Raman spectroscopy in vivo : Evidence on the structure of dipicolinate in intact spores of Bacillus megaterium

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RAMAN SPECTROSCOPY In m: DIPICOLINATE William

RESEARCH COMMUNICATIONS

EVIDENCE ON THE STRUCTURE OF

IN INTACT SPORES OF Bacillus

H. Woodruff,

Thomas G. Spiro

Mepaterium

and Charles

Gilvarg

Departments of Chemistry and Biochemical Sciences Princeton University, Princeton, New Jersey 08540 Received

March

5,1974

SUMMARY: Raman spectra of spores of g. megaterium are interpreted in terms of the predominant chemical component in the spores, the 2,6 pyridinedicarboxylate molecule (dipicolinate). The spectra show that the structure of dipicolinate within the spores is quite different from that of tridentate calcium dipicolinate, a previously proposed model for in-spore dipicolinate structure. Structural possibilities are discussed in light of the Raman evidence. INTRODUCTION:

Typical

by weight

of

2,6

cium

ion,

is

imputed

the

spores

structure point,

to heat

concerning

to indicate

late

(CaDPA)

thereof

(4).

and other

that (2,3)

spores

is

results

on intact present

been

gion

is

a third

that

some 30% of the

of

shown of

the

weight 2,6

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

cal-

acid of

protoplast of

the

of the

spore

pyridinedicarboxylate

with

stresses

(1).

of

The

an important Ultraviolet

spores

volume

197

together

resistance

spores

the bulk

to be the

10%

exists.

in

or as dipicolinic

approximately

the high

therefore

evidence

The location

*Abbreviations: sulfoxide = DMSO.

for

little

DPA is

which,

environmental

the

which

has recently only

contain *

to be responsible

spectroscopic

ted

spores

pyridinedicarboxylate,

of DPA within

infrared

Copyright All rights

bacterial

have as the

(H2DPA) the

been

che-

or an ester

(5).

protoplast,

interpre-

calcium

DPA within

spore

and

(6)

the

spore

As the

core

this

means

which

also

= DPA; dimethyl

re-

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DPA Dianlon

Wild TYPO spores

~-46 Spores

M-46 Germmate I

1

/

1500

1700

I

I

I300

1

I I00

1

900

cm“

of spores and germinated spores of Figure 1. Raman spectra Bacillus megaterium, including the spectrum of the dipicolinate dianon in 1 g aqueous NaOH. The discontinuities in the spectrum of wild type spores are due to instrument baseline readjustments. Spectra were obtained using a Coherent Radiation CR-5 Ar+ laser, a Spex 1401 double monochromator, and d.c. amplification of the Exciting wavelength "signal .from an ITT FW130 photomultiplier. Samples of the model compounds (Table was either 48801 or 4579a. I) were contained in 1 mm diameter capillaries, the solids being powder samples. Suspensions of the spores and germinate of Bacillus megaterium were filtered on 13 mm Millipore discs having The discs were mounted on the Spex rotating 0.45 Km pore size. sample accessory. contains

the

tributable trum

to this

of intact

of DPA in evidence simple

spore

the

form

acids

and cytoplasmic

one substance.

spores

indicates calcium

nucleic

of Bacillus

which that

chelate,

We have

obtained

megaterium.

predominates the

proteins,

DPA species

in

protoplast. the

or H2DPA, or an ester 198

the Raman spec-

The spectrum

the in

is at-

spores or amide

is

that

The Raman is not

the

of DPA.

Vol. 58, No. 1, 1974

BIOCHEMICAL

RESULTS AND DISCUSSION: germinated strain

spores (wild

the

resting

1520

cm

trum

of the

type)

the

spectrum

of

~-46 former the

germinated

these

model

strongest

spores other

among

occurs

this ring

spores

is

presented

NaOH.

The vibrations

are

given

in

Table

assignments

for

the

model

ring near

chemical

aromatic species

vibrations

ring

spores,

the

with

is one the

and assignI.

Assignments

carboxylic

acids

by the probably

the high

in

The

background due to the

of the

the

the in

ring

1007

resting

resting cm -1 . No spores.

and germinated

(1).

of

I).

"breathing"

at

resting

latter

spectrum

199

(Table

spectra

observed

the

little

symmetric

In the

between

of DPA in

995 cm-1 in

is

spectrum

of DPA vary

studied is

cm -1 .

1000

difference

absence

obscured

the

(12).

DPA are

is

with

spores.

of

which

The

peak centered

comparision

vibrations

peak at

spores

The DPA dianion

seen as a weak feature

the

wild-type

a broad

is

broad

resting

as much DPA (7).)

vibration

The primary

and

(The spec-

half

for

due to the

of these

mode which

of

1158,

about

examined

species

and pyridine

frequency

1007,

of the

1 are

to previous

The vibrations in

a local

that

1 _M aqueous

DPA species

made by analogy

(a-11,16)

at

background.

shows only

in Figure

of DPA in

model

for

used;

than

spores

of the 2. megaterium

ments were

only

were

and

The two spectra

peaks

(fluorescent)

is weaker

spectra

dianion

several

~-46.

of resting

995 cm-l.

