FEMS Microbiology Letters 44 (1987) 427-430
Published by Elsevier
427
FEM 02956
Thiosulfate reductase as a chlorate reductase
in Salmonella typhimurium
D a n i e l L. Riggs *, J a n e S. T a n g a n d E r i c k a L. B a r r e t t
Department of Food Science and Technology, University of California, Davis, CA, U.S.A.
Received 22 June 1987
Revision received and accepted 14 July 1987
Key words: Salmonella typhimurium; Chlorate sensitivity; Nitrate reductase; (chiC and phs mutants)
1. S U M M A R Y
The contribution of thiosulfate reductase to
chlorate sensitivity in Salmonella typhimurium was
examined. Electrophoresed extracts of nitrategrown cells of both the wild type and a chiC
mutant were shown to contain chlorate reductase
activity of the same relative mobility as a thiosulfate reductase activity which was present in the
chic mutant, but not in the wild-type grown under these conditions. A mutation is phs, which is
essential for t h i o s u l f a t e r e d u c t a s e b y S.
typhimurium, was shown to confer some chlorate
resistance in the wild-type background and to
increase the chlorate resistance obtained with a
chiC mutation. Finally, thiosulfate in the anaerobic
growth medium was shown to protect a chiC
mutant growing in the presence of chlorate, but it
did not protect the wild type. The results are
consistent with a picture in which thiosulfate reductase can function as a chlorate reductase in
Correspondence to: Ericka L. Barrett, Dept. of Food Science
and Technology, University of California, Davis, CA 95616,
U.S.A.
* Present address: Department of Biology, University of
California, La Jolla, CA 92093, U.S.A.
both the wild-type and chiC backgrounds, although its capacity to reduce thiosulfate is diminished by the presence of an active nitrate
reductase encoded by chiC.
2. I N T R O D U C T I O N
Selection for resistance to chlorate ion has been
used for m a n y years to obtain mutants of
Escherichia coli, S. typhimurium, and other Enterobacteriaceae with defects in anaerobic nitrate reduction [1-6]. The basis of the selection is the
inability of nitrate reductase mutants to reduce
chlorate to toxic chlorite. A m o n g the several chl
loci involved in chlorate resistance, chiC has been
identified as the site of the structural gene for
nitrate reductase in both E. coli [3,4,6,7] and S.
typhimurium [8]. However, full resistance to chlorate is rarely traced to single lesions in chiC [1,6,8].
Instead, the vast majority of chlorate resistant
mutants isolated are pleiotropic and contain lesions in one of the genes essential for the formation of all molybdoenzymes, e.g., formate dehydrogenase [1,5,9,10], trimethylamine oxide reductase [11-14], the secondary nitrate reductase
[8], and, in hydrogen sulfide-producing species
such as S. typhimurium and Proteus mirabilis,
0378-1097/87/$03.50 © 1987 Federation of European Microbiological Societies
428
thiosulfate reductase [2,5,9,10,12], and tetrathionate reductase [2,5,9,10].
S. typhimurium chiC mutants have been shown
to retain significant chlorate sensitivity [8,12].
Although one might surmise that this residual
chlorate sensitivity is due to the activity of some
of the other reductases, little is known about their
contribution to chlorate reduction. Stouthamer [10]
has presented evidence suggesting that Salmonella
and Proteus spp. contain a 'chlorate reductase C'
enzyme which m a y be distinct from the other
reductases [9,10]. Here, we present results suggesting that thiosulfate reductase contributes to chlorate reduction by S. typhimurium and might thus
be the previously described 'chlorate reductase C'.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacterials trains, media and growth conditions
The strains of S. typhimurium used are listed in
Table 1. Nutrient broth was from Difco Laboratories; it was routinely supplemented with 0.5%
NaC1. Nitrate broth consisted of nutrient broth
with 0.5% K N O 3. KCIO 3 was used at 0.1 m M
concentration. All incubations were anaerobic and
at 37 ° C. Anaerobic growth was achieved by incubating completely filled tubes or flasks as standing cultures. For growth experiments, each tube
contained a glass bead to permit mixing before
reading culture density.
Table 1
Bacterial strains
Strain
LT2
EB8
EB40
EB244
EB309
TC110
a
Genotype
wild type
chlcl128
chlC1130::TnlO
phs (am)
chiC1130::Tnl0 phs
fla56 HI-iM10 ilVA454
pit proA46 purC7
purl1590 rha-461
rpsL166 ch/A1110
Source of reference
B.N. Ames
[8]
[19]
[16]
EB40 x EB244 a
[12]
Tetracycline-resistant transductants of EB244 infected by
phage P22 grown on EB40 were selected using genetic techniques described previously [19].
