ayad and G. R. Barker, Biochem. Biophys. Res. Ccmmun., 1062 (1967) Bott,?. F., and G. A. Wilson, J. Bacterial., 94: 562 (1967) Charpak, M., and R. Dedoner, Comet. rend., 26Or 5638 (1965) Elsden, S. R. and J. G. Ormerod, Biochem. Jy63: 691 (1956) Erickson, R. and W. Braun, Bacterial. Proc., 60: (1968) Felkner, I. C., and 0. Wyss, Biochem. BiophysyRes. Commun., 32: 44 (1968) Felkner, I. C., and 0. Wyss, Fed. Proc., 24: 468 (1965) Felkner, I. C., and 0. Wyss, Biochem. Biophys. Res. Commun., 16: 94 (1964) Fortnagel P., and E. Freese, J. Bacterial., 95: 1431 (1968) Hanson, R. S., J. Blicharska and J. Szulmajs~r, Biochem. Biophys. Res. Commun., 17: 1 (1964) Hanson, R. S., 7. Blicharska, M. Arnaud and J. Szulmajster, Biochem. Biophys. Res. Commun., 17: 690 (1964) Jacob, F. and J. Mold, J. Mol. Biol., 3: 318 (1961) Lacks, S., Genetics, 53: 207 (1966) Maas, W. K. and E. McEll, Ann. Rev. Microbial., 18: 95 (1964) Marmur, J., J. Mol. Biol., 2: 208 (1961) 28:

907

Vol. 41, No. 4, 1970

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

McDonald, W. C., I. C. Felkner, A. Turetsky and T. S. Matney, J. Bacterial., 84: 1071 (1963) Nester, E. W., J. Bacterial., z: 867 (1964) Paigen, K., Biochem. et. Biophys. Acta., 77: 318 (1963) Rebello, J. L. and N. Strauss., J. Bacterzl., 98: 683 (1969) Schaeffer, P., and H. Ionesco, C. R. Acad. Sci.249: 481 (1959) Spizizen, J., B. Reilly and B. Dahl, Proc. of Intern. Congr. Genetics. llth, The Hague, 1: 31 (1963) Spizizen, J., Proc. Natl. Acad. Sci. U. S., 44: 1072 (1958) 89: 288 (1965) Strauss, N., J. Bacterial., Tomasz, A., J. Bacterial., m: 860 (1970) Thorne, C. B., J. Bacterial., 83: 106 (1962) Vogel, R. H. and H. J. Vogel, zochem. Biophys. Res. Commun., 18: 768 (1965) Wilson, G. A. and K. F. Bott, J. Bacterial., 95: 1439 (1968) -

908