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Catabolite repression in Bacillus subtilis
18
Biochimica et Biophysica Acta, 541 ( 1 9 7 8 ) 1 8 - - 3 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28537
CATABOLITE REPRESSION IN BACILLUS SUBTILIS
B A R B A R A DOWDS, L E S L E Y B A X T E R a n d M I C H A E L M c K I L L E N *
Department of Biochemistry, Trinity College, Dublin 2 (Republic of Ireland) (Received O c t o b e r 1 7 t h , 1 9 7 7 )
Summary The D-gluconate transport system of Bacillus subtilis is optimally induced by exposure of cells for 2 h to 5 mM D-gluconate in the growth medium. D-Gluconate transport is subject to catabolite repression, as distinct from inducer exclusion or catabolite inhibition, in a manner parallel to the repression of inducible histidase synthesis, suggesting that the repression is not specific to this transport system. Maximum repression with the repressing carbon source (10 mM) added to cells grown in either casein hydrolysate or amino acid medium is achieved within two doubling times. Urea, the only non-carbon source tested for a repressing effect, was found to act solely by inducer exclusion. The ability of a sugar carbon source to evoke catabolite repression appears to be unrelated to its suitability as a substrate for the sugar : phosphoenolpyruvate phosphotransferase system but nonetheless the conversion to a phosphorylated derivative of the sugar seems essential. Repressed cells fail to synthesize, or do so to a more limited extent, an as y e t unidentified phosphorylated comp o u n d (probably a highly phosphorylated nucleotide) which is accumulated in the medium of non-repressed cells. Mutant studies imply that inosinic acid synthesis is necessary for catabolite repression whereas the adenosine highly phosphorylated nucleotides required for spurulation are not.
Introduction
The nature of the molecular events controlling the initiation of bacterial sporulation continues to remain elusive [ 1 ]. Sporulation represents the simplest differentiating system and an understanding of the repression mechanism involved in the sporulation process may be aided by the study of another type of genetic regulation, namely catabolite repression. The general phenomenon of catabolite repression may operate [2] by * To
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19 inducer exclusion, 'true' catabolite repression (including transient repression [3] ) and catabolite inhibition. This paper is concerned with the effect of catabolite repressing compounds on the inducible D-gluconate transport system of Bacillus subtilis [4,5]. In order to check that the observed repression was not specific to the D-gluconate transport system, we tested the effect of some of the repressing compounds on histidase synthesis, which is genetically well-documented in this respect [6]. Lopez and Thoms [7] have suggested that in B. subtilis repressing compounds may exert their effects via one or more common metabolites, namely phosphorylated sugars. This idea is supported by the similarities between catabolite repression and suppression of sporulation, the latter process known to be inhibited by a build-up of phosphorylated intermediates of metabolism [8,9]. In all cases studied in bacteria, catabolite repression results from a single or very few regulatory systems acting on the expression of all genes subject to this form of control [2,10--13]. For example, in Escherichia coli most of such genes are regulated by the phosphotransferase system both directly and by its effect on the transport of inducer and the intracellular concentration of cyclic AMP which in turn regulate protein synthesis [14]. The siinilarities between the response of inducible enzyme and transport proteins studied in B. subtilis [6,7,15--18] indicate a regulation common to them all, but a mechanism involving cyclic AMP or cyclic GMP is very unlikely [18--25]. In addition, unlike the case for E. coli [26], Lopez and Thoms [7] found that sugar uptake by itself did not effect catabolite repression in B. subtilis. In this laboratory Baxter et al. [15] showed that structural analogues of D-glucose which both lack the capacity to repress and to act as phosphotransferase system substrates in E. coli [27] do in fact cause repression in B. subtilis. If neither the sugar: phosphotransferase system nor cyclic AMP is involved in catabolite repression in Bacillus, just how is this process mediated? There is much evidence pointing to a role for highly phosphorylated nucleotides in the regulation of gene expression in B. subtilis and other bacteria. For example, guanosine 5'-diphosphate 3'-diphosphate (ppGpp or MS1) modulates control at the level of transcription, of the histidine operon in Salmonella typhimurium [28] and other catabolic operons in E. coli [29], and both ppGpp and pppGpp (MSll) appear prior to the decrease in RNA synthesis following amino acid starvation of B. subtilis and stringent but not relaxed strains ofE. coli [30]. For an opposite (i.e., non-repressed} state such as sporulation to occur, synthesis of other highly phosphorylated nucleotides: the adenosine tetra- and pentaphosphates, appears to be necessary [31], while ribosomes from the cells committed to sporulation lack the capacity to synthesize MS1 and MSll. Certain eukaryotic RNA species whose synthesis may be switched on and off have highly phosphorylated adenosine and guanosine nucleotides incorporated into the 5'-end of the chains [32]. It may be that their presence stems from a primitive type of regulation. We suggest that substances capable of initiating catabolite repression are metabolised to their phosphorylated derivatives, that the information which establishes repression flows from, for example, sugar phosphate to UTP or ATP and from specific phospho-proteins to GTP depending on the particular control system. UTP, ATP and GTP might possibly regulate the level of highly phosphorylated nucleotides either by an alter-
20 ation in substrate concentration [33] or by feedback regulation of the synthesizing enzymes [34]. Finally, we may speculate that the alteration in highly phosphorylated nucleotide concentration modulates the synthesis of the RNA species which require this type of substrate for initiation of their synthesis. There is some direct evidence implicating highly phosphorylated nucleotides in the control of catabolite repression. Firstly, Yeung et al. [24] reported the extracellular accumulation of an unknown phosphorylated compound in B. megaterium, grown in glucose or galactose but not glycerol, whose rate of excretion depended on the carbon source. Secondly, pppAppp is known to be excreted into the medium at the beginning of sporulation of B. subtilis [35]. Evidence for the passage of the repressing information via purine metabolism comes from the coupled loss of the ability to sporulate and repress by mutants defective in inosinic acid synthesis. The early step in its synthesis, that is feedback inhibited by ATP and GTP, is implicated [36]. While it remains possible that highly phosphorylated nucleotides mediate catabolite repression in a manner similar to that of cyclic AMP, it seems more likely that the effect is slightly less specific since despite an intensive search, Coote [37] failed to isolate a mutant of B. subtilis resistant to catabolite repression. The apparently lower genetic specificity for catabolite repression in B. subtilis, in contrast to E. coli, might be explained by a possible lower specificity of highly phosphorylated nucleotide than of cyclic AMP function; this could arise by the compensation for loss of one highly phosphorylated nucleotide by an increase in another, such as the substitution of adenosine by uridine nucleotides both of which appear prior to sporulation in B. subtilis [38]. Materials and Methods Strains. B. subtilis SB-26 (trp C2 met C3), obtained from Dr. E. Nestor via Dr. N. Sueoka, is a derivative of the transformable 168 strain. The mutants in intermediary metabolism were strains 61372 and 61411 whose phenotypes were phosphoglucoisomerase- and phosphofructokinase-negative, respectively (gifts from Dr. E. Freese [8]) and BR 95 glp K 3--7 and Br 95 glp D 12--11 phenotypes, glycerol kinase and NAD-independent sn-glycerol-3-phosphate dehydrogenase-negative, respectively (obtained from Dr. V. Lindgren). Dr. H.J. Rhaese [31] supplied the sporulation mutants of B. subtilis JH 649 (trp C2 phe-1 spo O F221) and 50413 (trp C2 ind spo-) and their spo ÷ revertants. The B. megaterium mutants in purine metabolism, gin- 26 (U- glutamine-), glu- 11 (U- glutamate-) and put 562 (U- purine (class la)) and their revertants (U-) were provided by Dr. C.E. Elmerich [36]. Media and growth conditions. The mineral salts medium contained (in 1 1 of distilled water): K2HPO4, 14 g; KH2PO4, 6 g; MgSO4 • 7H20, 0.25 g; MnC12 • 4H20, 0.02 g; L-giutamic acid (monopotassium salt), 0.15 g; (NH4)2SO4, 2 g; tripotassium citrate H~O, 1 g. The medium was supplemented with 0.025 g L-tryptophan per 1, 0.03 g L-methionine per 1 and 0.5%, w/v, casein hydrolysate (Merck). Alternatively an altered form of the amino acid carbon source described by Willecke and Mindich [39] was used; the histidine was omitted
