- Email: [email protected]
Growth of Escherichia coli on selenate
573
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 2 5 7 9 3
G R O W T H OF E S C H E R I C H I A R. E. H U B E R * ,
C O L I ON S E L E N A T E
I. H. S E G E L AND R. S. C R I D D L E
Department of Biochemistry and Biophysics, University of California, Davis, Calif. ( U.S.A .) (Received J a n u a r y 9th, 1967)
SUMMARY
The growth characteristics of a selenium-tolerant sub-strain of Escherichia coli K - I 2 in synthetic medium containing o.oi M Na2SeO 4 were studied and compared to growth on sulfate-containing media. Growth curves in selenate medium displayed longer lag periods than comparable curves in sulfate medium. Growth in selenate medium was essentially linear rather than exponential. Efficiency of growth (g cells produced per g glucose utilized) on selenate was less than that on sulfate; selenategrown cells had a higher glycogen and total carbohydrate content than did the sulfate-grown cells. The sulfate contamination in o.oi M selenate was found to be less than 3" lO-5 M by a biological method. Biological and radioactive tracer studies suggest that the cells utilize selenium only if sulfur is also present and that selenium can replace only 30-40 % of the sulfur normally required. The maiority of I~sSelselenate utilized was incorporated into protein. The only organic form in which selenium was detected in appreciable quantities in protein was selenomethionine.
INTRODUCTION
Microorganisms have been shown to react to selenium compounds in a variety of ways. Inhibition of growth by selenium compounds is well documented and has been recently reviewed by ROSENFELD AND BEATH1. Complete replacement of some sulfur nutrients with their selenium analogs has also been claimed ~, 3. SHRIFT AND KELLY4 described experimental conditions under which Escherichia coli K - I 2 was adapted to grow in the presence of otherwise toxic levels of K2SeO 4 (o.oi M) and no added sulfate. These cells were found to grow exponentially after only a short lag period. Since the natural chemical environment of E. coli is generally very low in selenate, a strain of this organism adapted to grow in the presence of o.oi M selenate might be expected to have properties differing from normal. The experiments described in this paper were designed to compare some biological and chemical properties of selenium- and sulfur-grown cells, e.g., the relative efficiencies of sulfur and selenium uptake, growth yields, degree of replacement of sulfur b y selenium in cellular constituent, and identities of cellular compounds into which selenium was incorporated. * Shell P r e d o c t o r a l Fellow. P r e s e n t A d d r e s s : I n s t i t u t de Chimie Biologique, Facult6 des Sciences, P1. Victor H u g o , Marseille, France.
Biochim. Biophys. Acta, i41 (1967) .573-586
574
R.E.
HUBER, I. H. SEGEL, R. S. CRIDI)LE
MATERIALS AND METHODS
Bacteria The culture of E. coli K-I2 used was a kind gift from Dr. A. sHRIFTof the Kaiser Foundation Scientific Research Division, Richmond, Calif. This strain had been adapted to grow in the presence of high concentrations of K2SeO 4 (ref. 4). Reagents Na2SeO~, K~SeO~ and K~SeO3 were purchased from the K and K Laboratories. The selenium amino acids, selenomethionine and selenocystine, were purchased from the Sigma Chemical Company. Glucose and lactose were obtained from the Nutritional Biochemicals Corporation. Most of the other inorganic chemicals were purchased from the Mallinckrodt Chemical Works. Radioisotopes Radioactive 7'SeO3~- was obtained from the Oak Ridge National Laboratories. It was converted to 7'SeO42- b y the method of VIR~JPAKSI~AAND SHRIFTg. Although all the 7~Se that remained at the end of the oxidation procedure was 7*SeO,2- (as shown b y paper electrophoresis), recoveries varied from 5o-7o % indicating the loss of some volatile selenium compound during the oxidation. [3~S]Sulfate was purchased from the Oak Ridge National Laboratories. Media The basic liquid growth medium contained o.I M sodium phosphate buffer (pH 7.o), 1.5 g/1 NH4C1, 8 g/1 glucose or lactose, the desired sulfur or selenium source (generally o.oi M) and i.o ml/1 trace metals solution 6. Distilled water, subsequently passed through a mixed bed deionizing resin, was used in preparation of all media. The sugars were sterilized separately and added to the rest of the medium immediately before inoculation. An interchange of glucose or lactose as the carbon and energy source had essentially no effect on the total growth obtained. Solid medium was prepared by adding 3 To w/v agar to the above basic liquid medium. Growth conditions The cells were generally grown in submerged culture at 37 ° in a New Brunswick Scientific Company temperature-controlled gyrotory shaker operating at a speed of 200 rev./min and describing a 1-inch circle. Final cell densities obtained at temperatures from 28 ° to 37 ° were not significantly different. Usually 5 ° ml of media were used for growth in a 25o-ml erlenmeyer flask. The bacteria were maintained by transferring them to fresh liquid selenate-containing solid media every 2 or 3 days. The bacteria grown in the basic selenate-containing solid medium remained viable for periods up to one month when stored at 5°. Harvest Bacteria were harvested from liquid medium by centrifugation and washed with glucose- and sulfur (selenium)-free media before chemical analysis. Biochlm. Biophys. Asia, I4~ (1967) 573-586
575
GROWTH OF E. coli ON SELENATE
Measurement of growth Growth of the bacteria was followed by measuring turbidity at 45 ° mt~ in a Bausch and Lomb Spectronic 20 spectrophotometer. The extinction coefficients of selenium- and sulfur-grown cells were identical as verified b y dry weight measurements, cell nitrogen determinations b y a micro-kjeldahl method, and cell protein determinations b y the LOWR': method ~. 'Maximum growth' was generally measured after incubation Ior 7 days.
Glycogen and total carbohydrate determinations Cell glycogen was determined as described by SIGAL, CATTANEO AND SEGEL8. Total cell carbohydrate was measured in acid-hydrolysates of washed cells by an anthrone method 9.
Determination of radioactivity Samples containing either 3~S or ~nSe were counted in Packard Tri-Carb liquid scintillation counter. Samples containing both 3~S and 7nSe were counted in planchets, with and without an aluminum cover, in a Nuclear Chicago gas-flow counter. The radioactivity recorded with the aluminum cover in place resulted only from decay of ~nSe, a ~, emitter. The ratio of ~*Se radioactivity with cover/without cover was determined experimentally. Consequently, the radioactivity of the ~Se in the uncovered sample could be calculated. The radioactivity resulting from 35S decay could then be determined as total counts/rain in uncovered sample minus counts/rain of ~aSe in uncovered sample.
Determination of radioactive products formed in 7nSe-grown cells 75Se-grown cells were harvested, washed, and treated twice with IO % trichloroacetic acid at IOO°. The trichloroacetic acid-insoluble material (protein) was hydrolyzed b y three methods. (a) A portion of the cell protein was treated for 12 h at 37 ° with an equal weight of pancreatic trypsin (Washington Biochemical Corp.) in phosphate buffer (pH 8.3). (b) A second portion was treated for 12 h at 37 ° with an equal weight of pronase (Calbiochem. Corp.). (c) A third portion was hydrolyzed in 6 M HC1 at 1IO ° for 6 h. Suitable aliquots of each hydrolysate were chromatographed on W h a t m a n No. I paper, 4 ° cm × 3 cm. In some experiments, the samples at the origins were held for 60 sec above a solution containing 15 % H20 2 betore chromatography. This treatment resultedin the oxidation of selenocysteine and selenomethionine. The strips were developed with the upper layer of a butanol-acetic acid-water (lOO:25:125) solvent until the front had advanced about 25 cm past the origin. After development, the strips were dried and scanned on a Vanguard Autoscanner 8oo paper strip counter. Selenomethionine and selenocystine standards were tested for stability in hot trichloroacetic acid and were developed in the same way. After drying they were stained with ninhydrin dissolved in acetone to determine their location. RESULTS AND DISCUSSION
Adaptation of cells to selenate When the selenium-tolerant strain of E. coli was first received from Dr. A. SHRIFT, it failed to grow significantly in our synthetic medium containing O.Ol M Biochim. Biophys. Acla, 141 (1967) 573-586
576
R. E, HUBER, I, H. SEGEL, R. S. CRIDDLE
selenate and no ad d e d sulfur. However, the organism would grow in the presence of o.oz M selenate if sulfate were present. Two m e t h o d s were used to r e a d a p t the organism to g r o w t h in t h e m e d i a c o n t a i n i n g no ad d ed sulfur. Th e first m e t h o d inv o l v e d the transfer of t h e cells e v e r y 3 - 4 days to m e d i a containing progressively less sulfate u n t i l finally t h e r e q u i r e m e n t for a d d e d sulfur was eliminated. Th e second m e t h o d i n v o l v e d the periodic transfer of the cells to fresh m e d i u m containing o.oi M selenate, and no added sulfur regardless of how small the previous growth h ad been. B o t h m et h o d s were successful in a d a p t i n g the cells to grow in m e d i u m c o n t a i n i n g o.oi M selenate w i t h no a d d e d sulfate. Fig. I shows the results of the second m et h o d .
