Fractionation of nucleic acids from dormant and germinated Azotobacter cysts

Vol. 35, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FRACTIONATION

OF NUCLEIC ACIDS FROM DORMANT AZOTOBACTER CYSTS* Karl

Department

Received

E.

of at

Olson

and

Microbiology, Austin, Austin,

Orville

AND GERMINATED

Wyss

The University Texas 78712

of

Texas

May 5, 1969

Summary. Chromatography of nucleic acid extracts of Azotobacter vinelandii cysts and of phased germinated cysts was performed using methylated albumin-kieselguhr(MAK) columns. After 4 hours germination, DNA and ribosomal RNA peaks are seen to elute at higher NaCl concentrations than those of dormant cysts. The elution profile of transfer RNA did not change during cyst germination. Kvidence is presented which suggests that shifts in elution profiles are due to changes in nucleic acid configuration during the process of germination. This evidence may indicate an additional mechanism for cyst survival in a deleterious environment. The

normal

encapsulated homogeneous (1961),

through over

termed

a cyst.

in

The

the

The

vegetative physical

Parker

and

Socolofsky

(1966)

central

Wyss,

body

investigation nation process. differences

Lin

the

and

nucleic

was

to

have of

1968),

monitor the

of the

is

the

*This investigation was supported grant ~1-02830-10 and training Institutes of Health.

acids

acids which of

713

results

been

are

Wyss,

well 1962). of

several 1967;

Olson

documented

purpose during

indicates

by Public Health grant GM 00600-09

a

properties

no

dormant

of

medium.

Wyss,

there is

cyst

have

various

The

by

the

and

and

there cyst.

replica

a growth

cysts

(Olson

nucleic

presented

nucleic

in

Although

cysts

transcell

surrounded of

on

cyst. of

is

resting

Socolofsky

reported

the

Sadoff,

cell

is

of

1961;

Socolofsky

spherical

8 hours

properties

acids

Evidence between

6 to

Wyss,

composition

19681 on

which

thick

a fairly

and

a spherical

Germination

after

resistant and

concerning

research

body,

and

Neumann

a compact,

structure.

long,

wall

vegetative

into

central

(Socolofsky

isolated

reports

and

the

5 days of

cell

a rather

cell

by Wyss,

consists

coat

is

a thin

process

4 to

cyst

cell

with

described

stage of

cell,

documented

and

a four

double-layered

a normal

the

As

a period

vegetative

dense

vegetative rod

cytoplasm.

formed the

Azotobacter gram-negative

of the

the cyst

present germi-

qualitative

and

germinating

Service from the

research National

cysts

Vol. 35, No. 5, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHODS Organism.

A.

studies

for

were

grown

1952)

at

obtained as

the

the

in

Burk's

nitrogen-free

33 C with

0.5%

by

at

growth

carbon

ATCC 12837, of vegetative

vinelandii, production

cells

Cysts

Germination.

Phased

pensions

was

using

methods

reported

by

obtained

broth

33 C for

placed

15 minutes.

on a rotary

intervals

was

filter. viable

diluent

as

of

24

per

9 to

10 days3

dormant

of

temperature

the

ml

1 C for

was

then

added

At

and

were

at

zero

cyst and in

the

and

were Smith

dilution

growth

followed

at

and

optical

with

no.42

Wyss

(1969),

with

Burk's

and

Burk's

was

half-hour

removed

method

on

sterile

culture

calorimeter of

Wyss

50 minutes and

susshift

Lin

placed

time

culture

the

hours

with

(1965)

report

by

Cysts were 0,2$ n-butanol

washed

held

the

the

made

after

of

a Klett-Summerson

with were

after

Knight,

18 hours.

33 C.

at

with

counts

harvested

cysts

and

samples

accordance

cell

broth

shaker

recorded

In

medium

Vedehina

Sucrose

thereafter,

density

107

sucrose

and

source.

agar

germination

1 x

without

(Wilson

carbon

a modification and

organism used in these and cysts. Cells

broth

the

at

Zaitseva

Approximately

(1965). Burk's

were

collected

Phased

by

as

33 C on Burk's

were

the cells

salts

sucrose

source.

vegetative

was

with

agar

0.576

sucrose. Nucleic

Acid

7.5 hours

and

4,6,

Isolation.

nucleic

acid

nation,

suspensions

collected

Total from

by

suspended

at were

coat central 0.15M

two

final were the

plus

shaking was

tion with dissolved

of

and

washed

buffer

(pH

frozen

Burk's

ensure

25mM

The

pellet

of

maximum the

and bodies Tris

15 minutes, by

washed were

tris(hydroxy-

1 mg per lysis

After

mild of

and

then carried

of of

the

sodium

lauryl

shaking,

lyoozyme. the

central

sulfate the

EDTA + in 40 ml

nucleic

body to

a

acids

equal

volumes

Gierer

and

Schramm

(1.956).