21 and the other amino acids (tryptophan and methionine excepted) were present at three times the concentration recommended. All strains were grown as single colonies on tryptose blood agar base (TBAB) plates (Difco) at 37°C for 24 h and subsequently maintained at 4°C. The plates were appropriate supplemented for mutant-strains [36]. F o r growth in liquid media the cells were loop-inoculated from a fresh colony ( < 2 4 h) growing on a TBAB plate, into 5.0 ml of (37°C) growth medium. The medium was contained in a sterile metal-capped tube which was vigorously shaken overnight (12--14 h minimum) at 37°C in a reciprocating water bath shaker. The culture was then diluted by placing a 1-ml portion into either 15 or 25 ml of fresh prewarmed growth medium containing 5 mM D-gluconate, contained respectively in either 150- or 250-ml conical flasks fitted with nephelometer sidearms. (D-Gluconate optimally induces its own transport system at a concentration of 5 mM in the medium without altering the growth rate of the cells [4] ). The apparent absorbance at 650 nm of such diluted cultures was initially approx. 0.05 units. All cultures were shaken vigorously in a reciprocating water bath at 37°C (150 rev./min, 35 mm). The growth of the culture was routinely followed with an Eel colorimeter (OR 1 filter) using the nephelometer side arm of the flask. A 10 mM concentration of repressing c o m p o u n d was added at an Eel absorbance of 0.1, when the lag phase of growth had ended, since 10 mM glucose shortens the lag phase b u t does n o t alter the doubling rate of B. subtilis under the conditions described. The shaker was never stopped for more than 30 s and the cultures were never allowed to cool as the growth o f B . subtilis is particularly sensitive to changes in these parameters. An Eel value of 1.0 is virtually equivalent to an A650n m of 1.0 and a cell density of 4 • 108 cells/ml of culture. The cells were usually grown to early exponential phase (A6son m of 1.0), then rapidly harvested by membrane filtration (47 mm diameter, 0.6 pm pore size) and washed with one volume of prewarmed (37°C) resuspension medium (mineral salts medium containing 0.5% D-glucose and 0.007% chloramphenicol). The filter was then c u t in half, placed in a tube with the calculated volume of resuspension medium at 37°C to give a final apparent A650nm of 1.3 and then vortex-mixed for approx. 30 s. The exact apparent absorbance of the resuspension was then obtained at 650 nm on a 1 : 3 dilution of the cells. An absorbance of 1.0 at 650 nm is equivalent to 340 pg dry weight of cells per ml of suspension. The suspension was stored at 37°C for less than 45 min before assays were performed. Gluconate transport assay. The assay tube held 0.7 ml of resuspension medium at 37°C containing sodium D-[U-14C]gluconate (0.38 pCi). The reaction was initiated by the rapid addition of 0.6 ml of cell suspension to the assay tube. Transport was allowed to proceed for 30 s and was terminated by rapidly removing 1.0 ml of the suspension with an automatic pipette and filtering immediately on a premoistened membrane filter (25 mm diameter, 0.65 pm pore size) supported on the base only by a Swinnex-25 holder (Millipore Ltd.). The filter pad was immediately washed with 5 ml of resuspension medium (37°C) and then rapidly transferred to 10 ml of a toluene/ethanol (12 : 7, v/v) based scintillation fluid containing 0.4% PPO. Reaction termination and washing procedures were completed within 15 s and all assays were carried o u t in duplicate. The zero-time transport control was obtained by assaying resuspension
22 medium instead of cells under the standard conditions. All scintillation vials were swirled before being placed in a liquid scintillation spectrometer (Packard Instruments Ltd., Model 3375 or 3385) and counted using the preset '4C counting channel. Counting time was arranged so that the standard deviation of counting, for the anticipated lowest activity sample (excluding the zero-time transport control), was less than 1%. The zero-time transport control never exceeded the natural background count rate by more than 3-fold. The mean value for the initial velocity of transport within a particular experiment using the above procedure, was 71.8 + 1.4 pmol per min per gram dry weight of cells (10 assays). Variations in the measured initial velocity did occur from experim e n t to experiment (50--90 pmol/min per g dry weight of cells). Under the conditions of the standard transport assay the cells removed not more than 10% of the total radioactivity initially present in the assay mixture. The initial external D-gluconate concentration in all assays was 0.1 mM (~3 × Kin). Histidase assay. Cells were grown in amino acid medium since assay of urocanic acid is performed by its absorbance at 277 nm [6] and casein also absorbs at this wavelength. Cultures were grown to A65onm of 1.0, shaken in toluene over ice and centrifuged at 3600 rev./min for 15 min. The histidase assay was essentially that described by Chasin and Magasanik [6]. Labelling, extraction and chromatography of regulatory nucleotides. The procedure described by Rhaese et al. [41] was essentially followed. Cells were labelled with 0.5 mCi/ml [32P]orthophosphate added at A6s0 = 0.1, and grown till A6s0 = 1.0. 10 mM D-glucose as repressor was added to one flask of cells at A6s0 = 0.1. Extraction of nucleotides with formic acid was followed by filtration on membrane filters (pore size 0.6 pm) to yield an intracellular extract while the m e t h o d of Rhaese et al. [40] was used to obtain a " t o t a l " extract (intracellular extract plus medium). Thin-layer chromatography of the total and intracellular extracts was performed on polyethyleneimine-impregnated cellulose plates. One-dimensional chromatography was with 1.5 M sodium phosphate buffer, pH 3.4 [40]. Two-dimensional chromatography solvents were 3.3 M a m m o n i u m formate + 4.2% boric acid adjusted to pH 7.0 with NH4OH (first dimension) and 0.85 M KH2PO4, pH 3.4 (second dimension). In the latter case, between the two runs the plates were dried, soaked in methanol for 5 min, then in distilled water for 15 min and oven-dried [41]. 20-pl spots of each formic acid extract were applied to the chromatogram with blow drying. Blue Brand Medical X-ray film (tinted estar safety base duplitized) was exposed to the chromatography plates for 6--36 h depending on the length of storage time of the labelled extracts. The films were developed with Kodak DX80 developer. Chromatograms were marked and cut into 1 X 1 cm square sections and placed in scintillation vials to which were added 10 ml of 0.6% PPO in toluene scintillation fluid. The samples were then counted in a Packard TriCarb 3385 spectrometer using the preset 32p channel. Polyacrylamide gel electrophoresis. Formic acid was removed from 40-pl volumes of the extracts under a stream of nitrogen gas and the residue taken up in SDS solubilising solution [42]. 25 pl were then applied to a 10% polyacrylamide slab gel in Tris/SDS buffer at a constant current of 25 mA [42]. After electrophoresis the gel was wrapped in a monolayer of cling film (Propax) and autoradiography was performed as detailed in the thin-layer chromatography section above. Finally, the gel was stained with Coomassie blue [43].
23
Results
Influence of carbon source on activity of D-gluconate transport system Table I shows that the non-induced D-gluconate transport activity is approximately the same (2--4 pmol/min per g dry weight) whatever the carbon source used for growth, though activity may be slightly higher in cells grown on a rich carbon source such as casein hydrolysate (7 pmol/min per g dry weight). When cells were grown on D-gluconate as sole carbon source (induced cells) the initial transport velocity was 23 pmol/min per g dry weight of cells. When cells were induced in the presence of 5 mM D-gluconate and grown on a variety of carbon sources, D-gluconate transport activity was either repressed (D-glucose and malate) or elevated (succinate, D-ribose and casein hydrolysate) to varying extents in comparison to the activity obtained from cells grown in D-gluconate as sole carbon source. In no case did a carbon source cause total catabolite repression of transport activity (i.e., identical D-gluconate transport activity in the presence and in the absence of inducer). We selected 0.5% (w/v) casein hydrolysate as a carbon source for most purposes since it allowed a rapid growth rate (td = 30 min) whilst still permitting strong catabolite repression by other easily utilised carbon sources such as D-glucose (Table II). The amino acid medium was used as a defined carbon source to check the extent, if any, of repression produced by the casein hydrolysate medium.
Type of repression In order to show that the repression caused by D-glucose, for instance, resulted from the phenomena of catabolite repression or inhibition as opposed
TABLE I INFLUENCE OF GROWTH MEDIUM ON ACTIVITY OF D°GLUCONATE TRANSPORT
SYSTEM
SB-26 w a s g r o w n o n t h e m i n e r a l salts m e d i u m , s u p p l e m e n t e d w i t h t h e a p p r o p r i a t e c a r b o n s o u r c e s .
A d d i t i o n s to g r o w t h m e d i u m
D-Gluconate transport activity ( ~ m o l • m i n -1 • g-1 dry w e i g h t )
25 mM D-gluconate
23.0
25 m M D - g l u c o s e 25 m M D - g l u c o s e + 5 m M D - g l u c o n a t e
2.0 8.0
25 m M c i t r a t e 25 m M c i t r a t e + 5 m M D - g l u c o n a t e
2.0 22.0
25 mM succinate 25 m M s u c c i n a t e + 5 m M D - g l u c o n a t e
2.5 45.5
25 m M L - m a l a t e 25 mM L-malate + 5 mM D-gluconate
3.0 13.0
25 m M D - r i b o s e 25 m M Doribose + 5 m M D - g l u c o n a t e
not determined 32.0
A m i n o acid s u p p l e m e n t A m i n o acid s u p p l e m e n t + 5 m M D - g l u c o n a t e
4 24
0.5% casein h y d r o l y s a t e 0.5% casein h y d r o l y s a t e + 5 m M D-gluconate
7.0 70.0
24 T A B L E II R E P R E S S I O N BY V A R I O U S M E T A B O L I T E S O F D - G L U C O N A T E T R A N S P O R T IN B. S U B T I L I S SB-26 A d d i t i o n s t o i n d u c i n g m e d i u m at a final c o n c e n t r a t i o n o f 10 m M None L-Glutamate Succinate Citrate D-Ribose Galactose L-Malate D-Mannose Sorbitol Glycerol D-Fructose D-Glucose Mannitol
Percentage of non-repressed transport activity remaining 100 > 100 100 :> 1 0 0 > 100 60 54 31 25 21 13 8 8
to inducer exclusion, transport activity was compared in non-induced cells grown in the presence and absence of the repressing compound. As the initial velocity of D-gluconate transport was lower in the presence of 10 mM D-glucose than in its absence (5 and I pmol/min per g dry weight of cells), it may be concluded that D-glucose represses by catabolite repression and/or inhibition. However, inducer exclusion cannot be ruled out and indeed seems a probable component of the repression mechanism, since the degree of repression is much greater in induced than in non-induced cells. Malate (10 mM) was the only other repressing carbon source tested in this way. Apparently, malate, like Dglucose, causes some catabolite repression and/or inhibition (3.5 pmol/min per g dry weight of cells). It was assumed that the other c o m p o u n d s producing marked repression (Table II) would act in the same way as D-glucose and malate. In order to determine whether D-glucose was causing catabolite inhibition, cells, which had been induced for D-gluconate transport, were assayed by the standard assay procedure in the presence and absence of D-glucose. The same level of transport activity was observed whether or n o t D-glucose was present in the assay buffer, indicating that catabolite inhibition is not involved in the repression process.