3.0 o
A4|o mF*
o
o o o
~
, O
2.O,
LO.
•
O NUMBER
OF
' -~"O--~
TRANSFERS
Fig. z. Adaptation of cells to grow in synthetic medium containing o.oI M selenate and no added sulfur. Transfers were made every e days and growth was estimated a~ter 7 days by light absorb~ ance at 45° m~. An absorbance (A45om~) of I is equivalent to 30o mg]l of cells (dry weight basis).
A~5or,w |.~
,~ ,~o ,~ ,L ~, ~ 2o )o 2o ,~o ,~, ~o ~'o 7o ,~, ,~o ~, HOURS OF GROWTH
Fig. 2. Growth curves plotted on ~ semi-log scale. ~ & , ~ r o ~ h on o,oi M sulfate; ~ ~, g r o ~ h on o.oi M selenate. The selenate medium was inoculated with stafionary~phase, selenategrown cells. Growth was estimated by absorbance at 450 m~.
Biochim. Biophys. Acta, I4 z (I967} 573-586
577
GROWTH OF E. coli ON SELENATE
In the early stages of adaptation, the medium turned red indicating the formation of metallic selenium. As the adaptation progressed, less color development was observed in the medium and after complete adaptation, no red color developed. (Selenate-adapted cells still reduced large amounts of selenate to selenium under anaerobic conditions.) The maximum growth yields obtained in the selenate medium varied between 300 to 900 rag/1. The variation very likely resulted from variable sulfur contamination in the air, water and medium constituents. In the same medium containing excess sulfate, the maximum growth yield was generally 2 g/1. The cells were also adapted to grow in media containing o.oi M selenite, and no added sulfur. Adaptation to selenite was achieved only by the first method described above for selenate adaptation. Maximum growth yields in selenite ranged from I5o to 300 rag/1. Similar attempts to adapt the cells to grow in media containing o.oi M selonocystine or selenomethionine were unsuccessful. Growth curves
Figs. 2 and 3 show semi-log and linear plots of the growth of the seleniumtolerant strain in media containing either o.oi M selenate or o.oi M sulfate, There are two striking differences between growth of the organisms in selenate compared with sulfate. Firstly, cells in selenate media show extremely long lag periods, occasionally ranging up to 60 h before growth starts. Secondly, growth in selenate is essentially linear, rather than exponential. The doubling time in selenate media is about IO h compared to 3 h for cells growing in sulfate media. The long lag period in selenate media may reflect a requirement for some essential sulfur compound before growth can begin. In selenate media, the synthesis of any critical sulfur compound can only
2.0"
o
~ , ~ o , ~ o ~ o ~ s ~ ~o~s
o~
~o~
G~o~
Fig. 3. G r o w t h c u r v e s p l o t t e d on a l i n e a r scale. & ~ & , g r o w t h on o.oi M s u l f a t e ; ~ , on o .o i M selenate. G r o w t h was e s t i m a t e d b y l i g h t a b s o r b a n c e a t 45o m~ .
~owth
Biochim. Biophys. Acta, 141 (1967) 573-586
578
R.E.
HUBER, I. I~. SEGEL, R. S. CRIDDLE
take place from a small amount of contaminating sulfur. Furthermore, the rate of synthesis of sulfur metabolites in the presence of high intracellular levels of selenium analogs may be extremely slow. Growth was estimated by light absorbance rather than direct counts of viable cells. Consequently, a long lag period would also be observed if only a small portion of the cells in the inoculum were viable. Using this alternative explanation, extrapolation of the rising portion of the growth curve in Fig. 2 to o h would suggest that only about 5-1o % of the cells in the selenate-grown inoculum may have been viable. The linear growth observed in selenate medium is reminiscent of the type of bacterial growth observed in the presence of amino acid analogs 1°. The explanation for linear growth is unknown. Linear growth would result if at cell division, some essential and limiting compound was unequally distributed between the two daughter cells. Consequently, only one of the cells would continue to grow until the next cell division. SHRIFTAND KELLY~ observed only a short lag period and subsequent exponential growth with the same strain. These authors used K2SeO~ prepared from SeOe in their medium. However, when we substituted commercial KzSeO~ tot Na~SeO~ in our media, we observed no significant difference in either length of lag period, nature of the growth, or final cell densities attained.
Growth e~ciency As shown in Table I, the efficiency of growth in selenate medium was markedly depressed compared to that obtained in sulfate medium. Furthermore, as shown in Table II, selenate-grown cells have a significantly higher glycogen content than sulfate-grown cells. These two results--decreased growth efficiency and high glycogen content--suggest a condition of 'uncoupled growth'7, u, i.e., a condition in which energy is made available faster than it can be utilized for biosynthetic reactions. Decreased growth yields and increased glycogen content have been observed during linear growth of Aerobacter aerogens induced by p-fluorophenylalanine12 and during experimental growth of a leaky threonine-requiring strain of E. coli in the absence of exogenous threonine (A. Si~Rlrr, personal communication). In the present study, it appears that selenate interferes with biosynthetic process to a much greater extent than with energy metabolism. Thus, several characteristics of growth on selenate are very TABLE 1 GROWTH EFFICIENCY ON SULFATI~ AND S~LENAT]~ MEDIA Cells wcrc g r o w n a e r o b i c a l l y for 6 d a y s in the synthetic m e d i u m c o n t a i n i n g e i t h e r o . o i M Na2SO 4 a n d no a d d e d selenium, or o.or M Na~SeO4 a n d no a d d e d sulfur. The glucose c o n c e n t r a t i o n s were g r o w t h l i m i t i n g . G r o w t h yield is recorded as g d r y w e i g h t cells p r o d u c e d pe r i o o g glucose utilized.
Glucose utilized (mg[l)
250 5° 0 750 iooo
Growth yield (%) Sulfate medium
Selenate medium
26 35 38 36
x lo I7 22
Biochim. Biophys. Acta, 141 (1967) 573-586
GROWTH OF E. coli ON SELENATE
579
similar to those observed i n the presence of a m i n o acid analogs. The u n c o u p l e d growth could very likely result from the s u b s t i t u t i o n of seleno-amino acids for sulfur a m i n o acids in proteins. TABLE II TOTAL CARBOHYDRATE AND GLYCOGEN CONTENT OF CELLS
Cells were grown in synthetic media containing a limiting amount of glucose and either o.oi M SO4~- or o.oi M SeO~~-. After growth ceased, the cells were harvested and analyzed.
Medium
Total carbohydrate* (%)
Glycogen*(%)
Sulfate Selenate
3.o i I. 5
i .2 8.5
*
Dry weight basis.
Determination of sulfur contamination in the medium constituents Fig. 4 shows the effect of added sulfate in the m a x i m u m growth yield of the cells in the absence of selenate. The intercept on the A450m/, axis (at zero added SO42-) represents growth on sulfur c o n t a m i n a t i o n from the air, water a n d m e d i u m constituents. While the results varied slightly from e x p e r i m e n t to e x p e r i m e n t the total c o n t a m i n a t i n g sulfur was sufficient to support growth to an A450m~ of o.2, or 6o mg dry weight of cells per 1. The intercept in the ' a d d e d sulfate' axis represents the t o t a l c o n t a m i n a t i n g sulfur concentration, in terms of sulfate. The results can be expressed m a t h e m a t i c a l l y as shown in E q n . I where G represents the total observed growth, (Sa) is the c o n c e n t r a t i o n of added sulfate, (Se) is the total c o n c e n t r a t i o n of c o n t a m i n a t i n g sulfur (as sulfate), a n d f is a constant. Thus, when (Sa) = o, G = f(Se), a n d when G = o, (Se) = o. Therefore, (Se) -~ distance between intercept a n d origin. ~ = ~(S~) + f(S~)
(i)
2.5.
2¸0
~4somF
0¸5
/ I
'~
~
~'
~
~
~
Added Sulfate Concentration [ x JOt)
Fig. 4. Dependency of maximum growth yield on the concentration of SO~~- added to the basal medium. The A 45omy values have been corrected for the absorbance of the inoculum. The inoculum was grown in o.oz M SeO~~- medium to minimize intracellular sulfur reserves.