The spore

spectra

showing

contains

at approximately

of

are

similar,

spectra

Two strains strain

spores

RESEARCH COMMUNICATIONS

1 shows the

and a mutant

on top of a rising

because

of the

Figure

of a. megaterium.

spores

-1

AND BIOPHYSICAL

Therefore, germinated

the

spectra

modes

of the

the spores, of the various

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

Table Raman Spectra Saturated H2DPA in DMSO

and Assignments Solid H2DPA

Aqueous DPA'

RESEARCH COMMUNICATIONS

I

of Dipicolinate Solid CaDPA

Saturated Aqueous CaDPA

1753

Assignment, Symmetry* Isolated

1713

1614 .. 1628

1592 1576

1465

1447

C=O, A1

Associated 1600

1577

cm -1

Species,900-1800

1572

C=O,A1

1 + 6a**

1589

-

1580

C=C-C,

A1

1570

-

1580

C-C-C,

B1

1470

19a**

1439

1445

1447

Ionized

COO-, A1

1386

1395

1397

Ionized

COO-, A1

1328 1298

Associated

COOH,A1

1270

1273

Associated

COOH,A1

1160

1182

1192

1194

1155

1155

1156

15**

1087

1082

18a**

1195

9a*

1007

Isolated

998

999

1002

*

Based upon polarization solids. *Jr Pyridine assignments, nucleic ent

acid

bases

(14,15).

frequencies

the

1021

measurements

for

see Reference

12.

and aromatic

In contrast of

1017

to the

carboxylate

side

ring

vibrations,

groups

200

Ring,

solutions,

protein

C-O, A1

chains

of DPA differ

A1

assumed

which

are

the vibrational substantially

for

pres-

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

coo-

CoDPA

K

I

I

COOH

C;O---H

H,DPA Solid

I

2izo

c=o

,I’

1

1,

L 1600

I

I

,

I

I

1

,

,

,

, i

,

,

1600

1500

1400

1200

1100

”

1700

I300

1

C-r

,

1000

900

cm-l

Figure 2. Frequency correlation brations in DPA species, including megaterium.

among the ing

various

degrees

derivatives

the

tra

of the

ing

modes expected

resting

carboxyl

group

spores.

It

arized

intense

bands

spores

to the

for

that

to an extent

but

the

The DPA species salt.

This

at

the

is

two symmetric

DPA in

the

spore

which

causes

the

carboxyl

conclusion

spore is

spec-

of

protoplast

is

groups

on the

sub-

to be pol-

interaction following

is not to previous

the

the

the

DPA= and CaDPA on the

is clear

201

relationship and in

of this

contrary

by

stretch-

species

The nature

protoplasts

the

carboxylate

DPA model

the

these

groups

1158 and 1520 cm -1 in_

between

vary-

bonding.

among the

other.

due to the

carboxylate

2 shows the

Raman evidence in

upon

This

Figure

intermediate

hand and H2DPA on the

cium

DPA.

frequencies

appears

I).

and hydrogen

to an interaction

certain,

imposed

protonation,

We assign

ject

(Table

of polarization

complexation,

diagram for carboxylate viDPA in spores of Bacillus

one

is unpoint.

simple

cal-

interpretations

Vol. 58, No. 1, 1974

of

infrared

species

(2)

in

1750 cm served

-1

.

It

species

the is

reflect

spores

not

would

.from

strontium

the

salts

have

compounds the

that

involved

in

or amide

stretching

frequencies, in

frequencies

of calcium

ions

coordination

thereof. vibration

near

such as those

lanthanide

in

DPA

the

carboxylates

spore

Raman spectra

to DPA which found

in

ob-

the

is

quite

calcium

dif-

and

(18,19). of

of Table

as an example

The predominant

spectra.

H2 DPA or an ester

the

the

ring

low compared

spectrum.

(3)

been observed

tridentate

The intensity is anomalously

RESEARCH COMMUNICATIONS

carboxylate

spores,

possible

AND BIOPHYSICAL

show a carbonyl

a mode of binding

ferent

in

is

Intermediate

in

(8).

and ultraviolet

the

Any of these

BIOCHEMICAL

I,

the

breathing

to the ring

We interpret

carboxylate

ACKNOWLEDGEMENTS: The authors the U.S. Public Health Service (for W.H.W.) National Institutes GM-54135.

peaks.

mode is usually the

intensity

of Raman hypochromism a ring-stacking

mode of DPA in

(14),

interaction

the

in

For

the

spores the

strongest

reduction which

the

in

suggests spore

model

feature the

that

spores DPA is

protoplast.

acknowledge support of this work by Grants AM-09058 and GM-13498 and of Health Postdoctoral Fellowship

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Vol. 58, No. 1, 1974

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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203