Kinetics of induction, and repression of the D-gluconate transport system. McKillen and Rowntree [4] reported that the inclusion of 5 mM D-gluconate in the 0.5% (w/v) casein hydrolysate medium resulted in maximal induction of D-gluconate transport activity without significantly altering the growth rate of the culture. The time course for the induction and subsequent decay of D-gluconate transport activity was determined by taking replicate cultures of non-induced cells and adding D-gluconate (5 mM) at different times after inoculation, and harvesting the cells at an absorbance of 1.0. Fig. 1 shows the initial velocity of D-gluconate transport in relation to the time allowed for induction to take place. It can be seen that maximum induction (6-fold) with 5 mM gluconate has occurred 2 h (4 doubling times) after its addition to the previously non-induced culture. To determine the rate of decay of transport activ-
25
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2O
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~-~ %- - %-J
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0 1 2 3 4 18 Time allowed f o r induction before harvesting of celts [h) Fig. 1. I n d u c t i o n k i n e t i c s . 5 m M D-gluconatae was a d d e d to an e x p o n e n t i a l l y growing culture at different t i m e s b e f o r e h a r v e s t i n g . A t a n a b s o r b a n c e o f 1 . 0 , c e l s were filtered, r e s u s p e n d e d i n b u f f e z c o n t a i n i n g c h l o r a m p h e n i c o l and assayed for D-gluconate transport a c t i v i t y .
ity the cells were induced under standard conditions, full induction occurring before an absorbance of 0.5, then harvested, washed in non-inducing growth medium and resuspended in the latter to an A6s0 = 0.5. The cells were grown in this non-inducing medium to an absorbance of 1.0, then harvested and assayed for D-gluconate transport. The initial velocity for D-gluconate transport was reduced by one half to 35 pmol/min per g dry weight of cells. A control in which the induced cells were resuspended in standard inducing growth medium and grown for a further generation indicated that resuspension per se had no effect on the ability of the cells to transport gluconate at the standard rate of 70 pmol/min per g dry weight of cells. Repression kinetics were obtained on cells which were exposed to inducer at an absorbance of 0.05. Six parallel cultures were grown which in the absence of repressor would normally show a transport rate of a b o u t 70 pmol/min per g. Glucose was added to each at different times before harvesting. Fig. 2 shows that it began to exert its repressing effect after approx. 1 doubling time, this rising to a maximum when the glucose was present for t w o doubling times or more. The order of addition of D-gluconate and D-glucose did n o t affect the transport rate of the harvested cells so long as these c o m p o u n d s were added, at least 2 and 1 h, respectively, before harvesting.
Types of repressing and non-repressing compounds Table II shows the D-gluconate transport activity remaining in cells grown in casein hydrolysate medium with 5 mM gluconate, in the presence of a range
26
70
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E 5O E 4O
o 3o c 20 o
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Time allowed for repression before harvesting of cells (h) Fig. 2. Repression kinetics. A culture w a s i n d u c e d with 5 m M D-gluconate at an absorbance of 0.05. I 0 m M D-glucose w a s a d d e d a t d i f f e r e n t times before harvesting at a n absorbance of 1.0.
of metabolites all added at a final concentration of 10 mM (this concentration was low enough so as not to alter the growth rate of cells in casein hydrolysate or amino acid media). All of the repressing substances enter the glycolylic pathway and the citric acid cycle. The phosphotransferase system sugars tested (D-glucose and D-fructose) produced severe repression and in general unsubstituted hexoses and sugar alcohols produced much greater repression than 5-carbon sugars, amino acid or Krebs cycle intermediates. Phosphorylated sugars, such as glucose 6-phosphate and fructose 6-phosphate, repressed to a minor extent (results n o t shown) which may be explained by their inability to cross the cell membrane. In support of this contention is the fact that B. subtilis failed to grow on any phosphorylated sugar (i.e., D-ribose 5-phosphate, D-glucose 1-phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate, 2-phosphoglycerate and 6-phosphoglycerate) when tested for use as a sole carbon source at 0.5% concentration. Casein hydrolysate medium by itself seems to have little repressing activity; the most severe repressing c o m p o u n d (D-glucose) left only 17% of nonrepressed activity in cells grown in amino acid medium as compared to 5--11% in casein hydrolysate grown cells. Finally, since urea specifically inhibits the expression of catabolite repression sensitive operons in E. coli [44] we tested its effect on D-gluconate transport in B. subtilis. In the presence of 10 mM urea the percentage of nonrepressed D-gluconate transport activity remaining was 72 and 32--46% in cells grown in casein hydrolysate and amino acid media, respectively. We have now extended our preliminary studies attempting to localise the site of action of compounds causing catabolite repression [15] by the use of mutants blocked in the intermediary metabolism of certain carbohydrates. The positions of the metabolic blocks in the mutants used are described in Table III. It seems reasonable to conclude that the transport of glycerol across the
27 T A B L E III R E P R E S S I O N O F D - G L U C O N A T E T R A N S P O R T I N B. S U B T I L I S S T R A I N S D E F E C T I V E IN I N T E R MEDIARY METABOLISM
Mutant strain
Addition to medium
Transport rate
(10 m M )
g-1 dry wt.)
( # m o l • m i n - 1.
Non-repressed activity remaining (%)
61372
none
(phosphoglucoisomerase negative)
D-glucose
61411
none
(fructose-l-phosphate kinase negative)
D-fructose
BR 9 5 glp K 3-7
none
(glycerol kinase negative)
glycerol
BR 9 5 glp D 12-11
none
(NAD-indep endent sn-glycerol-3 -phosphate dehydrogenase negative)
glycerol
59 31
100
34 lS
100
43 44
100 >100
51 58
>100
53 54
100
Location of blocks in intermediary metabolism D-glucose - D-fructose
glycerol
glp K 3-7
> glucose 6-phosphate
613~72
> fructose 1-phosphate
61411 ~
> fructose 6-phosphate > fructose 1.6-diphosphate
> glycerol 3-phosphate glp D 1 2 - 1 1 > d i h y d r o x y a c e t o n e phosphate
membrane has no role to play in initiating catabolite repression. On the other hand both D-glucose and D-fructose initiate catabolite repression either by virtue of their transport across the membrane or on conversion to their phosphorylated derivatives or by a combination of the two alteratives. The fact that the repression severity caused by D-glucose is slightly lower in the case of both the phosphoglucoisomerase and fructose-l-phosphate kinase negative mutants in comparison to the wild type may reflect the fact that accumulation of hexose phosphates in these mutants decreases the uptake of the parent sugar by the cell. We have not been able to directly assess the importance of phosphorylated intermediates in mediating catabolite repression but the implication that phosphorylated compounds in general may be able to initiate catabolite repression is upheld by some recent results of Lopez and Thoms [7]. The D-glucose mediated catabolite repression was further investigated by examining the ability of D-glucose analogues to cause repression. Harwood et al. [27] found in E. coli that substituents about carbon atoms 1 or 2 of glucose were inhibitors of adenylate cyclase and also substrates of the sugar: phosphotransferase system whereas analogues with changes in other parts of the molecule exhibited neither property. When analogues were added to inducing medium at a final concentration of 10 mM the percentage of non-repressed transport activity remaining was as follows: 6-deoxy-D-glucose (78%), 2-deoxyD-glucose (69%), 3-O-methyl-D-glucose (34%) and ~-methyl-D-glucoside (30%). Our results, at least in B. subtilis, show no such relationship between position of substitution and repressing activity. This is hardly surprising if the assump-
28 tion is made that neither the sugar : phosphotransferase system nor cyclic AMP are involved in catabolite repression in B. subtilis. On the other hand the stereospecificity of the glucose molecule is of supreme importance since L-glucose was unable to produce any catabolite repression. It has not been determined whether L-glucose can be transported by these cells. In the case of the other analogues, the cells grew slowly on 0.4% 2-deoxy-D-glucose and a-methyl-Dglucoside as sole carbon sources but failed to grow on 6-deoxy-D-glucose. The severity of repression observed with these analogues appears to parallel closely the extent of growth the cells are capable of making on these substrates. A supposition that some of the repression is caused by the transport of a sugar or analogue across the membrane could be used to account for the ability of 6-deoxy-D-glucose to repress despite its failure to support growth.
Catabolite repression of histidase synthesis Potential repressing substrate (10 mM) and inducer (14 mM histidine) were added at an absorbance of 0.1 to cells growing in amino acid medium. The cells were harvested at an absorbance of 1.0 and assayed for production of urocanic acid from L-histidine. The percentage of non-repressed activity remaining in cells exposed to the various potential repressing substrates was 48% D-glucose), 54% (D-mannose) and 100% (urea). Though the figures are widely different for the extent of repression of histidase synthesis and gluconate transport, the order of severity of repression by the three substrates is the same, suggesting that catabolite repression is probably being mediated in the same way for the two inducible systems.
Molecular mediation of catabolite repression H o w could the proposed signal from the intracellular concentration, of a phosphorylated sugar(s) for instance, be passed on so as to result in a cessation of transcription of catabolite repression-sensitive genes? The possibility of detecting a difference in the concentration or pattern of phosphorylated metabolites in the cytoplasm or exogenously in growth medium between repressed and non-repressed cells was investigated. Both 'total' extracts (intracellular extract plus medium) and intracellular extracts derived from cells grown on [32P]orthophosphate were chromatographed and the only difference observed between D-glucose-repressed and non-repressed cells may be seen in Figs. 3 and 4. The various 32P-labelled compounds, separated on both one- and two-dimensional chromatograms, had identical R F values and were present in equal amounts in each case whether obtained from the repressed or nonrepressed intracellular extracts. However a difference was detected between the " t o t a l " extracts in that a much greater proportion of the [32P]phosphorus was incorporated into the origin spot in the non-repressed than in the D-glucoserepressed extracts. Rhaese and Groscurth [35], Rhaese et al. [38] and Cashel et al. [41] have determined the R F values for various nucleotides, including highly phosphorylated nucleotides on this type of separating system. In the first dimension the deoxyribonucleotides migrate further than their ribonucleotide analogues, while in the second dimension the order of migration from slowest to fastest is guanine, adenine, cytosine, uracil and thymine nucleotides. The second dimension also facilitates separation of nucleotides on the basis on
29 .......2 n d
d imens~on
r~
o
E
0.
~m---- O r i g i n spot
Fig. 3. Separation of 32 P-labelled c o m p o u n d s from fully induced cells g r o w n in casein-containing growth m e d i u m to which 10 m M glucose was added at an absorbance of 0.1 at the same time as the label. A "total" extract (intracellular extract plus m e d i u m in formic acid) was cbromatographed on polyethylenimine cellulose in t w o dimensions.