Biochim. Biophys. Acta, 141 (1967) 573-586
580
R . E . HUBER, I. H. SEGEL, R. S. CRIDDLE
The experiment described in Fig. 4 was repeated with the medium constituents present at IO % the usual concentration (but all the components including glucose were still present in excess). If (Sc)I represents the total contaminating sulfate in the original medium, and (So)0.1 represents the total contaminating sulfate in the diluted medium, the two equations shown below can be written. (Sc)m represents the contaminating sulfate from the medium constituents, and (Se)x represents the contaminating sulfate from all other sources. (s~)l
= (s~)m + (sc)x
(2)
and (So)0.1 = o.1(Sc)m + (So)x
(3)
(Se)l and (Sc)0.1 were determined experimentally as described above. (Sc)m and (Sc)x were then calculated b y solving the two simultaneous equations. The results of these experiments showed that the total sulfate contamination in the usual I X medium, (Se)I, was 5.2. IO-~ M, while the total sulfate contamination in the diluted medium, (Sc)0.1, was 2.06. IO-~ M (average of 6 experiments). Thus, about 60 % of the total sulfur contamination comes from the medium constituents, and 4 ° % from the water and air. Reagent grade sodium phosphates contain about 0.003-0.005 % sulfur as sulfate. This contamination accounts for almost all the sulfur present in the medium constituents as determined b y the bioassay technique described above. These results point out the difficulty in preparing completely sulfur-free media, particularly in areas where air contamination with sulfur components m a y be high.
Determination of the sulfur contamination in the Na2Se0 4 A further refinement of the above method was used to estimate the maximum amount of sulfur contamination that could be present in the o.oi M Na2SeO 4 and also to establish whether sulfur and selenium were being used in some fixed ratio. Four series of media were prepared. One series contained o.oi M selenate, another 0.0075 M selenate, a third 0.005 M selenate, and a fourth series contained no selenate. Various additions of limiting sulfate concentrations were added to the flasks in each series. The cultures were grown for 7 days to allow growth to go to completion. If the growth obtained in any one series resulted solely from the sulfur present (as contamination in the selenate and from the added sulfate), the intercept of the straight line with the abscissa obtained for each series should be an estimate of the total sulfur contamination in the media (Eqn. 4). If, however, the bacteria were actually using the selenate, as well as the sulfur, (contamination plus added), the intercept will give a high estimate of 'sulfur contamination' since it will also include the selenium (Eqn. 5)(Sc)tot = (Sc)m ~- (-~c)seo:-
(4)
'(Sc)'tot = (Sc)m ~- (Sc)se042_ ~v (Se)
(5)
The concentrations of selenate in the three selenate-containing series were far in excess of what the cells could possibly utilize. Consequently, any difference in growth between media containing the same amount of added sulfate, but different amounts of added selenate, must result from either (a) contaminating sulfate in the added Biochim. Biophys. Acta, 14i (1967) 573-586
GROWTH OF E .
coli
oN SELENATE
58I
selenate, or (b) contaminating sulfate plus selenate if selenium can only be used when some sulfur is also present i.e., if selenium can only be used to replace a part of the cells' sulfur requirement. This latter condition implies that selenium can be utilized only in a fixed maximum ratio to sulfur. Fig. 5 shows the results of such an experiment. If we assume first that the increase in growth in the presence of selenate results only from the sulfur contamination in the selenate, then we obtain a maximum sulfur contamination level of about 1.5-3.o.Io-*M in o.oi M SeO4e-. For example, an increase in selenate concentration from 0.005 M to o.oo75 M yielded an increase in total apparent sulfate contamination of 5.5" IO-S moles, which is equivalent to 2.2. IO-* M sulfur (as sulfate) in o.oi M selenate. SI-IRIFT13 showed by means of neutronactivation analysis, that his K~SeO 4 prepared from SeO~ contained 3.0" IO-* moles of sulfur per o.oi mole of selenium. While he considered this small amount ot sulfur insignificant, in our experiments this amount of sulfur is sufficient to account for about half of the total growth obtained in the presence of o.oi M selenate.
1.5 ,~.4~om~,£
intercept = 2 ~ x l o "Q J 1.0
/ ]r~tercept = t6.5 xtO "~
/
int*rc~~=~t.O*t0-*
0.5-
Y
J J
intercept = 3.3 x ~0" l t !
J I I
l 2
! 3
! 4
I 5
I 6
Added Sulfote Concentration (xlO ~)
Fig. 5. M a x i m u m g r o w t h as a function of added sulfate in the presence of selenate concentrations indicated. The intercept values on the minus X axis are also indicated. G r o w t h was estimated b y light absorbance at 45o m p .
The slope of the curve lacking selenate in Fig. 5 is 0.049 absorbance units per pmole of added sulfate, while the slopes of the curves for the selenate-containing series average 0.070 absorbance units per pmole of added sulfate. The greater increment of growth per increment of sulfate at any fixed selenate concentration indicates that selenate is being utilized. Furthermore, the facts that the curves for the selenatecontaining series are parallel, and have a constant spacing between them show that sulfur and selenium are being used in a fixed ratio. One /zmole of sulfur will support the growth of about 15 mg dry weight of cells in the absence of selenate, and 21 mg dry weight of cells in the presence of selenate. The ratio of selenium to sulfur utilization then is 6 : 15 or 0.4. We can now revise our estimate of the sulfur contamination in the sodium selenate. While the total apparent contamination was about 1.5-3.o. io -~ moles of sulfur per o.oi mole of selenium, the results shown in Fig. 5 suggest that Biochim. Biophys. Acta, 141 (1967) 573-586
582
R.E.
HUBER, I. H. SEGEL, R. S. CRIDDLE
40 % of the 'contamination-supported growth' was actually selenium-supported growth. The selenate then must contain about o.9-~.8, xo-~ moles of sulfur per o.o~ mole selenium (o.9-~.8. ~o-~ mole %). The experiments described in Fig. 5 were conducted at relatively high selenate concentrations (o.oo~5-o.o~ M). Fig. 6 shows the effect of varying selenate concentrations on the maximum growth obtained in synthetic media containing a limited
5.0.
2.0"
J
A+eot,~. 1.0
,
~
~
:
~
[
!
~
o.oo~
!
~
I
:
!
!
~
o.o~
]
~ ! ~ ! I
o.o~
o,o~
Se O~'conc~Ml Fig. 6. M a x i m u m g r o w t h as a function of added selenate in the presence of a limiting a m o u n t of sulfate concentration. The limiting sulfate concentration in each flask was 4" I°-S M. G r o w t h was estimated by light absorbance at 450 m#. TABLE III INCORPORATION
O F 3SS A N D
rSSe I N T O C E L L P R O T E I N
The cells were grown for 7 days in synthetic media containing either ~ S O ~ ~- (Expt. i), asSO4e- plus unlabeled SeOi ~- (Expt. z), or 7sSeO~- (Expt. 3). Cell protein was isolated as described in ~IATI~RIALS AND
METHODS.
Expt. 'Sulfur" source No. (M)
I 2 3
3sSOa z-* (4-5" Io-~) s~SO~~-* (4.5"1o -~) + SeOl ~- (lO -3) 7SSeO~ ~-** (5" IO-a)
Incorporation of label into protein
Replacement of Se for S
counts/rain per mg fzmoles/mg
t~moles/mg %
1.66- lOT
0.395
o
o.97" lO7 3.38. ~o4
o.231 o.13
o.164"** (o.I3)
o 41.6 *** 32.9 §
* Specific activity, 4.2. lOT c o u n t s / m i n per #mole, based on sulfate added (4" IO-S M), plus c o n t a m i n a t i n g sulfur (as sulfate) in the m e d i u m constituents (o. 5. io -s M). ** Specific activity, 2.6. ios c o u n t s / m i n per/~mole. *** Replacement for Se for S calculated in E x p t . 2 as depression of incorporation of 3~S, compared to control (Expt. i). § Moles of Se (Expt. 2)/moles S (Expt. I) × ioo.
Biochim. Biophys. Acta, i 4 i (r967) 573-586
GROWTH OF E. coli ON SELENATE
583
amount of sulfate (4" IO-~ M). The results suggest that selenate must be present in a relatively high concentration before it can effectively compete with sulfur.
Incorporation of 3~S and ~*Se into cell protein The bioassay technique described above suggested that when sulfur is limiting, selenium can replace about 40 % of the cell's sulfur requirement. The validity of the bioassay technique is supported by the results shown in Table I I I . I t can be seen in Expts. ~ and 2, that in the presence 'of limiting amounts of ~SO~ ~-, o.oi M SeO¢~-
~00
0
UNTREATED
~
TRYPSIN
~"
PRONAS'E
~
200 Ioo
--_
_
~1~
b
2OC
I(X o
._~ ~ ~ ,,- ~ ~o
b
IOC
t
•~ ~e
~
ACID HYDROLYSIS
~;
~.~ ~
~OC
8 ~o~ ~ I~¢
ACID HYDROLYSIS(OXIDIZED)
~
~
CM
~
FROM THE
ORIGIN
,'~
~'o
~'5
Fig. 7- D i s t r i b u t i o n of r a d i o a c t i v i t y a l o n g c h r o m a t o g r a p h y strips. O is t h e origin w hi l e F is t h e s o l v e n t front. E a c h s t r i p h a d a r a d i o a c t i v e m a r k e r a p p l i e d a b o u t 2 c m before t h e origin. The s t r i p s were s c a n n e d a t 12 i n c h / h w i t h a o.5-inch slit w i d t h . T A B L E IV ~sSe CONTENT OF CELL PROTEIN Cells g r o w n on o.oo 5 M ~*$eO~ ~- were h a r v e s t e d , washed, a n d t h e n d i s r u p t e d b y sonic oscillation. The d i a ] y z e d s o n i c a t e w a s p r e c i p i t a t e d w i t h lO% t r i c h l o r o a c e t i c a c i d a t IOO°. The p r e c i p i t a t e w a s w a s h e d w i t h t r i c h l o r o a c e t i c acid, d i a l y z e d a n d c o u n t e d . The t r i c h l o r o a c e t i c a c i d w a s t h e n dissolved in i M N a O H , p r e c i p i t a t e d a n d w a s h e d a second t i m e w i t h t r i c h l o r o a c e t i c acid.
