Chloramine Mutagenesis in Bacillus subtilis

Abstract. Chloramine (which occurs widely as a by-product of sanitary chlorina-
tion of water supplies) is shown to be a weak mutagen, when reversion of trpC to trp*
in Bacillus subtilis is used as an assay. Some DNA-repair mutants appear to be more
sensitive to chloramine, suggesting the involvement of DNA targets in bactericide.
The influence of plating media on survival of cells treated with chloramine suggests a
bacterial repair system acting upon potentially lethal lesions induced by chloramine.

Chloramine (NH-Cl). the reaction
product of chlorine and ammonia in aque-
ous solution, is widely used directly for,
or is generated as a by-product of, the dis-
infection of public water supplies and
swimming pools. Nevertheless. little is
known of the biological mechanism of ac-
tion of chlorine or of possible genetic ef-
fects. Our own study (/) has shown that
chloramine reacted with Bacillus subtilis
DNA in vivo and in vitro; however.
Boyle (2) did not obtain auxotrophs by
treating Escherichia coli cells with
chloramine; and a preliminary experi-
ment with bacteriophage lambda by Hay-
atsu (3) did not show mutagenicity of hy-
pochlorite. On the other hand, while this
manuscript was in preparation, Wlod-
kowski and Rosenkranz (4) reported that
sodium hypochlorite was a weak base-
substitution mutagen in Salmonella
typhimurium. We now report the mu-
tagenicity of chloramine. using reversion
of trpC to trp* in B. subtilis as an assay.

The strains used in this study were de-
rivatives of indole-requiring B. subtilis
strain 168 (5). As an index of the in-
volvement of DNA targets in bactericide
by chloramine, the sensitivity of differ-
ent B. subtilis strains carrying various

Fig. 1. Chloramine sen-

sitivity of different B.

subtilis strains. (a) @.

168 (uvr*, irpC); O, 10!
$B879 (uvr, trpC, his B);
(b) @, BDI70 (rec *, trp-
C, threo5), 0, BD191
(recB, trpC, threoS), A.
BDI93) (rec3,— trpC,
threoS). O, BD194 (rec-
A. trpC); (c) @. SB10S8
(pol*, pheA, trpC, his-
B); ©, SB1060 (polAS.
phe A, trpC, hisB). For
the chloramine treat-
ment, NaOC] was diluted
in 0.05M phosphate

10?

103

Fraction of survival

10

 

 

DNA-repair mutations was examined.
Cell concentrations of each mutant and
of its control for chloramine treatment
were adjusted to about the same. In
addition, the mixed cultures of strains
168 + SB&879 and = strains BDI70 +
BDi94 were treated with chloramine and
their survivors were sorted out by their
respective nutritional markers. The sur-
viving fractions of a representative test
for each mutant as a function of

chloramine doses are shown in Fig. 1. -

While uvr (SB879) and recB (BD191)
showed no evident increases in sensi-
tivity, rec3 (BD193), recA (BD194),
and polAS (SB1060) were consistently
more sensitive than their respective con-
trols. Another test of the polAS mutant
in a different strain, SB1059 (polAS5,
pheA, trpC), demonstrated the same
chloramine sensitivity as SB1060 did.
[This finding is consistent with that ob-
tained by Rosenkranz (6) which was also
reported in the course of these experi-
ments.) Our data suggested that at least
polAS*, and possibly recA* and rec3*,
are involved in the repair of chloramine-
induced damage in DNA.

Strain 168 (¢r7pC) was used to test the
influence of plating media on survival af-

 

 

   

 

 

 

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buffer, pH 7. to dif- (a 4
ferent concentrations. 0 18
Chloramine (NHC)

was formed by incubat-

ing eight parts of NaOCl

37 56 74 920 18 37 56 74920 18 37 56 74 92

NH4CI dose (yum)

and one part of 0.1M NH,Ci at 37°C for | hour. The prepared early stationary phase cells (/)
were diluted ten times into chloramine solution and treated for 30 minutes at 37°C. The reaction
was stopped by the addition of one volume of 0.02M sodium thiosulfate. Viable cell counts were
scored by averaging the colonies of two plates (amino acid media, see Table !) after incubation at
37°C for 2 days. The minimum number of colonies per plate was 161 + 8 percent.