Fig. 4. S e p a r a t i o n o f 3 2 p - l a b e l l e d c o m p o u n d s f r o m f u l l y i n d u c e d , n o n - r e p r e s s e d cells ( g z o w n in c a s e i n without the addition of glucose). Label added at an absorbance of 0.1. A "total" (intraceHular extract p l u s m e d i u m in f o r m i c a c i d ) w a s c h r o m a t o g r a p h e d o n p o l y e t h y l e n e i m i n e in t w o d i m e n s i o n s .
30 their number of constituent phosphate groups; the higher the number, the shorter the migration. Thus our origin spot may contain a very highly phosphorylated nucleotide(s) (>5 phosphate group and probably containing the guanine base [38]) and/or phosphorylated polysaccharide(s) and protein(s). The extracts were then applied to 10% polyacrylamide slab gels (Fig. 5). No Coomassie Brillant Blue staining components were detected on the gel and 32p_ labelled components appears to be only present at the origin and tracker dye positions on the gel slab. Furthermore, there was no qualitative difference detected between the cells grown under the two conditions for the two types of extract. Thus the difference observed on the thin-layer chromatograms may be attributable to either a highly phosphorylated nucleotide, a polynucleotide or to a phosphoprotein of molecular weight between 20 000 and 130 000.
D-Glucose catabolite repression in mutants defective in nucleotide metabolism B. subtilis mutants defective in the synthesis of highly phosphorylated nucleotides. Table IV shows the degree of catabolite repression of D-gluconate transport activity produced by D-glucose in mutants deficient in p p p A p p p synthetase. The JH 649 strain has a single-site mutation with respect to 649-R, but the difference between the repression of their respective D-gluconate transport activities is probably not significant especially in the light of the negligible difference detected between the 50413 strain and its revertant, 50413-R. The degree of catabolite repression of histidase synthesis is also essentially similar (67%) in the case of both the 50413 strain and its 50413-R revertant (results
I+
I-
T-
T+
I
P o s i t i o n of t r a c k e r dye after
electrophoresis Fig. 5. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f f o r m i c acid e x t r a c t s o f c a s e i n - g r o w n cells labelled w i t h 3 2 p a t a n a b s o r b a n c e o f 0 . 1 . R i g h t t o left: T +, T - , I - , I +. I: intracellular e x t r a c t ; T: " t o t a l " (intracellular e x t r a c t plus m e d i u m ) e x t r a c t ; +: g l u c o s e added; --: n o n - r e p r e s s e d ( g l u c o s e n o t a d d e d ) .
31 TABLE
IV
GLUCOSE S U B T I L IS
REPRESSION
OF
D-GLUCONATE
TRANSPORT
IN SPORULATION
MUTANTS
O F B.
A l l m u t a n t s , derived f r o m strain S B - 2 6 , w e r e o b t a i n e d f r o m Dr. H.J. R h a e s e [ 3 1 ] . R h a e s e et al. m e a s u r e d n u e l e o t i d e s y n t h e s i s in vitro w i t h r i b o s o m e s i s o l a t e d f r o m the appropriate stage o f g r o w t h . H P N s I a n d I I are n o t s e e n in vivo and m a y be u n s t a b l e p r e c u r s o r s o f H P N I V , p p p A p p p . H P N I V is s y n t h e s i s e d b y the e n z y m e , p p p A p p p s y n t h e t a s e w h i c h is d e f e c t i v e in the s p o O F m u t a n t . H P N , highly p h o s p h o r y l a t e d n u c l e o tide.
Mutant:
Genotype:
A b i l i t y to s y n t h e s i s e n u c l e o t i d e s T2 ppGpp + pppGpp { log g r o w t h ppApp
pppApp
( H P N I)
(HPN II)
JH649
649-R
50413
50413-R
trp C 2 p h e - 1 spoOF221
trp C2 phe-1 spo ÷
trp C2 ind spo-
trp C2 ind spo +
Yes Yes
No Yes
Yes Yes
No Yes
( T2 log g r o w t h
V e r y little No
Yes No
V e r y little No
Yes No
T2 ( log g r o w t h
No No
Yes No
No No
Yes No
80 8
55 10
65 8
68 6
10
18
12
9
V 0 in i n d u c e d g r o w t h m e d i u m ( # m o l • m i n - l • g - l dry w t . ) -- glucose + glucose
Percentage of non-repressed transport r e m a i n i n g after glucose addition
not shown in Table IV). It may be concluded that the adenosine highly phosphorylated nucleotides are not required for the expression of catabolite repression. B. Megaterium mutants defective in purine metabolism. A lower level of D-gluconate transport activity was observed in B. megaterium than in B. subtilis using the same conditions for cell growth, induction and transport assay. Nonetheless, this D-gluconate transport activity was subject to D-glucose catabolite repression (Table V). Despite the addition of sufficient adenine {0.02 g/l) to facilitate growth, the pur 562 mutant is clearly much less sensitive to catabolite repression than the wild type, gin- 26R1. The gin- 26 mutant was used in order to test whether repression is mediated at one of the control steps in purine synthesis where glutamine donates an amino group to the base. The D-glucose catabolite repression may be dependent to a certain extent on the presence of glutamine synthetase; the repression of D-gluconate transport activity is lower in the gln- mutant, grown in the presence of glutamine (0.5 g/l) than in its revertant, gin- 25R1. On the other hand, a mutant defective in glutamate synthase obtained from Dr. C. Elmerich [36] {results not shown) exhibited the same degree of repression as its wild type revertant strain of B. megaterium. These results imply that the early stages in inosinic acid synthesis (conversion of 5-phosphoribosyl-l-pyrophosphate to 5-aminoimidazole ribonucleotide) are necessary for catabolite repression, as they are for sporulation [36]. The possible requirement for glutamine though not glutamate is contentious
32 TABLE V GLUCOSE REPRESSSION OF D-GLUCONATE DEFECTIVE IN PURINE METABOLISM
TRANSPORT
IN MUTANTS
O F B. M E G A T E R I U M
All m u t a n t s w e r e o b t a i n e d f r o m Dr. C.E. E l m e r i c h a n d g r o w n in m e d i u m s u p p l e m e n t e d w i t h u r a c i l a n d adenine or glutamine [36].
Mutant: Genotype:
G i n - 26
G i n - 26 R I
Put 562
Sporulation only repressed in p r e s e n c e o f g l u t a m i n e . U- gin- (glutamine synthetase)
spo + U-
Sporulation repressed by adenine but not by uracil U - P u r - (class l a ) *
20.7 2.8
20.8 17.4
13.5
83.7
V 0 in i n d u c e d g r o w t h m e d i u m ( # m o l • rain - l • g - I dry wt.) -- glucose 15 + glucose 3.3
Percentage o f non-repressed transport remaining after g l u c o s e addition
22
* Class l a m u t a n t s a r e d e f i c i e n t in t h e c o n v e r s i o n o f 5 - p h o s p h o r i b o s y l - l - p y r o p h o s p h a t e imidazole ribonucleotide.
to 5-amino-
since the appropriate amino acid supplements had to be added in order to allow culture growth. Ideally the supplements should provide sufficient amino acid to facilitate growth while not allowing catabolite repression in the mutant defective in the production of amino acids involved in repression, but in practire this balance is difficult to achieve. Discussion
The only qualitative or quantitative difference between the formic acid extracts of repressed and non-repressed cells lies in the greater amount of label at the origin, of the extracellular material from the latter cells. If this represents a highly phosphorylated nucleotide, then its most likely identity would be pppGppp. Rhaese et al. [31], report that adenosine and guanosine highly phosphorylated nucleotides complement each other in their presence within the cell so that when one is present at a particular stage of growth or alternatively in a given m u t a n t strain, the other is absent. If this is so, and if the sporulation process is regarded as a removal of the repression exerted on the expression of certain genes, then it may be postulated that the guanosine highly phosphorylated nucleotides not present during sporulation are in fact necessary for the mediation of catabolite repression. The fact that ppGpp is known to alter transcription of selected operons o f E . coli [29] and S. typhimurium [28] supports this view. The complementary highly phosphorylated nucleotides, that is, those containing adenine, may then serve to alleviate the effects of repressing sugars and their metabolites. In the case of B. subtilis the guanosine highly phosphorylated nucleotides
33 may mediate catabolite repression either directly in the role of a negative effector or indirectly by draining the cell of a positive effector or substrate required for transcription. In the latter case it is possible that ATP effluxes from the cell and a decreased rate of synthesis of highly phosphorylated nucleotides occurs since it has been shown that both adenosine [9] and guanosine [34] highly phosphorylated nucleotides require ATP for their formation, ppGpp is synthesised in membrane vesicles of B. subtilis [9] and hence would constitute a plausible initiating signal from the external "rich" environment. Similarly, the adenosine tetra- and pentaphosphates are eligible signals of the external " p o o r " medium [9] and could act by antagonising the synthesis or activity of the guanosine nucleotide(s), pppGppp may then accumulate extracellularly, presumably to prevent a replenishment of the ppGpp and/or pppGpp pools. The mechanism whereby the concentrations of the highly phosphorylated nucleotides are controlled is likely to be extremely complicated. The results with the mutants defective in purine metabolism imply that the early stage in inosinic acid synthesis is important, independent of its role in the synthesis of ATP. In E. coli, ppGpp is known to inhibit IMP dehydrogenase and adenylosuccinate synthetase [34] which are involved in the formation of GMP and AMP from inosinic acid. Finally, ATP and GTP regulate not only their own synthesis but also that of the highly phosphorylated nucleotides. In summary, the sequence of events provoking a change in transcription of catabolite repression sensitive operons in response to transient changes in the nutrient status of the cell, appears to rely on the build-up of a sufficient pool of phosphorylated sugars [7--9,15] from which information involving a phosphate or phosphoryl group for example can be relayed to ATP and GTP and from thence to highly phosphorylated nucleotides. The nature of the final event which alters transcription is not yet known though some suggestions have been made above. Rhaese et al. postulate HPN III, a highly phosphorylated nucleotide containing uridine with a base to phosphate ratio of 1 : 4 as the nuclear signal, for intiation of sporulation, since it requires membrane and cytoplasmic factors for its synthesis and its appearance is also correlated with sporulation [9]. The identity of the final signal will only be elucidated by using an in vitro system of transcription. To demonstrate unequivocally whether or not ppGpp, pppGpp and/or pppGppp accumulation give rise to catabolite repression that is relieved by pppAppp would be a relatively simple matter since a m u t a n t of B. subtilis deficient in the guanosine highly phosphorylated nucleotides has been isolated [38] and the latter c o m p o u n d can now be synthesised and purified [9].