Fraction
~SSe content (counts/min per rng prote$n)
W h o l e cells Dialyzed sonicate D i a l y z e d t r i c h l o r o a c e t i c acid (precipitate) (rst) D i a l y z e d t r i c h l o r o a c e t i c acid (precipitate) (znd)
34 32 24 26
ooo ooo ooo ooo
Biochim. Biophys. Acta, i4~ (i967) 573-586
584
It. E. HUBER, I. H. SEGEL, R. S. CRIDDLE
will depress ~S incorporation b y 41.6 %. From this experiment alone, it is impossible to tell how much of the depression results from substitution of selenium for sulfur, and how much from dilution of the specific activity of the sulfate b y sulfur in the selenate. However, in Expt. 3, where the cells were grown in the presence of ~SeO, ~and no added sulfur (to maximize selenium incorporation), the selenium content of the cell protein was 32. 9 % of the usual sulfur content (Expt. I). The results confirm that selenium is utilized by the cells in place of part of the normal sulfur requirement.
Identification, of the 7*Se-labeled compounds in the protein fraction The results in Table IV show that about 7 ° % of the 7*Se in the cells and protein fraction is present in a form that cannot be washed or dialyzed away from the protein. In order to identify the labeled components in the protein fraction, the protein was hydrolyzed with trypsin, pronase, and acid as described earlier. The hydrolysates were chromatographed and the resulting chromatograms were scanned for radioactivity. The results are shown in Fig. 7. It can be seen that virtually all the radioactivity remains at the origin in the chromatograms of the untreated protein. Treatment with trypsin or pronase to produce peptides of various sizes results in a smear of radioactivity in the subsequent chromatograms. These results show that (a) the selenium was present in the protein in compounds linked by peptide bonds, and (b), the selenium was more or less randomly distributed in all cell proteins, i.e., there was no particularly selenium-rich peptide present. Acid hydrolysis yields a well-defined peak at about 13 cm from the origin. The radioactive peak migrated coincidently with authentic unlabeled selenomethionine. When the acid hydrolysate was exposed to H20 ~ prior to chromatograph, the bulk of the radioactivity subsequently migrated to about 5 cm from the origin, a position that corresponds to selenomethionine sulfoxide and sulfone 1~. No selenocysteine or selenocystine was detected. However, it
1.5
60~ °
° °
1.0
~ 4oo~
.~
__~___2. . . . . . ~---4
~
~ ~
l
.'''"
L, £ ~
"
~
A~om~
~ 0.5
~o~ ~ o
~ ~ ; ~ ;0 ,~ ~ ,~,~o,g ,io ,~ ,io ~ ~ ,~ ;o HOURS
Fig. 8. G r o w t h and u p t a k e of 7~Se and 3~S. The cells were grown in synthetic medium containing high specific activity, o.oi M ~SeO~ ~-, carrier-free 3~SO~e-, and no added sulfate. @--(D, g r o w t h determined b y absorbance at 45 ° m # ; × - - × , counts of 3~S per IO/~1 of cell suspension. The cells used were washed and resuspended at same cell density as in the g r o w t h m e d i u m ; & - - / X , counts of ~Se in o. 5 ml of cell suspension. The cells were washed as above. All counts are corrected to the value one ~vould observe w i t h o u t the a l u m i n u m cover.
Biochim. Biophys, Acta, 141 (1967) 573-586
GROWTH
OF
E. coli oN
SELENATE
585
should be emphasized t h a t selenocysteine and selenocystine are quite unstable to acid hydrolysis and could have been destroyed ~*, ~s. Simultaneous uptake of 7~Se042F i g . 8 shows the simultaneous uptake of 3sS and 75Se b y cells growing in synthetic m e d i u m containing o.oi M SeO~2- and no added sulfur. Fig. 9 shows the d a t a as the ratio of 35S/VSSe radioactivity per unit weight of cells.
OC 4¢ O ~0 •
A
,~ io ~, go ~o ~, ~o~o' ,I,
~o ,g ,,o ~..' '
•
i i ,~,,~ ~o ,oo ,,~
HOURS
Fig. 9. Ratio of [35S]sulfate counts to [VSSe]selenate counts taken up by the cells. Values are corrected for a constant cell weight. The ratio plot shows t h a t very early in the growth cycle, sulfur is preferentially used over selenium. The S/Se ratio passes through a m a x i m u m and then falls to a constant value indicating again t h a t sulfur and selenium are used in a fixed ratio. If, during the initial growth period, the available sulfur is largely depleted from the medium, then subsequent growth after 20 h (maximum S/Se) will depend upon autolysis and reutilization of liberated sulfur. This suggestion is consistent with linear growth and very long lag periods in high selenate medium. CONCLUSIONS Growth of selenium adapted E. coli cells on selenate media with no sulfur added has been shown to be due in large part to sulfur contamination from the media water and air. The E. coli can, however, utilize selenium in the presence of limiting sulfur to contribute to cell growth. W i t h excess selenate, a limiting ratio of about 40 % Se to 60 % S m a y be obtained. Biological estimates of S contamination show t h a t the media without added selenate contained 5.2" IO-~ M sulfur while the media containing o.oi M selenate were 2.27" lO -5 M in sulfur. It appears t h a t no growth will occur in the absence of some sulfur. Growth in the presence of selenium is characterized b y long lag periods, linear growth curves and a decreased efficiency. Total growth is m a r k e d l y reduced. These observations differ somewhat from those of SHRIFT AND KELLYa even t h o u g h the same strain of E. coli was used. Their growth patterns showed m u c h shorter lag times Biochim. Biophys. Acta, 141 (1967) 573-586
586
R.E.
HUBER, I. H. SEGEL, R. S. CRIDDLE
and exponential growth. The source of selenium used was different, however, and some differences m a y be accounted for in terms of different sulfur contamination. Uptake of radioactive [75Se]selenate showed that most of the selenium which is incorporated into the-cells under these growth conditions seems to be firmly bound to the protein and is released as trichloroacetic acid-soluble material only when the peptide bonds are hydrolized. Paper chromatography of acid hydrolysates of the protein strongly suggest the presence of selenomethionine but there is no evidence for the presence of selenocystine. ACKNOWLEDGEMENT
This investigation was supported in part by U.S. Public Health Service Grants GM-I2292 and GM-IooI 7. REFERENCES I I. ROSI~NFELD AND O. A. BEATH, Selenium, Academic Press, New York, I964, p. i 11. 2 H. G. MAUTNER AND W. H. H. GUNTHER, Biochim. Biophys. Acta, 36 (1959) 561. 3 D. B. C o w l s AND G. N. COHEN, Biochim. Biophys. Acta, 26 (1957) 252. 4 A. SHRIFT AND E. KELLY, Nature, 195 (1962) 732. 5 T. K. VIRUPAKSHA AND A. SHRIFT, Biochim. Biophys. Acta, 74 (1963) 319. 6 L. A. YAMAMOTO AND 1. H. SEGEL, Arch. Biochem. Biophys., i i 4 (1966) 523 . 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 N. SIGAL, J. CATTANEO AND I. H. SEGEL, Arch. Biochem. Biophys., lO8 (1964) 44 o. 9 J. FOND, F. L. SCHAI*I~ERAND P. L. KIRK, Arch. Biochem. Biophys., 45 (1953) 319 • IO M. H. RICHMOND, Bacteriol. Rev., 26 (1962) 398. i i J. C. S~NEZ, Bacteriol. Rev., 26 (1962) 9512 I. H. SEGEL, J. CATTANCO AND N. SIGAL, Syrup. on the Mechanisms of Regulation of Cellular Activities in Microorganisms, Proc. Intern. C.N.S.R., 1963, C.N.R.S., Paris, 1965. 13 J. SCALA AND H. H. WILLIAMS, J. Chromatog., 15 (1964) 546. 14 R. E. HUBER AND R. S. CRIDDLE, Biochim. Biophys. Aeta, 141 (1967) 587 . 15 R. E. HUBER AND R. S. CRIDDLE, Arch. Biochem. Biophys., in the press.