After

of 95% ethyl 5OmM sodium phosphate

alcohol, buffer

the

final

precipitate

was

(pH

6.3),

containing

O.lM

714

liquefied

ml of

cyst

released

twice in O.lM then suspended

with of

germi-

was

pellet

0,2, stop

medium,

rupture

complete

addition

2.0%.

salts

acid(EDTA)

to

0.2M

during

4 C in

7.4).

at To

cultures.

intervals

at

extracted

with

3 volumes in

cyst time

centrifugation,

4 ml

for

obtained

concentration method

over

was then resuspended pH 8.0. The central

buffer

extracted

germinating

cycles

Following

bodies NaCl at

suspension

were

5mM ethylenediaminetetraacetic freeze-thaw

mild

acids

various

poured

(Tris)

structure.

EDTA-NaCl After

the

centrifugation

in

through

phased

synthesis

methyl)aminomethane

nucleic

phenol according to the third precipita-

Vol. 35, No. 5, 1969

NaCl. of

In

25

MAK

all

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

experiments

optical

density

were

eluted

from 0.3 to 1.2M Twelve fractions tration man

units

columns(Sueoka

acids

approximately

and

at

and

Cheng,

under

mild

NaCl in of 3 ml

1 mg nucleic

255mp) 1962)

air

was at

applied

room

pressure

absorption

in

was

to

(a

2.0

manner

buffer each

measured

at

7.5

cm

Nucleic

a stepwise for

total

by

temperature.

50mM sodium phosphate each were collected

ultraviolet

acid

with

(pH 6.3). saline concen-

255rnr-I

in

a Beck-

DU spectrophotometer. RESULTS In

with iments The

order

to

regard

to

were

of

are by

an

viable

after

addition

viable sky

obtain

in

of

counts the

cell

counts. with

carbon

the The

observed in

microscope

earlier

to

a cyst

of

cells

at

cell

of

counts

6 and

9 hours

made

with

with

were

Wyss,

culture. as deter-

correlation

observations

observations

of

double

close

events exper-

described

number

Direct

were

cellular

germination,

previously

total

source.

chamber Phase

the

was

random cyst

germination

utilizing 1.

than

during

phased

Fig.

counting

rather

synthesis

experiment

cell

Petroff-Hauser agreement

to

presented

mined

sequential acid

designed

results

method

obtain

nucleic

in

Neumann

a

the

close

and

Socolof-

(1961).

J 5 s;

8.5

CELLS-o0.0.

6.0

i

r

o

Fig. were then Viable cell

-*-

2

4

6

8

IO HOURS

12

14

16

24

1. Phased germination of Azotobacter cysts. Cyst cultures phased by the temperature shift method: 1 C for 50 minutes 33 C for 15 minutes, followed by the addition of sucrose. and direct cell c.>unts reveale, a two-fold increase in numbers at 6 and 9 hours. Extracts

illustrated elution

in patterns

of

dormant

cysts

Fig. 2. (Midgley

Other and

had

a nucleic

bacterial McCarthy, 715

acid

r- ,stems 1962).

elution exhibit Peaks

pattern I,

similar III and

a IV

A 0.0

_

I DORMANT

//

CYSTS

1.0

/

2

g N

0.0

0.6

f 3

0.6

;

0.4

I

I

1

I

20

40

60

00

I

0.2

1

100

FRACTION

GERMINATED 4.0

CYSTS

HOURS

20

40

60

00

100

FRACTION C o.O

_

DOAMAN 1

CYSTS

--c

4 HAS. GERMINATION 2

0.6

---

/

.A

/

z

0

I 20

I 40

/ ”

I

-

pq

1

1.0

-0.0

z

’

I 00

I 100

0.2

FRA&ON Fig. 2. Elution patterns (from MAK columns) of nucleic acids extracted from dormant cysts (A) and 4 hour germinated cysts (B) Nucleic acids were determined by absorbance at 255 rnp. In these patterns, peak I represents sRNA; peak II, DNA; peak III, 16~ rRNAJ peak IV, 23s rRNA; and peak V, unidentified RNAJ 0, sodium chloride gradient. Peaks II, III and IV exhibited a shift in elution pattern after 4 hours germination as seen in graph C which is a composite of graphs A and B.