Acknowledgements We thank the National Science Council (Ireland) for partial funding of this work and also the Department of Education of Ireland for research maintenance awards to L.B. and B.D. We are indebted to Miss Mairin O'Sullivan for most of the results shown in Table I.
References 1 Piggot, P.J. and Coote, J.G. (1976) Bacteriol. Rev. 40, 908--962 2 Patgen, K. and Williams, B. (1970) Adv. Microb. Physiol. 4, 252--323
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Epstein, W., Rothman-Denes, L.B. and Hesse, J. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2300--2304, McKillen, M.N. and Rowntree, J.H. (1973) Biochem. Soc. Trans. 1 , 4 4 2 - - 4 4 5 Baxter, L., Torrie, S. and McKillen, M. (1974) Biochem. Soc. Trans. 2, 1370--1372 Chasin, L.A. and Magasanik, B. (1968) J. Biol. Chem. 243, 5165--5178 Lopez, J.M. and Thorns, B. (1977) J. Bacteriol. 1 2 9 , 2 1 7 - - 2 2 4 Freese, E., Yong, K.O.H., Freese, E.B., Diesterhaft, M.D., Prasad, C. (1972) Spores V, 212--221 Rhaese, H.J. and Groscurth, R. (1976) Proc. Natl. Acad. Sci. U.S. 7 3 , 3 3 1 - - 3 3 5 Pastan, I. and Adhya, S. (1976) Bacteriol. Rev. 40, 527--551 Prival, M.S. and Magasanik, B. (1971) J. Biol. Chem. 2 4 6 6288--6296 Silverstone, A.E., Goman, M. and Seaife, J.G. (1972) Mol. Gen. Genet. 118, 223--234 Rothman-Denes, L.B., Hesse, J.E. and Epstein, W. (1973) J. Bacteriol. 114, 1040--1044 Postma, P.W. and Roseman, S. (1976) Biochim. Biophys. A c t a 457, 213--257 Baxter, L., McKillen, M. and Syms, M. (1975) Biochem. Soc. Trans. 2, 1205--1207 Willecke, K. and Pardee, A.B. (1971) J. Biol. Chem. 246, 1032--1040 Fournier, R.E., McKiUen, M.N. and Pardee, A.B. (1972) J. Biol. Chem. 247, 5587--5595 Priest, F.G. (1975) Biochem. Biophys. Res. Commun. 63, 606--610 lde, M. (1971) Arch. Biochem. Biophys. 1 4 4 , 2 6 2 - - 2 6 8 Sarkar, N. and Paulus, H. (1975) J. Biol. Chem. 2 5 0 , 6 8 4 - - 6 9 0 Bernlohr, R.W., Haddox, M.K. and Goldberg, N.D. (1974) J. Biol. Chem. 249, 4 3 2 9 - - 4 3 3 4 Setlow, P. (1973) Biochem. Biophys. Res. Commun. 52, 365--372 Rickenberg, H.V. (1974) Annu. Rev. Bacteriol. 28, 353--369 Yeung, K.H., Chaloner-Taxsson, G. and Yamazaki, H. (1976) Can. J. Biochem. 54, 854--865 Syms, M.E. (1975) B.A. (Mod.) Project, Dept. of Biochemistry, Trinity College, University of Dublin McGinnis, J.F. and Paigen, K. (1973) J. Bacteriol. 114, 885--887 Harwood, J.P., Gazdar, C., Prasad, C., Peterkofsky, A., Curtsi, S. and Epstein, W. (1976) J. Biol. Chem. 251, 2462--2468 Stephens, J.C., Artz, S.W. and Ames, B.N. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4389--4393 Yang, H.L., Zubay, G., Urm, E., Reiness, G. and Cashel, M. (1974) Proc. Natl. Acad. Sci. U.S. 71, 63-67 Gallant, J. and Margason, G. (1972) J. Biol. Chem. 247, 2289--2294 Rhaese, H.J., Hoch, J.A. and Groscurth, R. (1977) Proc. Natl. Acad. Sci. U.S. 74, 1125--1129 Proudfoot, N.J. and Brownlee, G.G. (1974) Nature 2 5 2 , 3 5 9 - - 3 6 2 Sy, J. and Akers, H. (1976) Biochemistry 15, 4399--4403 Gallant, J., Irr, J. and Cashel, M. (1971) J. Biol. Chem. 246, 5812--5816 Rhaese, H.J. and Groscurth, R. (1974) FEBS Lett. 44, 87--93 Elmerich, C. and Aubert, J.P. (1975) Spores VI, 385--390 Coote, J.G. (1974) J. Bacteriol. 120, 1102--1108 Rhaese, H.J., Grade, R. and Dichtelmiiller, H. (1976) Eur. J. Biochem. 64, 205--213 Willecke, K. and Mindich, L. (1971) J. Bacteriol. 106, 514--518 Rhaese, H.J., Dichtelm611er, H. and Grade, R. (1975) Eur. J. Biochem. 56, 385--392 Cashel, M., Lazzarini, R.A. and Kalbacher, B. (1969) J. Chromatogr. 40, 103--109 Maizel, J.V., Jr. (1971) Methods Virol. V, 179--246 Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606--2617 Sanzey, B. and Ullman, A. (1976) Biochem. Biophys. Res. Commun. 71, 1062--1067
Biochimica et Biophysica Acta, 541 ( 1 9 7 8 ) 1 8 - - 3 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28537
CATABOLITE REPRESSION IN BACILLUS SUBTILIS
B A R B A R A DOWDS, L E S L E Y B A X T E R a n d M I C H A E L M c K I L L E N *
Department of Biochemistry, Trinity College, Dublin 2 (Republic of Ireland) (Received O c t o b e r 1 7 t h , 1 9 7 7 )
Summary The D-gluconate transport system of Bacillus subtilis is optimally induced by exposure of cells for 2 h to 5 mM D-gluconate in the growth medium. D-Gluconate transport is subject to catabolite repression, as distinct from inducer exclusion or catabolite inhibition, in a manner parallel to the repression of inducible histidase synthesis, suggesting that the repression is not specific to this transport system. Maximum repression with the repressing carbon source (10 mM) added to cells grown in either casein hydrolysate or amino acid medium is achieved within two doubling times. Urea, the only non-carbon source tested for a repressing effect, was found to act solely by inducer exclusion. The ability of a sugar carbon source to evoke catabolite repression appears to be unrelated to its suitability as a substrate for the sugar : phosphoenolpyruvate phosphotransferase system but nonetheless the conversion to a phosphorylated derivative of the sugar seems essential. Repressed cells fail to synthesize, or do so to a more limited extent, an as y e t unidentified phosphorylated comp o u n d (probably a highly phosphorylated nucleotide) which is accumulated in the medium of non-repressed cells. Mutant studies imply that inosinic acid synthesis is necessary for catabolite repression whereas the adenosine highly phosphorylated nucleotides required for spurulation are not.
Introduction
The nature of the molecular events controlling the initiation of bacterial sporulation continues to remain elusive [ 1 ]. Sporulation represents the simplest differentiating system and an understanding of the repression mechanism involved in the sporulation process may be aided by the study of another type of genetic regulation, namely catabolite repression. The general phenomenon of catabolite repression may operate [2] by * To
whom request for
reprints should
be sent.