Biochim. Biophys. Acta, 141 (1967) 573-586
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 2 5 7 9 3
G R O W T H OF E S C H E R I C H I A R. E. H U B E R * ,
C O L I ON S E L E N A T E
I. H. S E G E L AND R. S. C R I D D L E
Department of Biochemistry and Biophysics, University of California, Davis, Calif. ( U.S.A .) (Received J a n u a r y 9th, 1967)
SUMMARY
The growth characteristics of a selenium-tolerant sub-strain of Escherichia coli K - I 2 in synthetic medium containing o.oi M Na2SeO 4 were studied and compared to growth on sulfate-containing media. Growth curves in selenate medium displayed longer lag periods than comparable curves in sulfate medium. Growth in selenate medium was essentially linear rather than exponential. Efficiency of growth (g cells produced per g glucose utilized) on selenate was less than that on sulfate; selenategrown cells had a higher glycogen and total carbohydrate content than did the sulfate-grown cells. The sulfate contamination in o.oi M selenate was found to be less than 3" lO-5 M by a biological method. Biological and radioactive tracer studies suggest that the cells utilize selenium only if sulfur is also present and that selenium can replace only 30-40 % of the sulfur normally required. The maiority of I~sSelselenate utilized was incorporated into protein. The only organic form in which selenium was detected in appreciable quantities in protein was selenomethionine.
INTRODUCTION
Microorganisms have been shown to react to selenium compounds in a variety of ways. Inhibition of growth by selenium compounds is well documented and has been recently reviewed by ROSENFELD AND BEATH1. Complete replacement of some sulfur nutrients with their selenium analogs has also been claimed ~, 3. SHRIFT AND KELLY4 described experimental conditions under which Escherichia coli K - I 2 was adapted to grow in the presence of otherwise toxic levels of K2SeO 4 (o.oi M) and no added sulfate. These cells were found to grow exponentially after only a short lag period. Since the natural chemical environment of E. coli is generally very low in selenate, a strain of this organism adapted to grow in the presence of o.oi M selenate might be expected to have properties differing from normal. The experiments described in this paper were designed to compare some biological and chemical properties of selenium- and sulfur-grown cells, e.g., the relative efficiencies of sulfur and selenium uptake, growth yields, degree of replacement of sulfur b y selenium in cellular constituent, and identities of cellular compounds into which selenium was incorporated. * Shell P r e d o c t o r a l Fellow. P r e s e n t A d d r e s s : I n s t i t u t de Chimie Biologique, Facult6 des Sciences, P1. Victor H u g o , Marseille, France.
Biochim. Biophys. Acta, i41 (1967) .573-586
574
R.E.
HUBER, I. H. SEGEL, R. S. CRIDI)LE
MATERIALS AND METHODS
Bacteria The culture of E. coli K-I2 used was a kind gift from Dr. A. sHRIFTof the Kaiser Foundation Scientific Research Division, Richmond, Calif. This strain had been adapted to grow in the presence of high concentrations of K2SeO 4 (ref. 4). Reagents Na2SeO~, K~SeO~ and K~SeO3 were purchased from the K and K Laboratories. The selenium amino acids, selenomethionine and selenocystine, were purchased from the Sigma Chemical Company. Glucose and lactose were obtained from the Nutritional Biochemicals Corporation. Most of the other inorganic chemicals were purchased from the Mallinckrodt Chemical Works. Radioisotopes Radioactive 7'SeO3~- was obtained from the Oak Ridge National Laboratories. It was converted to 7'SeO42- b y the method of VIR~JPAKSI~AAND SHRIFTg. Although all the 7~Se that remained at the end of the oxidation procedure was 7*SeO,2- (as shown b y paper electrophoresis), recoveries varied from 5o-7o % indicating the loss of some volatile selenium compound during the oxidation. [3~S]Sulfate was purchased from the Oak Ridge National Laboratories. Media The basic liquid growth medium contained o.I M sodium phosphate buffer (pH 7.o), 1.5 g/1 NH4C1, 8 g/1 glucose or lactose, the desired sulfur or selenium source (generally o.oi M) and i.o ml/1 trace metals solution 6. Distilled water, subsequently passed through a mixed bed deionizing resin, was used in preparation of all media. The sugars were sterilized separately and added to the rest of the medium immediately before inoculation. An interchange of glucose or lactose as the carbon and energy source had essentially no effect on the total growth obtained. Solid medium was prepared by adding 3 To w/v agar to the above basic liquid medium. Growth conditions The cells were generally grown in submerged culture at 37 ° in a New Brunswick Scientific Company temperature-controlled gyrotory shaker operating at a speed of 200 rev./min and describing a 1-inch circle. Final cell densities obtained at temperatures from 28 ° to 37 ° were not significantly different. Usually 5 ° ml of media were used for growth in a 25o-ml erlenmeyer flask. The bacteria were maintained by transferring them to fresh liquid selenate-containing solid media every 2 or 3 days. The bacteria grown in the basic selenate-containing solid medium remained viable for periods up to one month when stored at 5°. Harvest Bacteria were harvested from liquid medium by centrifugation and washed with glucose- and sulfur (selenium)-free media before chemical analysis. Biochlm. Biophys. Asia, I4~ (1967) 573-586
575
GROWTH OF E. coli ON SELENATE
Measurement of growth Growth of the bacteria was followed by measuring turbidity at 45 ° mt~ in a Bausch and Lomb Spectronic 20 spectrophotometer. The extinction coefficients of selenium- and sulfur-grown cells were identical as verified b y dry weight measurements, cell nitrogen determinations b y a micro-kjeldahl method, and cell protein determinations b y the LOWR': method ~. 'Maximum growth' was generally measured after incubation Ior 7 days.
Glycogen and total carbohydrate determinations Cell glycogen was determined as described by SIGAL, CATTANEO AND SEGEL8. Total cell carbohydrate was measured in acid-hydrolysates of washed cells by an anthrone method 9.
Determination of radioactivity Samples containing either 3~S or ~nSe were counted in Packard Tri-Carb liquid scintillation counter. Samples containing both 3~S and 7nSe were counted in planchets, with and without an aluminum cover, in a Nuclear Chicago gas-flow counter. The radioactivity recorded with the aluminum cover in place resulted only from decay of ~nSe, a ~, emitter. The ratio of ~*Se radioactivity with cover/without cover was determined experimentally. Consequently, the radioactivity of the ~Se in the uncovered sample could be calculated. The radioactivity resulting from 35S decay could then be determined as total counts/rain in uncovered sample minus counts/rain of ~aSe in uncovered sample.
Determination of radioactive products formed in 7nSe-grown cells 75Se-grown cells were harvested, washed, and treated twice with IO % trichloroacetic acid at IOO°. The trichloroacetic acid-insoluble material (protein) was hydrolyzed b y three methods. (a) A portion of the cell protein was treated for 12 h at 37 ° with an equal weight of pancreatic trypsin (Washington Biochemical Corp.) in phosphate buffer (pH 8.3). (b) A second portion was treated for 12 h at 37 ° with an equal weight of pronase (Calbiochem. Corp.). (c) A third portion was hydrolyzed in 6 M HC1 at 1IO ° for 6 h. Suitable aliquots of each hydrolysate were chromatographed on W h a t m a n No. I paper, 4 ° cm × 3 cm. In some experiments, the samples at the origins were held for 60 sec above a solution containing 15 % H20 2 betore chromatography. This treatment resultedin the oxidation of selenocysteine and selenomethionine. The strips were developed with the upper layer of a butanol-acetic acid-water (lOO:25:125) solvent until the front had advanced about 25 cm past the origin. After development, the strips were dried and scanned on a Vanguard Autoscanner 8oo paper strip counter. Selenomethionine and selenocystine standards were tested for stability in hot trichloroacetic acid and were developed in the same way. After drying they were stained with ninhydrin dissolved in acetone to determine their location. RESULTS AND DISCUSSION
Adaptation of cells to selenate When the selenium-tolerant strain of E. coli was first received from Dr. A. SHRIFT, it failed to grow significantly in our synthetic medium containing O.Ol M Biochim. Biophys. Acla, 141 (1967) 573-586
576
R. E, HUBER, I, H. SEGEL, R. S. CRIDDLE
selenate and no ad d e d sulfur. However, the organism would grow in the presence of o.oz M selenate if sulfate were present. Two m e t h o d s were used to r e a d a p t the organism to g r o w t h in t h e m e d i a c o n t a i n i n g no ad d ed sulfur. Th e first m e t h o d inv o l v e d the transfer of t h e cells e v e r y 3 - 4 days to m e d i a containing progressively less sulfate u n t i l finally t h e r e q u i r e m e n t for a d d e d sulfur was eliminated. Th e second m e t h o d i n v o l v e d the periodic transfer of the cells to fresh m e d i u m containing o.oi M selenate, and no added sulfur regardless of how small the previous growth h ad been. B o t h m et h o d s were successful in a d a p t i n g the cells to grow in m e d i u m c o n t a i n i n g o.oi M selenate w i t h no a d d e d sulfate. Fig. I shows the results of the second m et h o d .