716

Vol. 35, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

contained

RRA and

minations

by

the

minations

by

either

1956)

(Keck, other

16s

Similar

elution

hour

intervals

each

interval

separate

the

and

This while

method

were

extracted

and

the

When it

was

peaks

concentration

is II,

and

results that than

III than

peaks the

in

Fig.

and IV elute the analogous

II,

III

analogous Peak

at found

least be

culture

elutes

at

in and

IV

1962). at

acids

to

and IV peaks I

shown III

extracted Nucleic

a 4 hour

2.

been

Cheng,

at approximately peak from dormant

A _

and

reaction

peaks

samples

were

from

DNA deter-

has

chromatographed

did

illustrated

It

germination.

acids

noticed

for

obtained

nucleic

concentrations finding

of

RNA deter-

Ceriotti

RNA and

(Sueoka

obtained

start

the

(1968). transfer

respectively

by

1957) and of

Burton

were the

revealed

modification

contains

RNA,

after

DNA as (Schneider,

of I

patterns

times

tographed,

Keck

peak

23s

reproducible, saline

contained method

the

that

contain

II

orcinol

or

systems

peak

2 from

three closely were

chroma-

eluted at higher 0 and 2 hours. at

0.6M

saline

O.lM higher saline cysts, Nucleic

B PEAK I

2b

40

8OC-25

MIN

20

4.0

0.8

FRACTION

Ic 0.6 -1

0 PEAK

m

n

SOC-

25MIN

_

E" z 0.4 N 2 p 0.2

0.8 0 z"

0

0

,0.6 80

100

80

100

FRACTION

Fig. 3. Co-chromatography (from MAK columns) of pooled fractions from analogous peaks of dormant cyst and 7.5 hour germinated cyst nucleic acid extracts, Peak I eluted at 0.6M NaCl before and after heating (A,B). Peak IV eluted as two distinct peaks prior to heating (C), but only one peak was detected after heating the pooled fractions (D).

717

Vol. 35, No. 5, 1969

acid

extracts

log-phase

from in

significant Fig.

2,

at

tative

cell

no

fifth

profiles

were

lowing

experiments

analogous arations

are

heated

at

these

a single and

80

C for was

this

in

and

peak

I

as

from

and

folthe

results

prep-

of

of

two

fractions As

results in

elution

a

distinct

differences

pooled

cysts

peak. in

cyst

Detection

chromatographed.

illustrated

vegethere

this

germinated

configurational

Identical

in

the

The

were

rewere

seen

then

in

obtained

germinated

in

first

containing

hour

Similar

25 minutes detected.

of

configuration,

3.

Fig.

that

seen

and

changes

fractions

no

RNA but

character that

When were

was

contain

chromatographed.

presented

manner,

IV, hours

7.5

and

patterns.

although

in

peak

7.5

to

and

As

after

in The

cysts

contention

elution

peak III,

behave

the

there

6 and and

differences

again

profiles.

hypothesis

performed.

and

experiment in

of

dormant

pooled

supported

sulted

II

from

were

typical

result

elution profiles.

found

nature

a working

were

peaks

at

V was

the

with

the

eluted

cultures

employed,

elution

appeared

concerning

conjunction

were

various

Peak

cyst

similar

which

also

preparations.

information In

the

peak, and

germinated

procedure in

4 hours

hour

exhibited

extraction

a small

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7.5

cells

the

differences

detected

peaks

6 and

4,

vegetative

variations

is

BIOCHEMICAL

cysts

3,

Fig. for

peaks

did

not

3.

Fig.

DISCUSSION The in

an

Azotobacter

Parker

and

The

cyst.

the

nucleic

of

the

central

period,

yet

of

nation various

cyst

can

coat

to

of

structure

is

properties

communication adapt

survive

studies

resistance

this

also

than

the

undergoes it

of

suggests

the

that

conditions

in

the

of

elute

4S

a

were

detected.

elution

a one from

does pooled After 718

not

6 to

dormant change and heating

cysts in

and

re-chromatographed, the

been cysts

DNA and

saline

elution

pooled

germi-

and

the

higher

form-

have

cysts

4 hours, step

The

this

8 hour

profiles dormant

nuclear

formation.

during

the

After at

peaks

RNA peak

during

the

cyst

compaction

from

germination. to

peaks were

MAR column extracte-d

analogous

The

size

(1961),

during

from in

acids seen

Socolofsky

compaction

increases

during

and

decreases

Changes

analogous peaks

Neumann

body

nucleic

times

cysts.