3.2. Electrophoretic methods
Cells were grown anaerobically in nitrate broth,
and extracts were prepared as previously described [8]. Nitrate reduction was stained by the
method of Lund and DeMoss [15]. Thiosulfate
and chlorate reductions were stained using the
same procedure, except that Na2S20 3 or KC103,
respectively, was substituted for nitrate. Protein
was stained with 0.1% Coomassie blue R-250 in a
5 : 1 : 5 solution of water, glacial acetic acid, and
methanol.
4. R E S U L T S
4.1. Chlorate reductases detected by electrophoresis
We showed previously that chiC mutants grown
in nitrate retain significant sensitivity to chlorate
[8]. To identify the reductases contributing to
chlorate sensitivity under these conditions, we
stained electrophoresed extracts of chic mutant
EB8 and wild-type LT2 for chlorate reduction,
and then compared the relative mobilities of the
bands revealed to the relative mobilities of known
reductases. Nitrate-grown EB8 was found to contain 2 chlorate reductase activities which corresponded to the secondary nitrate reductase and to
thiosulfate reductase, respectively (Fig. 1). These
reductase bands were not revealed in electrophoresed extracts of LT2 stained using nitrate or
thiosulfate as electron acceptors, although they
did appear when chlorate was used. This result
indicates that, although anaerobic growth in the
presence of nitrate may interfere with the activity
of these reductases in the wild type, it does not
completely repress their synthesis.
4.2. Chlorate resistance conferred by mutations in
phs
Expression of phs is essential for thiosulfate
reductase activity in S. typhimurium [16]. If thiosulfate reductase contributes to chlorate sensitivity, then mutations in phs should confer some
chlorate resistance. The contribution of phs was
evaluated by measuring growth of phs and chlC
single and double mutants in the presence of
chlorate (Table 2). Unsupplemented nutrient broth
was used in these experiments to avoid repression
429
TSR
PROTEIN
NR
ACTIVITY CR ACTIVITY
ACTIVITY
Table 2
Effect of phs mutation on chlorate sensitivity
Strain
LT2
EB40
EB244
EB309
TC110
IP
I
I
LT2
EB8
LT2
EB8
I
EB8
Relevant genotype
LT2
EB8
Fig. 1. Nitrate, thiosulfate, and chlorate reductases in extracts
of nitrate-grown cells subjected to electrophoresis in 4%
acrylamide. Activity stains using methyl viologen as electron
donor: NR, nitrate reductase; TSR, thiosulfate reductase; CR,
chlorate reductase. N o band appeared in extracts of LT2
stained using thiosulfate. Enzyme abbreviations in protein
stain: N R m, minor nitrate reductase [8]; N R M, major nitrate
reductase (chic product); TSR, thiosulfate reductase [16].
A650 after 6 h incubation a
wild type
chlC::TnlO
phs
chlC:: T n l 0 phs
chlA
No
KCIO 3
With
KC103
(%) b
0.129
0.093
0.131
0.105
0.154
0.074
0.062
0.084
0.077
0.145
(57)
(67)
(64)
(73)
(94)
a Inoculated (1%) tubes were filled completely and incubated
as standing cultures. The inocula consisted of overnight
standing cultures grown in unsupplemented nutrient broth.
Values reported are averages from 2 separate experiments,
each performed in duplicate.
b ~ = A650 with chlorate/Ar5 o without chlorate× 100. Experimental error: _2%.
0.20 LT2
0.15
of thiosulfate reductase by sugars [18] and to
avoid competitive inhibition of chlorate reduction
by other anaerobic electron acceptors specific for
the chlorate-reducing enzymes. Because anaerobic
growth in unsupplemented nutrient broth is very
poor, the background culture density resulting
from aerobic growth before residual oxygen is
depleted is proportionately great. However, as
shown in Table 2, wild-type culture density
achieved in this medium is still significantly
lowered by chlorate, while a chlA mutant culture
is only slightly affected. The phs mutation did
confer some resistance to chlorate in the wild-type
background, and it also slightly increased chlorate
resistance resulting from a chiC mutation. This
result suggests that thiosulfate reductase is partly
responsible for the chlorate sensitivity characteristic of mutants deficient in the major nitrate reductase, although enzymes encoded by genes other
than chiC and phs must also be able to act as
additional chlorate reductases.