3.0 o
A4|o mF*
o
o o o
~
, O
2.O,
LO.
•
O NUMBER
OF
' -~"O--~
TRANSFERS
Fig. z. Adaptation of cells to grow in synthetic medium containing o.oI M selenate and no added sulfur. Transfers were made every e days and growth was estimated a~ter 7 days by light absorb~ ance at 45° m~. An absorbance (A45om~) of I is equivalent to 30o mg]l of cells (dry weight basis).
A~5or,w |.~
,~ ,~o ,~ ,L ~, ~ 2o )o 2o ,~o ,~, ~o ~'o 7o ,~, ,~o ~, HOURS OF GROWTH
Fig. 2. Growth curves plotted on ~ semi-log scale. ~ & , ~ r o ~ h on o,oi M sulfate; ~ ~, g r o ~ h on o.oi M selenate. The selenate medium was inoculated with stafionary~phase, selenategrown cells. Growth was estimated by absorbance at 450 m~.
Biochim. Biophys. Acta, I4 z (I967} 573-586
577
GROWTH OF E. coli ON SELENATE
In the early stages of adaptation, the medium turned red indicating the formation of metallic selenium. As the adaptation progressed, less color development was observed in the medium and after complete adaptation, no red color developed. (Selenate-adapted cells still reduced large amounts of selenate to selenium under anaerobic conditions.) The maximum growth yields obtained in the selenate medium varied between 300 to 900 rag/1. The variation very likely resulted from variable sulfur contamination in the air, water and medium constituents. In the same medium containing excess sulfate, the maximum growth yield was generally 2 g/1. The cells were also adapted to grow in media containing o.oi M selenite, and no added sulfur. Adaptation to selenite was achieved only by the first method described above for selenate adaptation. Maximum growth yields in selenite ranged from I5o to 300 rag/1. Similar attempts to adapt the cells to grow in media containing o.oi M selonocystine or selenomethionine were unsuccessful. Growth curves
Figs. 2 and 3 show semi-log and linear plots of the growth of the seleniumtolerant strain in media containing either o.oi M selenate or o.oi M sulfate, There are two striking differences between growth of the organisms in selenate compared with sulfate. Firstly, cells in selenate media show extremely long lag periods, occasionally ranging up to 60 h before growth starts. Secondly, growth in selenate is essentially linear, rather than exponential. The doubling time in selenate media is about IO h compared to 3 h for cells growing in sulfate media. The long lag period in selenate media may reflect a requirement for some essential sulfur compound before growth can begin. In selenate media, the synthesis of any critical sulfur compound can only
2.0"
o
~ , ~ o , ~ o ~ o ~ s ~ ~o~s
o~
~o~
G~o~
Fig. 3. G r o w t h c u r v e s p l o t t e d on a l i n e a r scale. & ~ & , g r o w t h on o.oi M s u l f a t e ; ~ , on o .o i M selenate. G r o w t h was e s t i m a t e d b y l i g h t a b s o r b a n c e a t 45o m~ .
~owth
Biochim. Biophys. Acta, 141 (1967) 573-586
578
R.E.
HUBER, I. I~. SEGEL, R. S. CRIDDLE
take place from a small amount of contaminating sulfur. Furthermore, the rate of synthesis of sulfur metabolites in the presence of high intracellular levels of selenium analogs may be extremely slow. Growth was estimated by light absorbance rather than direct counts of viable cells. Consequently, a long lag period would also be observed if only a small portion of the cells in the inoculum were viable. Using this alternative explanation, extrapolation of the rising portion of the growth curve in Fig. 2 to o h would suggest that only about 5-1o % of the cells in the selenate-grown inoculum may have been viable. The linear growth observed in selenate medium is reminiscent of the type of bacterial growth observed in the presence of amino acid analogs 1°. The explanation for linear growth is unknown. Linear growth would result if at cell division, some essential and limiting compound was unequally distributed between the two daughter cells. Consequently, only one of the cells would continue to grow until the next cell division. SHRIFTAND KELLY~ observed only a short lag period and subsequent exponential growth with the same strain. These authors used K2SeO~ prepared from SeOe in their medium. However, when we substituted commercial KzSeO~ tot Na~SeO~ in our media, we observed no significant difference in either length of lag period, nature of the growth, or final cell densities attained.
Growth e~ciency As shown in Table I, the efficiency of growth in selenate medium was markedly depressed compared to that obtained in sulfate medium. Furthermore, as shown in Table II, selenate-grown cells have a significantly higher glycogen content than sulfate-grown cells. These two results--decreased growth efficiency and high glycogen content--suggest a condition of 'uncoupled growth'7, u, i.e., a condition in which energy is made available faster than it can be utilized for biosynthetic reactions. Decreased growth yields and increased glycogen content have been observed during linear growth of Aerobacter aerogens induced by p-fluorophenylalanine12 and during experimental growth of a leaky threonine-requiring strain of E. coli in the absence of exogenous threonine (A. Si~Rlrr, personal communication). In the present study, it appears that selenate interferes with biosynthetic process to a much greater extent than with energy metabolism. Thus, several characteristics of growth on selenate are very TABLE 1 GROWTH EFFICIENCY ON SULFATI~ AND S~LENAT]~ MEDIA Cells wcrc g r o w n a e r o b i c a l l y for 6 d a y s in the synthetic m e d i u m c o n t a i n i n g e i t h e r o . o i M Na2SO 4 a n d no a d d e d selenium, or o.or M Na~SeO4 a n d no a d d e d sulfur. The glucose c o n c e n t r a t i o n s were g r o w t h l i m i t i n g . G r o w t h yield is recorded as g d r y w e i g h t cells p r o d u c e d pe r i o o g glucose utilized.
Glucose utilized (mg[l)
250 5° 0 750 iooo
Growth yield (%) Sulfate medium
Selenate medium
26 35 38 36
x lo I7 22
Biochim. Biophys. Acta, 141 (1967) 573-586
GROWTH OF E. coli ON SELENATE
579
similar to those observed i n the presence of a m i n o acid analogs. The u n c o u p l e d growth could very likely result from the s u b s t i t u t i o n of seleno-amino acids for sulfur a m i n o acids in proteins. TABLE II TOTAL CARBOHYDRATE AND GLYCOGEN CONTENT OF CELLS
Cells were grown in synthetic media containing a limiting amount of glucose and either o.oi M SO4~- or o.oi M SeO~~-. After growth ceased, the cells were harvested and analyzed.
Medium
Total carbohydrate* (%)
Glycogen*(%)
Sulfate Selenate
3.o i I. 5
i .2 8.5
*
Dry weight basis.
Determination of sulfur contamination in the medium constituents Fig. 4 shows the effect of added sulfate in the m a x i m u m growth yield of the cells in the absence of selenate. The intercept on the A450m/, axis (at zero added SO42-) represents growth on sulfur c o n t a m i n a t i o n from the air, water a n d m e d i u m constituents. While the results varied slightly from e x p e r i m e n t to e x p e r i m e n t the total c o n t a m i n a t i n g sulfur was sufficient to support growth to an A450m~ of o.2, or 6o mg dry weight of cells per 1. The intercept in the ' a d d e d sulfate' axis represents the t o t a l c o n t a m i n a t i n g sulfur concentration, in terms of sulfate. The results can be expressed m a t h e m a t i c a l l y as shown in E q n . I where G represents the total observed growth, (Sa) is the c o n c e n t r a t i o n of added sulfate, (Se) is the total c o n c e n t r a t i o n of c o n t a m i n a t i n g sulfur (as sulfate), a n d f is a constant. Thus, when (Sa) = o, G = f(Se), a n d when G = o, (Se) = o. Therefore, (Se) -~ distance between intercept a n d origin. ~ = ~(S~) + f(S~)
(i)
2.5.
2¸0
~4somF
0¸5
/ I
'~
~
~'
~
~
~
Added Sulfate Concentration [ x JOt)
Fig. 4. Dependency of maximum growth yield on the concentration of SO~~- added to the basal medium. The A 45omy values have been corrected for the absorbance of the inoculum. The inoculum was grown in o.oz M SeO~~- medium to minimize intracellular sulfur reserves.