ter chloramine treatment and the effect
of chloramine on mutation. Ultraviolet
irradiation was employed to give com-
parative results. In ultraviolet-irradiated
cells, the survival on nutrient agar was
much lower than that observed on all
the other media (Table 1). Minimal
medium, however, showed the lowest
survival after chloramine treatment.
Since chloramine readily reacted with
amino acids and proteis (2), the sup-
plementation of nutrients after chlora-
mine treatment seemed to be essential in
the recovery of the cells. Among the nu-
trient-supplemented media, however,
amino acid supplements (AA, CH-trp) af-
forded more efficient repair than nutrient
agar for both ultraviolet-irradiated and
chloramine-treated cells. The tests for
the prototrophs (SB!9 and SB850, trp*)
gave similar results. This phenomenon
was similar to that observed in ultravio-
let irradiation of E. coli (7), and it was
suggested that the higher survival on syn-
thetic medium than on complex medium
resulted from delaying the growth of the
irradiated cells until repair of ultraviolet
damage occurred. The differential recov-
eries of chloramine-treated cells also sug-
gested a bacterial repair system acting
upon potentially lethal lesions, similar to

 

 

 

 

 

 

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Percentage survival

Fig. 2. Relative ¢rp* reversion frequencies and
absolute trp* revertants plotted against the
percentage of survival after chloramine treat-
ment (@) and ultraviolet irradiation (©). The
revertants and survival were scored on CH and
CH+trp media, respectively. Cell counts on
CH+trp medium were 2.26 x 10° to 2.61 x
10° for controls.

Reprinted from SCIENCE, 11 June 1976,volume 192,pages 1141-1143
Table |. Effects of different media on survival and on ¢rpC to irp* reversion after treatment with
chloramine or ultraviolet irradiation. The media used were as follows: NA, nutrient agar (Dif-
co); MM, Spizizen minimal medium + 0.5 percent sucrose + 1.75 percent agar; MM+trp.
MM + tryptophan (25 zg/ml); CH, MM + 4 mg of casein hydrolyzate per milliliter (Nutritional
Biochemicals); CH+trp, CH + tryptophan (25 pg/ml); AA-trp, MM plus all the amino
acids (25 ug/ml) except tryptophan; AA, MM plus all the amino acids (25 ug/ml). Viable cells
were counted as described in Fig. 1. The revertants were counted after 4 days of incubation

 

 

 

at 37°C.
. + 1 *
Treatment Survival (%) trp* reversion
NA MM+trp CH+trp AA MM CH AA~trp
NH.Cl
None 95.11 95.74 100+ 100.53 fH 8 6
: ( 5.1) (3.5) ( 2.6)
18 uM 78.19 70.29 98.94 90.43 10 9 i]
( $5.3) ( 4.0) ( 5.4)
37 uM 39.88 28.09 56.25 64.19 4 14 1
( 6.3) (iA) ( 7.6)
56 pM 0.69 0.25 2.47 13.67 2 32 24
(315) ( 574) (78)
74 pM 0.0037 0.0013 0.042 0.27 0 8 6
(- ) (8547) (983)
Ultraviolet
None 101.15 100.38 100+ 98.08 10 12 13
( 5.1) ( 4.6) ( 4.7)
1 min 23.53 71.33 65.89 65.87 8 13 12
( 3.9) (7.7) ( 7.6)
3 min 4.70 17.59 15.63 20.65 4 4 9
( 8.7) (9.8) (15.2)
6 min 0.30 2.76 2.10 5.13 3 2 3 :
(41.6) (36.4 ) (22.3)
9 min 0.0068 0.38 0.38 1.00 1 1 2
(- ) (- ) (762)

 

*Upper numbers are actual numbers of revertants scored; lower numbers in parentheses are relative fre-
quencies per 10° survivors. For ultraviolet treatment the prepared cells were suspended in phosphate buffer

(3.5 ml) and held at 37°C for 30 minutes; they were then treated with ultraviolet (5 erg
tCell counts are 2.26 x 10° for NH,C! treatment and 2.61 = 10!

petri dishes (5 cm in diameter).
violet irradiation.