4.3. Protection by thiosulfate
Nitrate was shown previously to protect LT2,
o_1Of f8
o 0.10c.O
o.o -
,o;
/
0
2
-•
Hours
4
•
6
8
10
0.20
EB40
0.15
/ 0 -----~
~
o0o
o.o5
0.00
:::.!.
'
:
.
• - - I
',
2
,
Hours
i
4
,
i
6
,
J
8
10
Fig. 2. Effect of thiosulfate on anaerobic growth in the presence of chlorate. Growth experiment performed as outlined in
Table 2, note a. Circles, nutrient broth; triangles, nutrient
broth with 0.1 m M thiosulfate; open symbols, no chlorate;
filled symbols, 0.1 m M KCIO 3 in growth medium.
430
b u t n o t chiC m u t a n t s a g a i n s t the effects of chlorate [8]. If thiosulfate r e d u c t a s e in the chiC m u t a n t
were a m a j o r c h l o r a t e r e d u c i n g enzyme, then its
n a t u r a l s u b s t r a t e (thlosulfate) m i g h t offer similar
protection. W e f o l l o w e d the g r o w t h of w i l d - t y p e
LT2 a n d a chiC m u t a n t s with a n d w i t h o u t chlorate a n d thiosulfate (Fig. 2). T h i o s u l f a t e o f f e r e d
c o n s i d e r a b l e p r o t e c t i o n to the chiC m u t a n t . It
failed to p r o t e c t the wild t y p e in which c h l o r a t e
r e d u c t i o n c o u l d also b e p e r f o r m e d b y the chiC-enc o d e d n i t r a t e reductase, which is p r e s e n t d u r i n g
a n a e r o b i c c o n d i t i o n s even in the a b s e n c e of nitrate.
In E. coli, n i t r a t e r e d u c t a s e activity u n d e r
a n a e r o b i c c o n d i t i o n s in the a b s e n c e o f n i t r a t e is
5 - 1 0 % of the activity f o u n d in the presence of
n i t r a t e [6].
5. D I S C U S S I O N
U s i n g three s e p a r a t e e x p e r i m e n t a l a p p r o a c h e s ,
we have s h o w n that thiosulfate r e d u c t a s e conlt~ibutes to t h e c h l o r a t e s e n s i t i v i t y o f S.
"typhimurium. A c h l o r a t e r e d u c t a s e activity l o c a t e d
in e l e c t r o p h o r e s e d extracts of a chiC m u t a n t exh i b i t e d the s a m e relative m o b i l i t y as thiosulfate
reductase; a phs m u t a t i o n was f o u n d to increase
c h l o r a t e resistance; a n d thiosulfate was shown to
p r o t e c t the chiC m u t a n t against the effects of
chlorate. S t o u t h a m e r a n d c o - w o r k e r s [9,10] d e m o n s t r a t e d a ' c h l o r a t e r e d u c t a s e C ' p r e s e n t in cells
of S. typhimurium a n d P. mirabilis g r o w n
a n a e r o b i c a l l y w i t h o u t an electron acceptor. Like
t h i o s u l f a t e r e d u c t a s e in P. mirabilis [18], ' c h l o r a t e
r e d u c t a s e C ' was n o t i n h i b i t e d b y azide [10]. T h a t
it was f o u n d in extracts o f n i t r a t e - g r o w n cells in
the a b s e n c e o f thiosulfate r e d u c t a s e activity a r g u e d
a g a i n s t the i d e n t i t y of this e n z y m e with thiosulfate
r e d u c t a s e at that time. H o w e v e r , the results pres e n t e d in Fig. 1 suggest that thiosulfate r e d u c t a s e
is a c t u a l l y p r e s e n t in n i t r a t e - g r o w n w i l d - t y p e cells,
b u t that it is a l t e r e d in such a w a y that it reduces
c h l o r a t e b u t n o t thiosulfate. A p o s s i b l e e x p l a n a tion for such b e h a v i o r is that the thiosulfate red u c t a s e p r o d u c e d in n i t r a t e - g r o w n chiC + cells
lacks a c y t o c h r o m e that is r e q u i r e d for thiosulfate
r e d u c t i o n , b u t is u n n e c e s s a r y for c h l o r a t e reduction b y the s a m e enzyme.
ACKNOWLEDGEMENTS
T h e s e studies were s u p p o r t e d b y Public H e a l t h
Service G r a n t s AI-15144 a n d AI-22685 f r o m the
n a t i o n a l I n s t i t u t e s o f H e a l t h a n d b y funds from
the C a l i f o r n i a A g r i c u l t u r a l E x p e r i m e n t Station.
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