Biochim. Biophys. Acta, 141 (1967) 573-586
580
R . E . HUBER, I. H. SEGEL, R. S. CRIDDLE
The experiment described in Fig. 4 was repeated with the medium constituents present at IO % the usual concentration (but all the components including glucose were still present in excess). If (Sc)I represents the total contaminating sulfate in the original medium, and (So)0.1 represents the total contaminating sulfate in the diluted medium, the two equations shown below can be written. (Sc)m represents the contaminating sulfate from the medium constituents, and (Se)x represents the contaminating sulfate from all other sources. (s~)l
= (s~)m + (sc)x
(2)
and (So)0.1 = o.1(Sc)m + (So)x
(3)
(Se)l and (Sc)0.1 were determined experimentally as described above. (Sc)m and (Sc)x were then calculated b y solving the two simultaneous equations. The results of these experiments showed that the total sulfate contamination in the usual I X medium, (Se)I, was 5.2. IO-~ M, while the total sulfate contamination in the diluted medium, (Sc)0.1, was 2.06. IO-~ M (average of 6 experiments). Thus, about 60 % of the total sulfur contamination comes from the medium constituents, and 4 ° % from the water and air. Reagent grade sodium phosphates contain about 0.003-0.005 % sulfur as sulfate. This contamination accounts for almost all the sulfur present in the medium constituents as determined b y the bioassay technique described above. These results point out the difficulty in preparing completely sulfur-free media, particularly in areas where air contamination with sulfur components m a y be high.
Determination of the sulfur contamination in the Na2Se0 4 A further refinement of the above method was used to estimate the maximum amount of sulfur contamination that could be present in the o.oi M Na2SeO 4 and also to establish whether sulfur and selenium were being used in some fixed ratio. Four series of media were prepared. One series contained o.oi M selenate, another 0.0075 M selenate, a third 0.005 M selenate, and a fourth series contained no selenate. Various additions of limiting sulfate concentrations were added to the flasks in each series. The cultures were grown for 7 days to allow growth to go to completion. If the growth obtained in any one series resulted solely from the sulfur present (as contamination in the selenate and from the added sulfate), the intercept of the straight line with the abscissa obtained for each series should be an estimate of the total sulfur contamination in the media (Eqn. 4). If, however, the bacteria were actually using the selenate, as well as the sulfur, (contamination plus added), the intercept will give a high estimate of 'sulfur contamination' since it will also include the selenium (Eqn. 5)(Sc)tot = (Sc)m ~- (-~c)seo:-
(4)
'(Sc)'tot = (Sc)m ~- (Sc)se042_ ~v (Se)
(5)
The concentrations of selenate in the three selenate-containing series were far in excess of what the cells could possibly utilize. Consequently, any difference in growth between media containing the same amount of added sulfate, but different amounts of added selenate, must result from either (a) contaminating sulfate in the added Biochim. Biophys. Acta, 14i (1967) 573-586
GROWTH OF E .
coli
oN SELENATE
58I
selenate, or (b) contaminating sulfate plus selenate if selenium can only be used when some sulfur is also present i.e., if selenium can only be used to replace a part of the cells' sulfur requirement. This latter condition implies that selenium can be utilized only in a fixed maximum ratio to sulfur. Fig. 5 shows the results of such an experiment. If we assume first that the increase in growth in the presence of selenate results only from the sulfur contamination in the selenate, then we obtain a maximum sulfur contamination level of about 1.5-3.o.Io-*M in o.oi M SeO4e-. For example, an increase in selenate concentration from 0.005 M to o.oo75 M yielded an increase in total apparent sulfate contamination of 5.5" IO-S moles, which is equivalent to 2.2. IO-* M sulfur (as sulfate) in o.oi M selenate. SI-IRIFT13 showed by means of neutronactivation analysis, that his K~SeO 4 prepared from SeO~ contained 3.0" IO-* moles of sulfur per o.oi mole of selenium. While he considered this small amount ot sulfur insignificant, in our experiments this amount of sulfur is sufficient to account for about half of the total growth obtained in the presence of o.oi M selenate.
1.5 ,~.4~om~,£
intercept = 2 ~ x l o "Q J 1.0
/ ]r~tercept = t6.5 xtO "~
/
int*rc~~=~t.O*t0-*
0.5-
Y
J J
intercept = 3.3 x ~0" l t !
J I I
l 2
! 3
! 4
I 5
I 6
Added Sulfote Concentration (xlO ~)
Fig. 5. M a x i m u m g r o w t h as a function of added sulfate in the presence of selenate concentrations indicated. The intercept values on the minus X axis are also indicated. G r o w t h was estimated b y light absorbance at 45o m p .
The slope of the curve lacking selenate in Fig. 5 is 0.049 absorbance units per pmole of added sulfate, while the slopes of the curves for the selenate-containing series average 0.070 absorbance units per pmole of added sulfate. The greater increment of growth per increment of sulfate at any fixed selenate concentration indicates that selenate is being utilized. Furthermore, the facts that the curves for the selenatecontaining series are parallel, and have a constant spacing between them show that sulfur and selenium are being used in a fixed ratio. One /zmole of sulfur will support the growth of about 15 mg dry weight of cells in the absence of selenate, and 21 mg dry weight of cells in the presence of selenate. The ratio of selenium to sulfur utilization then is 6 : 15 or 0.4. We can now revise our estimate of the sulfur contamination in the sodium selenate. While the total apparent contamination was about 1.5-3.o. io -~ moles of sulfur per o.oi mole of selenium, the results shown in Fig. 5 suggest that Biochim. Biophys. Acta, 141 (1967) 573-586
582
R.E.
HUBER, I. H. SEGEL, R. S. CRIDDLE
40 % of the 'contamination-supported growth' was actually selenium-supported growth. The selenate then must contain about o.9-~.8, xo-~ moles of sulfur per o.o~ mole selenium (o.9-~.8. ~o-~ mole %). The experiments described in Fig. 5 were conducted at relatively high selenate concentrations (o.oo~5-o.o~ M). Fig. 6 shows the effect of varying selenate concentrations on the maximum growth obtained in synthetic media containing a limited
5.0.
2.0"
J
A+eot,~. 1.0
,
~
~
:
~
[
!
~
o.oo~
!
~
I
:
!
!
~
o.o~
]
~ ! ~ ! I
o.o~
o,o~
Se O~'conc~Ml Fig. 6. M a x i m u m g r o w t h as a function of added selenate in the presence of a limiting a m o u n t of sulfate concentration. The limiting sulfate concentration in each flask was 4" I°-S M. G r o w t h was estimated by light absorbance at 450 m#. TABLE III INCORPORATION
O F 3SS A N D
rSSe I N T O C E L L P R O T E I N
The cells were grown for 7 days in synthetic media containing either ~ S O ~ ~- (Expt. i), asSO4e- plus unlabeled SeOi ~- (Expt. z), or 7sSeO~- (Expt. 3). Cell protein was isolated as described in ~IATI~RIALS AND
METHODS.
Expt. 'Sulfur" source No. (M)
I 2 3
3sSOa z-* (4-5" Io-~) s~SO~~-* (4.5"1o -~) + SeOl ~- (lO -3) 7SSeO~ ~-** (5" IO-a)
Incorporation of label into protein
Replacement of Se for S
counts/rain per mg fzmoles/mg
t~moles/mg %
1.66- lOT
0.395
o
o.97" lO7 3.38. ~o4
o.231 o.13
o.164"** (o.I3)
o 41.6 *** 32.9 §
* Specific activity, 4.2. lOT c o u n t s / m i n per #mole, based on sulfate added (4" IO-S M), plus c o n t a m i n a t i n g sulfur (as sulfate) in the m e d i u m constituents (o. 5. io -s M). ** Specific activity, 2.6. ios c o u n t s / m i n per/~mole. *** Replacement for Se for S calculated in E x p t . 2 as depression of incorporation of 3~S, compared to control (Expt. i). § Moles of Se (Expt. 2)/moles S (Expt. I) × ioo.
Biochim. Biophys. Acta, i 4 i (r967) 573-586
GROWTH OF E. coli ON SELENATE
583
amount of sulfate (4" IO-~ M). The results suggest that selenate must be present in a relatively high concentration before it can effectively compete with sulfur.
Incorporation of 3~S and ~*Se into cell protein The bioassay technique described above suggested that when sulfur is limiting, selenium can replace about 40 % of the cell's sulfur requirement. The validity of the bioassay technique is supported by the results shown in Table I I I . I t can be seen in Expts. ~ and 2, that in the presence 'of limiting amounts of ~SO~ ~-, o.oi M SeO¢~-
~00
0
UNTREATED
~
TRYPSIN
~"
PRONAS'E
~
200 Ioo
--_
_
~1~
b
2OC
I(X o
._~ ~ ~ ,,- ~ ~o
b
IOC
t
•~ ~e
~
ACID HYDROLYSIS
~;
~.~ ~
~OC
8 ~o~ ~ I~¢
ACID HYDROLYSIS(OXIDIZED)
~
~
CM
~
FROM THE
ORIGIN
,'~
~'o
~'5
Fig. 7- D i s t r i b u t i o n of r a d i o a c t i v i t y a l o n g c h r o m a t o g r a p h y strips. O is t h e origin w hi l e F is t h e s o l v e n t front. E a c h s t r i p h a d a r a d i o a c t i v e m a r k e r a p p l i e d a b o u t 2 c m before t h e origin. The s t r i p s were s c a n n e d a t 12 i n c h / h w i t h a o.5-inch slit w i d t h . T A B L E IV ~sSe CONTENT OF CELL PROTEIN Cells g r o w n on o.oo 5 M ~*$eO~ ~- were h a r v e s t e d , washed, a n d t h e n d i s r u p t e d b y sonic oscillation. The d i a ] y z e d s o n i c a t e w a s p r e c i p i t a t e d w i t h lO% t r i c h l o r o a c e t i c a c i d a t IOO°. The p r e c i p i t a t e w a s w a s h e d w i t h t r i c h l o r o a c e t i c acid, d i a l y z e d a n d c o u n t e d . The t r i c h l o r o a c e t i c a c i d w a s t h e n dissolved in i M N a O H , p r e c i p i t a t e d a n d w a s h e d a second t i m e w i t h t r i c h l o r o a c e t i c acid.