19 inducer exclusion, 'true' catabolite repression (including transient repression [3] ) and catabolite inhibition. This paper is concerned with the effect of catabolite repressing compounds on the inducible D-gluconate transport system of Bacillus subtilis [4,5]. In order to check that the observed repression was not specific to the D-gluconate transport system, we tested the effect of some of the repressing compounds on histidase synthesis, which is genetically well-documented in this respect [6]. Lopez and Thoms [7] have suggested that in B. subtilis repressing compounds may exert their effects via one or more common metabolites, namely phosphorylated sugars. This idea is supported by the similarities between catabolite repression and suppression of sporulation, the latter process known to be inhibited by a build-up of phosphorylated intermediates of metabolism [8,9]. In all cases studied in bacteria, catabolite repression results from a single or very few regulatory systems acting on the expression of all genes subject to this form of control [2,10--13]. For example, in Escherichia coli most of such genes are regulated by the phosphotransferase system both directly and by its effect on the transport of inducer and the intracellular concentration of cyclic AMP which in turn regulate protein synthesis [14]. The siinilarities between the response of inducible enzyme and transport proteins studied in B. subtilis [6,7,15--18] indicate a regulation common to them all, but a mechanism involving cyclic AMP or cyclic GMP is very unlikely [18--25]. In addition, unlike the case for E. coli [26], Lopez and Thoms [7] found that sugar uptake by itself did not effect catabolite repression in B. subtilis. In this laboratory Baxter et al. [15] showed that structural analogues of D-glucose which both lack the capacity to repress and to act as phosphotransferase system substrates in E. coli [27] do in fact cause repression in B. subtilis. If neither the sugar: phosphotransferase system nor cyclic AMP is involved in catabolite repression in Bacillus, just how is this process mediated? There is much evidence pointing to a role for highly phosphorylated nucleotides in the regulation of gene expression in B. subtilis and other bacteria. For example, guanosine 5'-diphosphate 3'-diphosphate (ppGpp or MS1) modulates control at the level of transcription, of the histidine operon in Salmonella typhimurium [28] and other catabolic operons in E. coli [29], and both ppGpp and pppGpp (MSll) appear prior to the decrease in RNA synthesis following amino acid starvation of B. subtilis and stringent but not relaxed strains ofE. coli [30]. For an opposite (i.e., non-repressed} state such as sporulation to occur, synthesis of other highly phosphorylated nucleotides: the adenosine tetra- and pentaphosphates, appears to be necessary [31], while ribosomes from the cells committed to sporulation lack the capacity to synthesize MS1 and MSll. Certain eukaryotic RNA species whose synthesis may be switched on and off have highly phosphorylated adenosine and guanosine nucleotides incorporated into the 5'-end of the chains [32]. It may be that their presence stems from a primitive type of regulation. We suggest that substances capable of initiating catabolite repression are metabolised to their phosphorylated derivatives, that the information which establishes repression flows from, for example, sugar phosphate to UTP or ATP and from specific phospho-proteins to GTP depending on the particular control system. UTP, ATP and GTP might possibly regulate the level of highly phosphorylated nucleotides either by an alter-
20 ation in substrate concentration [33] or by feedback regulation of the synthesizing enzymes [34]. Finally, we may speculate that the alteration in highly phosphorylated nucleotide concentration modulates the synthesis of the RNA species which require this type of substrate for initiation of their synthesis. There is some direct evidence implicating highly phosphorylated nucleotides in the control of catabolite repression. Firstly, Yeung et al. [24] reported the extracellular accumulation of an unknown phosphorylated compound in B. megaterium, grown in glucose or galactose but not glycerol, whose rate of excretion depended on the carbon source. Secondly, pppAppp is known to be excreted into the medium at the beginning of sporulation of B. subtilis [35]. Evidence for the passage of the repressing information via purine metabolism comes from the coupled loss of the ability to sporulate and repress by mutants defective in inosinic acid synthesis. The early step in its synthesis, that is feedback inhibited by ATP and GTP, is implicated [36]. While it remains possible that highly phosphorylated nucleotides mediate catabolite repression in a manner similar to that of cyclic AMP, it seems more likely that the effect is slightly less specific since despite an intensive search, Coote [37] failed to isolate a mutant of B. subtilis resistant to catabolite repression. The apparently lower genetic specificity for catabolite repression in B. subtilis, in contrast to E. coli, might be explained by a possible lower specificity of highly phosphorylated nucleotide than of cyclic AMP function; this could arise by the compensation for loss of one highly phosphorylated nucleotide by an increase in another, such as the substitution of adenosine by uridine nucleotides both of which appear prior to sporulation in B. subtilis [38]. Materials and Methods Strains. B. subtilis SB-26 (trp C2 met C3), obtained from Dr. E. Nestor via Dr. N. Sueoka, is a derivative of the transformable 168 strain. The mutants in intermediary metabolism were strains 61372 and 61411 whose phenotypes were phosphoglucoisomerase- and phosphofructokinase-negative, respectively (gifts from Dr. E. Freese [8]) and BR 95 glp K 3--7 and Br 95 glp D 12--11 phenotypes, glycerol kinase and NAD-independent sn-glycerol-3-phosphate dehydrogenase-negative, respectively (obtained from Dr. V. Lindgren). Dr. H.J. Rhaese [31] supplied the sporulation mutants of B. subtilis JH 649 (trp C2 phe-1 spo O F221) and 50413 (trp C2 ind spo-) and their spo ÷ revertants. The B. megaterium mutants in purine metabolism, gin- 26 (U- glutamine-), glu- 11 (U- glutamate-) and put 562 (U- purine (class la)) and their revertants (U-) were provided by Dr. C.E. Elmerich [36]. Media and growth conditions. The mineral salts medium contained (in 1 1 of distilled water): K2HPO4, 14 g; KH2PO4, 6 g; MgSO4 • 7H20, 0.25 g; MnC12 • 4H20, 0.02 g; L-giutamic acid (monopotassium salt), 0.15 g; (NH4)2SO4, 2 g; tripotassium citrate H~O, 1 g. The medium was supplemented with 0.025 g L-tryptophan per 1, 0.03 g L-methionine per 1 and 0.5%, w/v, casein hydrolysate (Merck). Alternatively an altered form of the amino acid carbon source described by Willecke and Mindich [39] was used; the histidine was omitted
21 and the other amino acids (tryptophan and methionine excepted) were present at three times the concentration recommended. All strains were grown as single colonies on tryptose blood agar base (TBAB) plates (Difco) at 37°C for 24 h and subsequently maintained at 4°C. The plates were appropriate supplemented for mutant-strains [36]. F o r growth in liquid media the cells were loop-inoculated from a fresh colony ( < 2 4 h) growing on a TBAB plate, into 5.0 ml of (37°C) growth medium. The medium was contained in a sterile metal-capped tube which was vigorously shaken overnight (12--14 h minimum) at 37°C in a reciprocating water bath shaker. The culture was then diluted by placing a 1-ml portion into either 15 or 25 ml of fresh prewarmed growth medium containing 5 mM D-gluconate, contained respectively in either 150- or 250-ml conical flasks fitted with nephelometer sidearms. (D-Gluconate optimally induces its own transport system at a concentration of 5 mM in the medium without altering the growth rate of the cells [4] ). The apparent absorbance at 650 nm of such diluted cultures was initially approx. 0.05 units. All cultures were shaken vigorously in a reciprocating water bath at 37°C (150 rev./min, 35 mm). The growth of the culture was routinely followed with an Eel colorimeter (OR 1 filter) using the nephelometer side arm of the flask. A 10 mM concentration of repressing c o m p o u n d was added at an Eel absorbance of 0.1, when the lag phase of growth had ended, since 10 mM glucose shortens the lag phase b u t does n o t alter the doubling rate of B. subtilis under the conditions described. The shaker was never stopped for more than 30 s and the cultures were never allowed to cool as the growth o f B . subtilis is particularly sensitive to changes in these parameters. An Eel value of 1.0 is virtually equivalent to an A650n m of 1.0 and a cell density of 4 • 108 cells/ml of culture. The cells were usually grown to early exponential phase (A6son m of 1.0), then rapidly harvested by membrane filtration (47 mm diameter, 0.6 pm pore size) and washed with one volume of prewarmed (37°C) resuspension medium (mineral salts medium containing 0.5% D-glucose and 0.007% chloramphenicol). The filter was then c u t in half, placed in a tube with the calculated volume of resuspension medium at 37°C to give a final apparent A650nm of 1.3 and then vortex-mixed for approx. 30 s. The exact apparent absorbance of the resuspension was then obtained at 650 nm on a 1 : 3 dilution of the cells. An absorbance of 1.0 at 650 nm is equivalent to 340 pg dry weight of cells per ml of suspension. The suspension was stored at 37°C for less than 45 min before assays were performed. Gluconate transport assay. The assay tube held 0.7 ml of resuspension medium at 37°C containing sodium D-[U-14C]gluconate (0.38 pCi). The reaction was initiated by the rapid addition of 0.6 ml of cell suspension to the assay tube. Transport was allowed to proceed for 30 s and was terminated by rapidly removing 1.0 ml of the suspension with an automatic pipette and filtering immediately on a premoistened membrane filter (25 mm diameter, 0.65 pm pore size) supported on the base only by a Swinnex-25 holder (Millipore Ltd.). The filter pad was immediately washed with 5 ml of resuspension medium (37°C) and then rapidly transferred to 10 ml of a toluene/ethanol (12 : 7, v/v) based scintillation fluid containing 0.4% PPO. Reaction termination and washing procedures were completed within 15 s and all assays were carried o u t in duplicate. The zero-time transport control was obtained by assaying resuspension
22 medium instead of cells under the standard conditions. All scintillation vials were swirled before being placed in a liquid scintillation spectrometer (Packard Instruments Ltd., Model 3375 or 3385) and counted using the preset '4C counting channel. Counting time was arranged so that the standard deviation of counting, for the anticipated lowest activity sample (excluding the zero-time transport control), was less than 1%. The zero-time transport control never exceeded the natural background count rate by more than 3-fold. The mean value for the initial velocity of transport within a particular experiment using the above procedure, was 71.8 + 1.4 pmol per min per gram dry weight of cells (10 assays). Variations in the measured initial velocity did occur from experim e n t to experiment (50--90 pmol/min per g dry weight of cells). Under the conditions of the standard transport assay the cells removed not more than 10% of the total radioactivity initially present in the assay mixture. The initial external D-gluconate concentration in all assays was 0.1 mM (~3 × Kin). Histidase assay. Cells were grown in amino acid medium since assay of urocanic acid is performed by its absorbance at 277 nm [6] and casein also absorbs at this wavelength. Cultures were grown to A65onm of 1.0, shaken in toluene over ice and centrifuged at 3600 rev./min for 15 min. The histidase assay was essentially that described by Chasin and Magasanik [6]. Labelling, extraction and chromatography of regulatory nucleotides. The procedure described by Rhaese et al. [41] was essentially followed. Cells were labelled with 0.5 mCi/ml [32P]orthophosphate added at A6s0 = 0.1, and grown till A6s0 = 1.0. 10 mM D-glucose as repressor was added to one flask of cells at A6s0 = 0.1. Extraction of nucleotides with formic acid was followed by filtration on membrane filters (pore size 0.6 pm) to yield an intracellular extract while the m e t h o d of Rhaese et al. [40] was used to obtain a " t o t a l " extract (intracellular extract plus medium). Thin-layer chromatography of the total and intracellular extracts was performed on polyethyleneimine-impregnated cellulose plates. One-dimensional chromatography was with 1.5 M sodium phosphate buffer, pH 3.4 [40]. Two-dimensional chromatography solvents were 3.3 M a m m o n i u m formate + 4.2% boric acid adjusted to pH 7.0 with NH4OH (first dimension) and 0.85 M KH2PO4, pH 3.4 (second dimension). In the latter case, between the two runs the plates were dried, soaked in methanol for 5 min, then in distilled water for 15 min and oven-dried [41]. 20-pl spots of each formic acid extract were applied to the chromatogram with blow drying. Blue Brand Medical X-ray film (tinted estar safety base duplitized) was exposed to the chromatography plates for 6--36 h depending on the length of storage time of the labelled extracts. The films were developed with Kodak DX80 developer. Chromatograms were marked and cut into 1 X 1 cm square sections and placed in scintillation vials to which were added 10 ml of 0.6% PPO in toluene scintillation fluid. The samples were then counted in a Packard TriCarb 3385 spectrometer using the preset 32p channel. Polyacrylamide gel electrophoresis. Formic acid was removed from 40-pl volumes of the extracts under a stream of nitrogen gas and the residue taken up in SDS solubilising solution [42]. 25 pl were then applied to a 10% polyacrylamide slab gel in Tris/SDS buffer at a constant current of 25 mA [42]. After electrophoresis the gel was wrapped in a monolayer of cling film (Propax) and autoradiography was performed as detailed in the thin-layer chromatography section above. Finally, the gel was stained with Coomassie blue [43].
