ACTION OF FORMALDEHYDE ON MICROORGANISMS

III. BACTERICIDAL ACTION OF SUBLETHAL CONCENTRATIONS OF FORMALDEHYDE

ON AEROBACTER AEROGENES
W. BROCK NEELY
Biochemical Research Laboratory, The Dow Chemical Company, Midland, Michigan
Received for publication 5 April 1963

ABSTRACT methionine in A. aerogenes treated with formalde-

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NEELY, W. BROCK (The Dow Chemical Co., hyde.
Midland, Mich.). Action of formaldehyde on mi- MATERIALS AND METHODS
croorganisms. III. Bactericidal action of sublethal
concentrations of formaldehyde on Aerobacter Culture. The organism, A. aerogenes ATCC
aerogenes. J. Bacteriol. 86:445-448. 1963.- 8308, was cultured and maintained as described
Bacteriostatic concentrations of formaldehyde by Neely (1963a). Mutant 24 of A. aerogenes
were found to be bactericidal during the initial A3(0) was generously supplied by F. M. Harold,
growth period. This bactericidal activity was due Department of Experimental Chemistry, Na-
to the primary induction of unbalanced growth by tional Jewish Hospital, Denver, Colo. This
formaldehyde. The subsequent formation of 1,3- particular mutant grew only if methionine or
thiazane-4-carboxylic acid prevented the syn- homocysteine were added to the medium. In
thesis of methionine. This dual activity of form- the present case, the mutant was maintained on
aldehyde resulted in an inhibition of both the minimal medium of Warren, Ells, and Camp-
nuclear and cytoplasmic syntheses, and created bell (1960) supplemented with methionine (30
the normal bacteriostatic condition that has been ,ug/ml). Unless otherwise stated, the use of the
observed before with sublethal concentrations of term A. aerogenes will mean the ATCC 8308 cul-
formaldehyde. ture.
Analytical techniques. The procedures for meas-
uring growth, viability, and C1402 evolution were
In our previous investigation dealing with the similar to those described previously (Neely,
action of formaldehyde on microorganisms (Neely 1963a). The chromatographic identification of
1963a), it was shown that formaldehyde had an 1 3-thiazane-4-carboxylic acid has been reported
initial bactericidal action when used in bacterio- (Neely, 1963b). To follow the formation of 1,3-
static concentrations. This action seemed worthy thiazane-4-carboxylic acid, the following tech-
of further investigation. The subsequent identifi- niques were adopted. A portion (30 ml) of the
cation of 1,3-thiazane-4-carboxylic acid in Aero- chemically defined medium of Warren et al.
bacter aerogenes treated with formaldehyde (Neely, (1960) was inoculated with a 24-hr culture of A.
1963b) provided a lead to the understanding of aerogenes. The resulting culture was adjusted
this particular action. The formation of the with water so that when the appropriate formal-
above thiazane derivative by microorganisms dehyde was added the total volume would be 40
effectively removes homocysteine from any ml. The solution was then placed in a water bath
further biological reaction. It may be assumed (30 C) and aerated.
that homocysteine is the precursor for the bio- When turbidity measurements indicated that
synthesis of methionine, for there is good evidence logarithmic growth had started, the formaldehyde
that this assumption is correct (Harold, 1962). was added. Samples (5 ml) were taken at periodic
Then it logically follows that the synthesis of this intervals and centrifuged immediately. The cells
essential amino acid will be prevented in the were washed twice with 2-ml portions of physio-
presence of formaldehyde. With this background, logical saline, and the supernatant and washings
I decided to investigate both the formation of were made up to 10 ml with water. The cells were
1, 3-thiazane-4-carboxylic acid and the action of then extracted twice with 2-ml portions of hot
445
446

The methyl ester of methionine was prepared

from the directions of Dekker, Taylor, and Fruton
(1949). The product had a mp of 150 to 151 C as
compared with the literature value of 151 C. In
addition, the infrared spectra had a strong ab-
sorption at 5.8 ,u, supporting the presence of an
ester linkage.
RESULTS
The formation and disappearance of 1,3-
thiazane-4-carboxylic acid by A. aerogenes treated
with 50 ,ug/ml of labeled formaldehyde are
shown in Fig. 1. Superimposed on this graph is
the C1402 evolution pattern from labeled formal-
dehyde and the decrease in total C14-activity
during growth. Finally, the appearance and
disappearance of a new constituent produced by
the organism is illustrated. This latter material
on chromatography in the butanol solvent moved
only slightly from the origin after 18 hr of
chromatography.
Experiments were designed to see what effect
the addition of methionine would have on the
formaldehyde-treated organism. The first ex-
periments with methionine were unsuccessful in
that no effect was observed. With labeled
methionine, it was discovered that A. aerogenes
was unable to incorporate preformed methionine
NEELY