Fraction
~SSe content (counts/min per rng prote$n)
W h o l e cells Dialyzed sonicate D i a l y z e d t r i c h l o r o a c e t i c acid (precipitate) (rst) D i a l y z e d t r i c h l o r o a c e t i c acid (precipitate) (znd)
34 32 24 26
ooo ooo ooo ooo
Biochim. Biophys. Acta, i4~ (i967) 573-586
584
It. E. HUBER, I. H. SEGEL, R. S. CRIDDLE
will depress ~S incorporation b y 41.6 %. From this experiment alone, it is impossible to tell how much of the depression results from substitution of selenium for sulfur, and how much from dilution of the specific activity of the sulfate b y sulfur in the selenate. However, in Expt. 3, where the cells were grown in the presence of ~SeO, ~and no added sulfur (to maximize selenium incorporation), the selenium content of the cell protein was 32. 9 % of the usual sulfur content (Expt. I). The results confirm that selenium is utilized by the cells in place of part of the normal sulfur requirement.
Identification, of the 7*Se-labeled compounds in the protein fraction The results in Table IV show that about 7 ° % of the 7*Se in the cells and protein fraction is present in a form that cannot be washed or dialyzed away from the protein. In order to identify the labeled components in the protein fraction, the protein was hydrolyzed with trypsin, pronase, and acid as described earlier. The hydrolysates were chromatographed and the resulting chromatograms were scanned for radioactivity. The results are shown in Fig. 7. It can be seen that virtually all the radioactivity remains at the origin in the chromatograms of the untreated protein. Treatment with trypsin or pronase to produce peptides of various sizes results in a smear of radioactivity in the subsequent chromatograms. These results show that (a) the selenium was present in the protein in compounds linked by peptide bonds, and (b), the selenium was more or less randomly distributed in all cell proteins, i.e., there was no particularly selenium-rich peptide present. Acid hydrolysis yields a well-defined peak at about 13 cm from the origin. The radioactive peak migrated coincidently with authentic unlabeled selenomethionine. When the acid hydrolysate was exposed to H20 ~ prior to chromatograph, the bulk of the radioactivity subsequently migrated to about 5 cm from the origin, a position that corresponds to selenomethionine sulfoxide and sulfone 1~. No selenocysteine or selenocystine was detected. However, it
1.5
60~ °
° °
1.0
~ 4oo~
.~
__~___2. . . . . . ~---4
~
~ ~
l
.'''"
L, £ ~
"
~
A~om~
~ 0.5
~o~ ~ o
~ ~ ; ~ ;0 ,~ ~ ,~,~o,g ,io ,~ ,io ~ ~ ,~ ;o HOURS
Fig. 8. G r o w t h and u p t a k e of 7~Se and 3~S. The cells were grown in synthetic medium containing high specific activity, o.oi M ~SeO~ ~-, carrier-free 3~SO~e-, and no added sulfate. @--(D, g r o w t h determined b y absorbance at 45 ° m # ; × - - × , counts of 3~S per IO/~1 of cell suspension. The cells used were washed and resuspended at same cell density as in the g r o w t h m e d i u m ; & - - / X , counts of ~Se in o. 5 ml of cell suspension. The cells were washed as above. All counts are corrected to the value one ~vould observe w i t h o u t the a l u m i n u m cover.
Biochim. Biophys, Acta, 141 (1967) 573-586
GROWTH
OF
E. coli oN
SELENATE
585
should be emphasized t h a t selenocysteine and selenocystine are quite unstable to acid hydrolysis and could have been destroyed ~*, ~s. Simultaneous uptake of 7~Se042F i g . 8 shows the simultaneous uptake of 3sS and 75Se b y cells growing in synthetic m e d i u m containing o.oi M SeO~2- and no added sulfur. Fig. 9 shows the d a t a as the ratio of 35S/VSSe radioactivity per unit weight of cells.
OC 4¢ O ~0 •
A
,~ io ~, go ~o ~, ~o~o' ,I,
~o ,g ,,o ~..' '
•
i i ,~,,~ ~o ,oo ,,~
HOURS
Fig. 9. Ratio of [35S]sulfate counts to [VSSe]selenate counts taken up by the cells. Values are corrected for a constant cell weight. The ratio plot shows t h a t very early in the growth cycle, sulfur is preferentially used over selenium. The S/Se ratio passes through a m a x i m u m and then falls to a constant value indicating again t h a t sulfur and selenium are used in a fixed ratio. If, during the initial growth period, the available sulfur is largely depleted from the medium, then subsequent growth after 20 h (maximum S/Se) will depend upon autolysis and reutilization of liberated sulfur. This suggestion is consistent with linear growth and very long lag periods in high selenate medium. CONCLUSIONS Growth of selenium adapted E. coli cells on selenate media with no sulfur added has been shown to be due in large part to sulfur contamination from the media water and air. The E. coli can, however, utilize selenium in the presence of limiting sulfur to contribute to cell growth. W i t h excess selenate, a limiting ratio of about 40 % Se to 60 % S m a y be obtained. Biological estimates of S contamination show t h a t the media without added selenate contained 5.2" IO-~ M sulfur while the media containing o.oi M selenate were 2.27" lO -5 M in sulfur. It appears t h a t no growth will occur in the absence of some sulfur. Growth in the presence of selenium is characterized b y long lag periods, linear growth curves and a decreased efficiency. Total growth is m a r k e d l y reduced. These observations differ somewhat from those of SHRIFT AND KELLYa even t h o u g h the same strain of E. coli was used. Their growth patterns showed m u c h shorter lag times Biochim. Biophys. Acta, 141 (1967) 573-586
586
R.E.
HUBER, I. H. SEGEL, R. S. CRIDDLE
and exponential growth. The source of selenium used was different, however, and some differences m a y be accounted for in terms of different sulfur contamination. Uptake of radioactive [75Se]selenate showed that most of the selenium which is incorporated into the-cells under these growth conditions seems to be firmly bound to the protein and is released as trichloroacetic acid-soluble material only when the peptide bonds are hydrolized. Paper chromatography of acid hydrolysates of the protein strongly suggest the presence of selenomethionine but there is no evidence for the presence of selenocystine. ACKNOWLEDGEMENT
This investigation was supported in part by U.S. Public Health Service Grants GM-I2292 and GM-IooI 7. REFERENCES I I. ROSI~NFELD AND O. A. BEATH, Selenium, Academic Press, New York, I964, p. i 11. 2 H. G. MAUTNER AND W. H. H. GUNTHER, Biochim. Biophys. Acta, 36 (1959) 561. 3 D. B. C o w l s AND G. N. COHEN, Biochim. Biophys. Acta, 26 (1957) 252. 4 A. SHRIFT AND E. KELLY, Nature, 195 (1962) 732. 5 T. K. VIRUPAKSHA AND A. SHRIFT, Biochim. Biophys. Acta, 74 (1963) 319. 6 L. A. YAMAMOTO AND 1. H. SEGEL, Arch. Biochem. Biophys., i i 4 (1966) 523 . 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 N. SIGAL, J. CATTANEO AND I. H. SEGEL, Arch. Biochem. Biophys., lO8 (1964) 44 o. 9 J. FOND, F. L. SCHAI*I~ERAND P. L. KIRK, Arch. Biochem. Biophys., 45 (1953) 319 • IO M. H. RICHMOND, Bacteriol. Rev., 26 (1962) 398. i i J. C. S~NEZ, Bacteriol. Rev., 26 (1962) 9512 I. H. SEGEL, J. CATTANCO AND N. SIGAL, Syrup. on the Mechanisms of Regulation of Cellular Activities in Microorganisms, Proc. Intern. C.N.S.R., 1963, C.N.R.S., Paris, 1965. 13 J. SCALA AND H. H. WILLIAMS, J. Chromatog., 15 (1964) 546. 14 R. E. HUBER AND R. S. CRIDDLE, Biochim. Biophys. Aeta, 141 (1967) 587 . 15 R. E. HUBER AND R. S. CRIDDLE, Arch. Biochem. Biophys., in the press.
Biochim. Biophys. Acta, 141 (1967) 573-586