23
Results
Influence of carbon source on activity of D-gluconate transport system Table I shows that the non-induced D-gluconate transport activity is approximately the same (2--4 pmol/min per g dry weight) whatever the carbon source used for growth, though activity may be slightly higher in cells grown on a rich carbon source such as casein hydrolysate (7 pmol/min per g dry weight). When cells were grown on D-gluconate as sole carbon source (induced cells) the initial transport velocity was 23 pmol/min per g dry weight of cells. When cells were induced in the presence of 5 mM D-gluconate and grown on a variety of carbon sources, D-gluconate transport activity was either repressed (D-glucose and malate) or elevated (succinate, D-ribose and casein hydrolysate) to varying extents in comparison to the activity obtained from cells grown in D-gluconate as sole carbon source. In no case did a carbon source cause total catabolite repression of transport activity (i.e., identical D-gluconate transport activity in the presence and in the absence of inducer). We selected 0.5% (w/v) casein hydrolysate as a carbon source for most purposes since it allowed a rapid growth rate (td = 30 min) whilst still permitting strong catabolite repression by other easily utilised carbon sources such as D-glucose (Table II). The amino acid medium was used as a defined carbon source to check the extent, if any, of repression produced by the casein hydrolysate medium.
Type of repression In order to show that the repression caused by D-glucose, for instance, resulted from the phenomena of catabolite repression or inhibition as opposed
TABLE I INFLUENCE OF GROWTH MEDIUM ON ACTIVITY OF D°GLUCONATE TRANSPORT
SYSTEM
SB-26 w a s g r o w n o n t h e m i n e r a l salts m e d i u m , s u p p l e m e n t e d w i t h t h e a p p r o p r i a t e c a r b o n s o u r c e s .
A d d i t i o n s to g r o w t h m e d i u m
D-Gluconate transport activity ( ~ m o l • m i n -1 • g-1 dry w e i g h t )
25 mM D-gluconate
23.0
25 m M D - g l u c o s e 25 m M D - g l u c o s e + 5 m M D - g l u c o n a t e
2.0 8.0
25 m M c i t r a t e 25 m M c i t r a t e + 5 m M D - g l u c o n a t e
2.0 22.0
25 mM succinate 25 m M s u c c i n a t e + 5 m M D - g l u c o n a t e
2.5 45.5
25 m M L - m a l a t e 25 mM L-malate + 5 mM D-gluconate
3.0 13.0
25 m M D - r i b o s e 25 m M Doribose + 5 m M D - g l u c o n a t e
not determined 32.0
A m i n o acid s u p p l e m e n t A m i n o acid s u p p l e m e n t + 5 m M D - g l u c o n a t e
4 24
0.5% casein h y d r o l y s a t e 0.5% casein h y d r o l y s a t e + 5 m M D-gluconate
7.0 70.0
24 T A B L E II R E P R E S S I O N BY V A R I O U S M E T A B O L I T E S O F D - G L U C O N A T E T R A N S P O R T IN B. S U B T I L I S SB-26 A d d i t i o n s t o i n d u c i n g m e d i u m at a final c o n c e n t r a t i o n o f 10 m M None L-Glutamate Succinate Citrate D-Ribose Galactose L-Malate D-Mannose Sorbitol Glycerol D-Fructose D-Glucose Mannitol
Percentage of non-repressed transport activity remaining 100 > 100 100 :> 1 0 0 > 100 60 54 31 25 21 13 8 8
to inducer exclusion, transport activity was compared in non-induced cells grown in the presence and absence of the repressing compound. As the initial velocity of D-gluconate transport was lower in the presence of 10 mM D-glucose than in its absence (5 and I pmol/min per g dry weight of cells), it may be concluded that D-glucose represses by catabolite repression and/or inhibition. However, inducer exclusion cannot be ruled out and indeed seems a probable component of the repression mechanism, since the degree of repression is much greater in induced than in non-induced cells. Malate (10 mM) was the only other repressing carbon source tested in this way. Apparently, malate, like Dglucose, causes some catabolite repression and/or inhibition (3.5 pmol/min per g dry weight of cells). It was assumed that the other c o m p o u n d s producing marked repression (Table II) would act in the same way as D-glucose and malate. In order to determine whether D-glucose was causing catabolite inhibition, cells, which had been induced for D-gluconate transport, were assayed by the standard assay procedure in the presence and absence of D-glucose. The same level of transport activity was observed whether or n o t D-glucose was present in the assay buffer, indicating that catabolite inhibition is not involved in the repression process.
Kinetics of induction, and repression of the D-gluconate transport system. McKillen and Rowntree [4] reported that the inclusion of 5 mM D-gluconate in the 0.5% (w/v) casein hydrolysate medium resulted in maximal induction of D-gluconate transport activity without significantly altering the growth rate of the culture. The time course for the induction and subsequent decay of D-gluconate transport activity was determined by taking replicate cultures of non-induced cells and adding D-gluconate (5 mM) at different times after inoculation, and harvesting the cells at an absorbance of 1.0. Fig. 1 shows the initial velocity of D-gluconate transport in relation to the time allowed for induction to take place. It can be seen that maximum induction (6-fold) with 5 mM gluconate has occurred 2 h (4 doubling times) after its addition to the previously non-induced culture. To determine the rate of decay of transport activ-
25
80
'T
70 _-.'." ......
:~: 6o =.
50
"E
40
2O
~,
~q I
I
I
I
~-~ %- - %-J
I
0 1 2 3 4 18 Time allowed f o r induction before harvesting of celts [h) Fig. 1. I n d u c t i o n k i n e t i c s . 5 m M D-gluconatae was a d d e d to an e x p o n e n t i a l l y growing culture at different t i m e s b e f o r e h a r v e s t i n g . A t a n a b s o r b a n c e o f 1 . 0 , c e l s were filtered, r e s u s p e n d e d i n b u f f e z c o n t a i n i n g c h l o r a m p h e n i c o l and assayed for D-gluconate transport a c t i v i t y .
ity the cells were induced under standard conditions, full induction occurring before an absorbance of 0.5, then harvested, washed in non-inducing growth medium and resuspended in the latter to an A6s0 = 0.5. The cells were grown in this non-inducing medium to an absorbance of 1.0, then harvested and assayed for D-gluconate transport. The initial velocity for D-gluconate transport was reduced by one half to 35 pmol/min per g dry weight of cells. A control in which the induced cells were resuspended in standard inducing growth medium and grown for a further generation indicated that resuspension per se had no effect on the ability of the cells to transport gluconate at the standard rate of 70 pmol/min per g dry weight of cells. Repression kinetics were obtained on cells which were exposed to inducer at an absorbance of 0.05. Six parallel cultures were grown which in the absence of repressor would normally show a transport rate of a b o u t 70 pmol/min per g. Glucose was added to each at different times before harvesting. Fig. 2 shows that it began to exert its repressing effect after approx. 1 doubling time, this rising to a maximum when the glucose was present for t w o doubling times or more. The order of addition of D-gluconate and D-glucose did n o t affect the transport rate of the harvested cells so long as these c o m p o u n d s were added, at least 2 and 1 h, respectively, before harvesting.
Types of repressing and non-repressing compounds Table II shows the D-gluconate transport activity remaining in cells grown in casein hydrolysate medium with 5 mM gluconate, in the presence of a range
26
70
~"
(5C
E 5O E 4O
o 3o c 20 o
~o J a ,
0
,
I
I
,
,
I
2
Time allowed for repression before harvesting of cells (h) Fig. 2. Repression kinetics. A culture w a s i n d u c e d with 5 m M D-gluconate at an absorbance of 0.05. I 0 m M D-glucose w a s a d d e d a t d i f f e r e n t times before harvesting at a n absorbance of 1.0.
of metabolites all added at a final concentration of 10 mM (this concentration was low enough so as not to alter the growth rate of cells in casein hydrolysate or amino acid media). All of the repressing substances enter the glycolylic pathway and the citric acid cycle. The phosphotransferase system sugars tested (D-glucose and D-fructose) produced severe repression and in general unsubstituted hexoses and sugar alcohols produced much greater repression than 5-carbon sugars, amino acid or Krebs cycle intermediates. Phosphorylated sugars, such as glucose 6-phosphate and fructose 6-phosphate, repressed to a minor extent (results n o t shown) which may be explained by their inability to cross the cell membrane. In support of this contention is the fact that B. subtilis failed to grow on any phosphorylated sugar (i.e., D-ribose 5-phosphate, D-glucose 1-phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate, 2-phosphoglycerate and 6-phosphoglycerate) when tested for use as a sole carbon source at 0.5% concentration. Casein hydrolysate medium by itself seems to have little repressing activity; the most severe repressing c o m p o u n d (D-glucose) left only 17% of nonrepressed activity in cells grown in amino acid medium as compared to 5--11% in casein hydrolysate grown cells. Finally, since urea specifically inhibits the expression of catabolite repression sensitive operons in E. coli [44] we tested its effect on D-gluconate transport in B. subtilis. In the presence of 10 mM urea the percentage of nonrepressed D-gluconate transport activity remaining was 72 and 32--46% in cells grown in casein hydrolysate and amino acid media, respectively. We have now extended our preliminary studies attempting to localise the site of action of compounds causing catabolite repression [15] by the use of mutants blocked in the intermediary metabolism of certain carbohydrates. The positions of the metabolic blocks in the mutants used are described in Table III. It seems reasonable to conclude that the transport of glycerol across the
27 T A B L E III R E P R E S S I O N O F D - G L U C O N A T E T R A N S P O R T I N B. S U B T I L I S S T R A I N S D E F E C T I V E IN I N T E R MEDIARY METABOLISM
Mutant strain
Addition to medium
Transport rate
(10 m M )
g-1 dry wt.)