well-known findings with ultraviolet irra-
diation.
The results of two typical experiments
. on the effect of chloramine and ultravio-
let irradiation on the yield of trpC to trp*
reversion are shown in Table 1. In ul-
traviolet-irradiated cells plated on differ-
ent media and in chloramine-treated cells
plated on minimal medium, there was en-
hancement of mutation frequencies; how-
ever, the absolute number of revertants
was reduced owing to the superimposed
lethality. The use of supplemented media
for the chloramine-treated cells, in con-
trast, yielded small but consistent in-
creases of total numbers of trp* rever-
tants (and implies marked enhancement
of trp* reversion rates). This result re-
sembled that observed in S. typhimurium
and E. coli (8): that the yield of trp~ to
trp* reversion induced by ultraviolet ir-
radiation was profoundly enhanced by
certain conditions of incubation after
. treatment.
The trpC marker of B. subtilis is a
stable locus with a low spontaneous re-

sec"! mm*) in plastic
for ultra-

version frequency of 2 to 9 per 10° cells.
Absolute increase of revertants was ob-
served only at survivals between 0.1 and
10 percent. However, chloramine kill
was difficult to control because of the
presence of other variables (such as cell
concentration, amino acid or protein resi-
dues in cell samples, and the cleanliness
of glassware) and the same dose of chlora-
mine did not always give reproducible
kill and reversion. These small increases
in absolute numbers of trp* revertants re-
inforced the evidence for mutagenesis by
chloramine, as judged by the relative in-
creases among the survivors. The com-
bined results of several experiments
were analyzed statistically (Fig. 2). Com-
paring the regression coefficiencies (rela-
tive reversion frequencies and _per-
centage of survivals) of the two killing
agents, chloramine (b = —43.62) had a
significantly (d = 3.99) higher effect, for
a given kill, than ultraviolet did
(b = —0.66), in enhancing the trp* re-
version. The chloramine sensitivity of 12
trp.” revertants was also studied, and

they were as sensitive to chloramine as
the trpC cells.

The effect of chloramine on the yield
of trpC to trp~ of other different strains
carrying various mutation pertinent to
DNA repair was also studied on CH me-
dium. The preliminary experiments
showed that the rrp* yields of polAS
(SB1060), rec3 (BD193). recA (BD194),

- and uvr (SB879) were not significantly

different from the yields of polAS* rec*
control strains (168, BD170) when
treated with chloramine. Strain recB
(BD191), however, had a comparatively
lower reversion frequency whether the
cells were chlorinated or not. No in-
crease of absolute number of revertants
in chloramine-treated cells was seen.
Similar phenomena have been reported
also in studying the mutability of recom-
bination-deficient and ultraviolet-sensi-
tive strains of E. coli toward ultraviolet
light (9).

The trp* revertants of 168 have been
studied for the transforming activity of
the linkage group (aroB* trpC* hisB*).
Of 75 chloramine-induced revertants, 24
percent showed reduced trp* trans-
forming activity compared to wild type
trp*. Spontaneous reversions showed
about 8 percent such transforming-defec-
tive types. These may represent more
complex genetic changes than base sub-
stitution.

KATHERINE Lu SHIH*
JOSHUA LEDERBERG
Department of Genetics,
Stanford University School of Medicine,
Stanford, California 94305

References and Notes

. K. L. Shih and J. Lederberg. J. Bacteriol. 125,

934 (1976).

2. W. C. Boyle, thesis. California Institute of
Technology, Pasadena (1963).

3. H. S. Hayatsu..S. Pan. T. Ukita. Chem. Pharm.
Bull. (Tokyo) 19, 2189 (1971),

4. T. J. Wiodkowski and H. S. Rosenkranz, Muta-
tion Res. 3, 39 (1975).

S. J. Spizizen, Proc. Natl. Acad. Sci. U.S.A. 44,
1072 (1958).

6. H. S. Rosenkranz. Mutation Res. 21, 171 (1973).

7. T. Alper and N. E. Gillies, J. Gen. Microbiol.
18. 461 (1958); ibid. 22, 113 (1960).

8. E. M. Witkin. Cold Spring Harbor Symp.
Quant. Biol. 21, 123 (1956); in Repair from
Genetic Radiation Damage. F. H. Sobel, Ed.
(Pergamon, London, 1962). p. 151, C. H. Clark,
Mutation Res. 8, 35 (1969). .

9. R. J. Hill, Photochem. Photobiol. 4, 563 (1965);
E. M. Witkin, Mutation Res. 16, 235 (1972);
_-____ and D. L. George. Genetics 73 (Suppl.).
91 (1973).

10. Supported by NIH grants GM-00295-14 and CA-
16896. We thank Dr. R. Davidoff-Abelson for
providing strains BDI70 to BDI94.

* Present address: Biological Adaptation Branch,

Ames Research Center, National Aeronautics

and Space Administration, Moffett Field. Calif.

94035.

22 January 1976

Copyright © 1976 by the American Association for the Advancement of Science