Wyss,

cell

RNA are

tration

distinct

the

from

ribosomal

When

by

process.

detected

nated

may

the

and

in

which The

environment. observed

material

at

that

survival

cyst

cell

growth.

indicate

the

the

resting

normal

presented

of

As

ative

for

for

evidence

acids

deleterious

a unique

(1966)

responsible

the

is

unsuitable

Socolofsky

primarily

size

cyst

environment

concen-

2 hour

germi-

pattern. two fractions,

Vol. 35, No. 5, 1969

only

BIOCHEMICAL

one peak was apparent.

time under

mild

acid

conditions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Heating

the nucleic

acids

has been observed

for

a short

by Bock (1967)

rupture a small number of the hydrogen bonds. It is suggested our findings that rupture of hydrogen bonds unique to the cyst

to from DNA

and ribosomal RNA occurs, which results in a configuration similar to those of vegetative cells and late germinated cysts. These results suggest that during the encystment process and subsequent compaction of the central body and nuclear material, the configuration of the DNA and ribosomal RNA is modified. Such a change in configuration protecting the nucleic terious environment.

could contribute a survival advantage by acids from possible denaturation in a deleThe transfer RNA did not exhibit this effect

suggesting

somewhat smaIler

that

these

molecules

are not modified

during the encystment process. Change in elution profiles in other bacterial systems have also been attributed to changes in configuration, quite possibly in the secondary and/or tertiary structure of the polynucleotide chains (Kaji and Tanaka, 1967; Stern, Zutra and Littauer, 1969). Stafford and Donnellan (1968) have reported photochemical evidence for differences spore and vegetative cell DNA. eluting

in the configuration

The detection, after 4 hours germination, after the 23s RNA peak is of interest.

of

of a small peak This peak is an RNA

peak and may represent messenger RNA. Doi and Igarashi (1964) studied spore messenger RNA fractions and these were found to elute after the 23s RNA. Failure to detect this peak prior to 4 hours germination may indicate low messenger RNA content in the dormant cyst and during early germination. The results of our investigation indicate that in addition to the survival advantage conferred by the protective cyst coat structure, other advantageous events may occur during cyst formation. Changes in nucleic acid configuration in conjunction with central body compaction may also represent an additional survival mechanism. Such changes in the intracellular environment may enhance the ability of the cyst to survive in a deleterious external environment. Quantitative changes in the nucleic acida during the germination process will be reported in a future communication.

REFERENCES Bock, R.M. 1967. In Methods in Enzymology, 218, Academic Press, Inc., New York. 719

Vol.

XII,

Part

A,

Vol. 35, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In Methods in Enzymology, Vol. XII, Part B, Press, Inc., New York. Doi, R.H. and Igarashi, R.T. 1964. Proc. Nat. Acad. Sci. 52,755. 177,702. Gierer, A. and Schramm, G. 1956. .Nature H. and Tanaka, Y. 1967. Biochim. Biophys. Acta =,642. Kaji, Keck, K. 1956. Arch. Biochem. Biophys. 6~~446. Lin, L. and Wyss, 0. 1965. Tex. Rep. Biol. Med. 2J,474. Lin, L. and Sadoff, H.L. 1968. J. Bacterial. %,2336. Midgley, J,E,M, and McCarthy, B-J, 1962. Biochim. Biophys. Acta Burton,

K. 1968. 163 > Academic

61,696. Olson, K-E. and Wyss, 0, 1967. Tex. J. Science 1 ,433. Olson, K.E. and Wyss, 0. 1968. Bacterial. Proc. -38,22. Parker, L.T. and Socolofsky, M.D. 1966. J. Bacterial. $0,297. Schneider, W.C. 1957. In Methods in Enzymology, Vol. III, 680. Academic Press, Inc., New York. Smith, D-D, and Wyss, 0. 1969. Antonie van Leeuwenhoek ,84. Socolofsky, M.D. and Wyss, 0. 1961. 3. Bacterial. f&94 F . Socolofsky, M.D. and Wyss, 0. 1962. J. Bacterial. 84,119. Stafford, R.S. and Donnellan, J.E. 1968. Proc. Nat. Acad. Sci.

2,822.

R., Zutra, L.E. and Littauer, U.2, 1969. Biochem. 8,313. N. and Cheng, T,Y+ 1962. J. Mol. Biol. 4,161. P.W. and Knight, S.G. 1952. Experiments in Bacterial Physioloa. Burgess Publishing Co., Minneapolis. Wyss, O., Neumann, M.G. and Socolofsky, M.D. 1961. J. Biophys. Biochem. Cytol. 2,555. G.N. and Vedenina, I.Ya. 1965. Microbiol.(DSSR) 2,945. Zaitseva, (English translation). Stern, Sueoka, Wilson,

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