2500

70

60

50I

40

30/

20
,0
water, and the hot-water extracts were made Fig. 2 and 3 indicate. Figure 2 is a dose-response
up to 5 ml. Samples of the various fractions curve demonstrating the dramatic effect that in-
were counted in a Tri-Carb liquid scintillation creased concentration of methione ester has on
spectrometer (Packard Instrument Co.) as well the ability of the microorganism to metabolize
as chromatographed on paper (Whatman no. 1), formaldehyde. The previous results (Neely,
by use of the top layer of a n-butanol-acetic acid- 1963a) indicated that the shape of the C14O0
water (4:1:5) solvent. For greater accuracy and evolution curve was parallel to the viable count.
sensitivity in measuring the C14 activity in the Figure 2 indicates that, at the highest concentra-
chromatogram, a 1-in. paper strip was sectioned tion of methionine, the viable count decreased
into 1-in. squares and counted by means of liquid and failed to recover during the time of the ex-
scintillation. The results are reported as counts periment. The viable-count and turbidity data.
per min per sample of bacterial extract chro- in Fig. 3 verified this interpretation.
matographed.
cific activity of 10 mc/mmole) and the C'4-la-
beled methionine (specific activity of 5 mc/
mmole) were supplied by the New England
Nuclear Corp., Boston, Mass.
In an effort to determine that A. aerogenes was
Chemicals. The C'4-labeled formaldehyde (spe- utilizing the methyl ester of methionine as a

rIk

2000 ..Unknown

8i0 SI t
200, 1000.

H0A4

-
Ol

*.'
b

01-.- I

3
o~~~~~~~~~~
2
| C1"402 Evolution

6
l.2 60

4 6
4ivj

8
1d)
-90

-50

~~~~~~~~~0.6
30

FIG. 1. Results of assaying for 1, 3-thiazine-4-

material from Aerobacter aerogenes treated with 60

,ug/ml of C14-labeled formaldehyde (0.6 ,uc/ml).
C"02

'iX~~~I
/

10 12
HOURS

carboxylic acid, decrease in total C14-activity, forma-

tion of C1402 , and the appearance of an unidentified

METr/ YL ESrER OF MIET/ON/NE

molesSmi
0
125
50
I

14
J. BACTERIOL.

16

Curve
is
-

20
20

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0
from the medium. The initial C'4-activity was 0 3 6 9 12 15 is
HOURS
21 24 27 30 33

similar to the activity found in the supernatant

after 24 hr of growth. Futhermore, the final
activity was found by paper chromatography to
reside completely in the methionine.
The experiments utilizing the methyl ester of
methionine were more successful, as the results in
FIG. 2. C1402 evolution by Aerobacter aerogenes
treated with labeled formaldehyde (0.6 lAc/4 ml) at a
concentration of 60 ,ug/ml and various concentra-
tions of the methyl ester of methionine. The C1402
calibration is similar to that reported previously
(Neely, 1963a).
VOL. 86, 1963 ACTION OF FORMALDEHYDE ON A. AEROGENES 447
by this agent prevent both cytoplasmic and
1300
.0 ureI 3 - Methonine, methyl eCter
nuclear syntheses and bring about a bacteriostatic
50,.tmoleshtl 80

8. ^
x
SOg50p/ml of HCHO0
2 E4-50#qu/et
of HCHO 70
E
0 / t 2- Vioble Count
situation.
ID 639{
.6
-- t\ 34-0ptic DensiDeosty 0 The experiments of Cohen and Barner (1956)
~ ~~/20
I.- also illustrated that sulfanilamide can become a
o bactericidal agent when the deficiency of meta-
j .4 /{ 400
bolites is made specific for the nuclear constituent.
This situation occurs when all the metabolites
~~~~~ ~
-~~~~~~~~~~~~~1- ~ ~ ~ ~ 0-
formed by the folic acid cycle are added back to
C
the medium, with the exception of thymine. This
>
set of circumstances causes the organism to die
0 200 300 400 500 60 200 by unbalanced growth in a manner analogous to

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MINUTES the thymine-requiring mutant of E. coli.
FIG. 3. Turbidity and viable-count data for If the action of formaldehyde on A. aerogenes
Aerobacter aerogenes treated with formaldehyde (50 is examined in relation to the above discussion,
,ug/ml) and the methyl ester of methionine (50 all of these observations fall into a similar pattern.
,umoles/ml). The formaldehyde was added at the time One of the actions of formaldehyde, which is
indicated by the arrow. probably connected with nuclear synthesis, is to
inhibit cell division (Neely 1963a). Assuming that
source of methionine, experiments were performed this is the sole action of formaldehyde during the
with the mutant strain A3(0). It was discovered early phase, then a situation occurs which causes
that the ester was equally good as the free amino death of the bacteria by unbalanced growth.
acid in supporting growth of this particular However, another competing reaction of formal-
auxotroph. To rule out the possibility that the dehyde is the formation of 1, 3-thiazane-4-car-
results in Fig. 2 and 3 were due in some way to boxylic acid, the formation of which reaches a
the ester group, the experiments using the methyl maximum shortly after the loss in viability occurs.
ester of methionine were repeated with the This reaction prevents the synthesis of methio-
methyl ester of glycine. (A sample of this material nine, an essential metabolite connected with
was supplied by H. C. White of the E. C. Britton cytoplasmic synthesis. Consequently, during the
Research Laboratory, The Dow Chemical Co., remaining action of formaldehyde, both nuclear
Midland, Mich.) This addition gave results which and cytoplasmic syntheses are inhibited with the
were comparable with the control experiments in resultant achievement of a bacteriostatic con-
Fig. 2 and 3. dition.
If this mechanism is correct, then the addition
DISCUSSION of preformed methionine to the culture should
Barner and Cohen (1954) and Cohen and accentuate the bactericidal action of formalde-
Barner (1956) have shown that a thymine-re- hyde. In the present case it was discovered that
quiring strain of Escherichia coli 15T- loses the A. aerogenes would not utilize preformed methio-
power to multiply or "dies" when it is permitted nine (see Results). However, the addition of the
to metabolize and grow in the absence of exog- methyl ester of methionine to the medium did
enous thymine. The effect was described by cause an increased drop in viability as com-
these authors as death by unbalanced growth, a pared with formaldehyde alone. The experi-
situation where nuclear synthesis was inhibited ments with the methionine-requiring mutant of
while cytoplasmic syntheses and growth con- A. aerogenes A3(0) demonstrated that the methyl
tinued. ester of this amino acid was satisfactory as a
Cohen and Barner (1956) also examined the source of methionine. This would confirm that
action of sulfanilamide on microorganisms. The the observed action of the methionine ester on
normal action of sulfanilamide is one of interfer- formaldehyde-treated A. aerogenes was nutri-
ring with the folic acid cycle. This in turn causes tional and not due to some other phenomenon.
the inhibition of the one-carbon transfer mech- The inability of the methyl ester of glycine to
anism, a mechanism which is essential for the increase the bactericidal action of formaldehyde
biosynthesis of a large number of metabolites. It eliminated the activity of the methionine ester
is evident that the multiple deficiencies produced as being due to the ester group.
448 NEELY
The presence of the new C14-labeled component,
which moved only slightly on chromatography in
the butanol solvent, is being examined and the
results will be reported at a later date.
LITERATURE CITED
BARNER, H. D., AND S. S. COHEN. 1954. The induc-
tion of thymine synthesis by T2 infection of a
thymine requiring mutant of Escherichia coli.
J. Bacteriol. 68:80-88.
COHEN, S. S., AND H. D. BARNER. 1956. Studies on
the induction of thymine deficiency and on the
effects of the thymine and thymidine ana-
logues in Escherichia coli. J. Bacteriol. 71:
588-597.
DEKKER, C. A., S. P. TAYLOR, JR., AND J. S.
J. BACTERIOL.
FRUTON. 1949. Synthesis of peptides of methi-
onine and their cleavage by proteolytic en-
zymes. J. Biol. Chem. 180:155-173.
HAROLD, F. M. 1962. Accumulation of cystathi-
onine in a homocysteine-requiring mutant of
Aerobacter aerogenes. J. Bacteriol. 84:382-383.
NEELY, W. B. 1963a. Action of formaldehyde on
microorganisms. I. Correlation of activity
with formaldehyde metabolism. J. Bacteriol.
85:1028-1031.
NEELY, W. B. 1963b. Action of formaldehyde on
microorganisms. II. Formation of 1,3-thi-
azane-4-carboxylic acid in Areobacter aero-
Downloaded from http://jb.asm.org/ on November 30, 2019 by guest
genes treated with formaldehyde. J. Bacteriol.
85:1420-1422.
WARREN, R. A. J., A. F. ELLS, AND J. J. R. CAMP-
BELL. 1960. Endogenous respiration of Pseudo-
monas aeruginosa. J. Bacteriol. 79:875-879.