( # m o l • m i n - 1.
Non-repressed activity remaining (%)
61372
none
(phosphoglucoisomerase negative)
D-glucose
61411
none
(fructose-l-phosphate kinase negative)
D-fructose
BR 9 5 glp K 3-7
none
(glycerol kinase negative)
glycerol
BR 9 5 glp D 12-11
none
(NAD-indep endent sn-glycerol-3 -phosphate dehydrogenase negative)
glycerol
59 31
100
34 lS
100
43 44
100 >100
51 58
>100
53 54
100
Location of blocks in intermediary metabolism D-glucose - D-fructose
glycerol
glp K 3-7
> glucose 6-phosphate
613~72
> fructose 1-phosphate
61411 ~
> fructose 6-phosphate > fructose 1.6-diphosphate
> glycerol 3-phosphate glp D 1 2 - 1 1 > d i h y d r o x y a c e t o n e phosphate
membrane has no role to play in initiating catabolite repression. On the other hand both D-glucose and D-fructose initiate catabolite repression either by virtue of their transport across the membrane or on conversion to their phosphorylated derivatives or by a combination of the two alteratives. The fact that the repression severity caused by D-glucose is slightly lower in the case of both the phosphoglucoisomerase and fructose-l-phosphate kinase negative mutants in comparison to the wild type may reflect the fact that accumulation of hexose phosphates in these mutants decreases the uptake of the parent sugar by the cell. We have not been able to directly assess the importance of phosphorylated intermediates in mediating catabolite repression but the implication that phosphorylated compounds in general may be able to initiate catabolite repression is upheld by some recent results of Lopez and Thoms [7]. The D-glucose mediated catabolite repression was further investigated by examining the ability of D-glucose analogues to cause repression. Harwood et al. [27] found in E. coli that substituents about carbon atoms 1 or 2 of glucose were inhibitors of adenylate cyclase and also substrates of the sugar: phosphotransferase system whereas analogues with changes in other parts of the molecule exhibited neither property. When analogues were added to inducing medium at a final concentration of 10 mM the percentage of non-repressed transport activity remaining was as follows: 6-deoxy-D-glucose (78%), 2-deoxyD-glucose (69%), 3-O-methyl-D-glucose (34%) and ~-methyl-D-glucoside (30%). Our results, at least in B. subtilis, show no such relationship between position of substitution and repressing activity. This is hardly surprising if the assump-
28 tion is made that neither the sugar : phosphotransferase system nor cyclic AMP are involved in catabolite repression in B. subtilis. On the other hand the stereospecificity of the glucose molecule is of supreme importance since L-glucose was unable to produce any catabolite repression. It has not been determined whether L-glucose can be transported by these cells. In the case of the other analogues, the cells grew slowly on 0.4% 2-deoxy-D-glucose and a-methyl-Dglucoside as sole carbon sources but failed to grow on 6-deoxy-D-glucose. The severity of repression observed with these analogues appears to parallel closely the extent of growth the cells are capable of making on these substrates. A supposition that some of the repression is caused by the transport of a sugar or analogue across the membrane could be used to account for the ability of 6-deoxy-D-glucose to repress despite its failure to support growth.
Catabolite repression of histidase synthesis Potential repressing substrate (10 mM) and inducer (14 mM histidine) were added at an absorbance of 0.1 to cells growing in amino acid medium. The cells were harvested at an absorbance of 1.0 and assayed for production of urocanic acid from L-histidine. The percentage of non-repressed activity remaining in cells exposed to the various potential repressing substrates was 48% D-glucose), 54% (D-mannose) and 100% (urea). Though the figures are widely different for the extent of repression of histidase synthesis and gluconate transport, the order of severity of repression by the three substrates is the same, suggesting that catabolite repression is probably being mediated in the same way for the two inducible systems.
Molecular mediation of catabolite repression H o w could the proposed signal from the intracellular concentration, of a phosphorylated sugar(s) for instance, be passed on so as to result in a cessation of transcription of catabolite repression-sensitive genes? The possibility of detecting a difference in the concentration or pattern of phosphorylated metabolites in the cytoplasm or exogenously in growth medium between repressed and non-repressed cells was investigated. Both 'total' extracts (intracellular extract plus medium) and intracellular extracts derived from cells grown on [32P]orthophosphate were chromatographed and the only difference observed between D-glucose-repressed and non-repressed cells may be seen in Figs. 3 and 4. The various 32P-labelled compounds, separated on both one- and two-dimensional chromatograms, had identical R F values and were present in equal amounts in each case whether obtained from the repressed or nonrepressed intracellular extracts. However a difference was detected between the " t o t a l " extracts in that a much greater proportion of the [32P]phosphorus was incorporated into the origin spot in the non-repressed than in the D-glucoserepressed extracts. Rhaese and Groscurth [35], Rhaese et al. [38] and Cashel et al. [41] have determined the R F values for various nucleotides, including highly phosphorylated nucleotides on this type of separating system. In the first dimension the deoxyribonucleotides migrate further than their ribonucleotide analogues, while in the second dimension the order of migration from slowest to fastest is guanine, adenine, cytosine, uracil and thymine nucleotides. The second dimension also facilitates separation of nucleotides on the basis on
29 .......2 n d
d imens~on
r~
o
E
0.
~m---- O r i g i n spot
Fig. 3. Separation of 32 P-labelled c o m p o u n d s from fully induced cells g r o w n in casein-containing growth m e d i u m to which 10 m M glucose was added at an absorbance of 0.1 at the same time as the label. A "total" extract (intracellular extract plus m e d i u m in formic acid) was cbromatographed on polyethylenimine cellulose in t w o dimensions.
Fig. 4. S e p a r a t i o n o f 3 2 p - l a b e l l e d c o m p o u n d s f r o m f u l l y i n d u c e d , n o n - r e p r e s s e d cells ( g z o w n in c a s e i n without the addition of glucose). Label added at an absorbance of 0.1. A "total" (intraceHular extract p l u s m e d i u m in f o r m i c a c i d ) w a s c h r o m a t o g r a p h e d o n p o l y e t h y l e n e i m i n e in t w o d i m e n s i o n s .
30 their number of constituent phosphate groups; the higher the number, the shorter the migration. Thus our origin spot may contain a very highly phosphorylated nucleotide(s) (>5 phosphate group and probably containing the guanine base [38]) and/or phosphorylated polysaccharide(s) and protein(s). The extracts were then applied to 10% polyacrylamide slab gels (Fig. 5). No Coomassie Brillant Blue staining components were detected on the gel and 32p_ labelled components appears to be only present at the origin and tracker dye positions on the gel slab. Furthermore, there was no qualitative difference detected between the cells grown under the two conditions for the two types of extract. Thus the difference observed on the thin-layer chromatograms may be attributable to either a highly phosphorylated nucleotide, a polynucleotide or to a phosphoprotein of molecular weight between 20 000 and 130 000.
D-Glucose catabolite repression in mutants defective in nucleotide metabolism B. subtilis mutants defective in the synthesis of highly phosphorylated nucleotides. Table IV shows the degree of catabolite repression of D-gluconate transport activity produced by D-glucose in mutants deficient in p p p A p p p synthetase. The JH 649 strain has a single-site mutation with respect to 649-R, but the difference between the repression of their respective D-gluconate transport activities is probably not significant especially in the light of the negligible difference detected between the 50413 strain and its revertant, 50413-R. The degree of catabolite repression of histidase synthesis is also essentially similar (67%) in the case of both the 50413 strain and its 50413-R revertant (results
I+
I-
T-
T+
I
P o s i t i o n of t r a c k e r dye after
electrophoresis Fig. 5. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f f o r m i c acid e x t r a c t s o f c a s e i n - g r o w n cells labelled w i t h 3 2 p a t a n a b s o r b a n c e o f 0 . 1 . R i g h t t o left: T +, T - , I - , I +. I: intracellular e x t r a c t ; T: " t o t a l " (intracellular e x t r a c t plus m e d i u m ) e x t r a c t ; +: g l u c o s e added; --: n o n - r e p r e s s e d ( g l u c o s e n o t a d d e d ) .
31 TABLE
IV
GLUCOSE S U B T I L IS
REPRESSION
OF
D-GLUCONATE
TRANSPORT
IN SPORULATION
MUTANTS
O F B.
A l l m u t a n t s , derived f r o m strain S B - 2 6 , w e r e o b t a i n e d f r o m Dr. H.J. R h a e s e [ 3 1 ] . R h a e s e et al. m e a s u r e d n u e l e o t i d e s y n t h e s i s in vitro w i t h r i b o s o m e s i s o l a t e d f r o m the appropriate stage o f g r o w t h . H P N s I a n d I I are n o t s e e n in vivo and m a y be u n s t a b l e p r e c u r s o r s o f H P N I V , p p p A p p p . H P N I V is s y n t h e s i s e d b y the e n z y m e , p p p A p p p s y n t h e t a s e w h i c h is d e f e c t i v e in the s p o O F m u t a n t . H P N , highly p h o s p h o r y l a t e d n u c l e o tide.
Mutant:
Genotype:
A b i l i t y to s y n t h e s i s e n u c l e o t i d e s T2 ppGpp + pppGpp { log g r o w t h ppApp
pppApp
( H P N I)
(HPN II)
JH649
649-R
50413
50413-R
trp C 2 p h e - 1 spoOF221
trp C2 phe-1 spo ÷
trp C2 ind spo-
trp C2 ind spo +
Yes Yes
No Yes
Yes Yes
No Yes
( T2 log g r o w t h
V e r y little No
Yes No
V e r y little No
Yes No
T2 ( log g r o w t h
No No
Yes No
No No
Yes No
80 8
55 10
65 8
68 6
10
18
12
9
V 0 in i n d u c e d g r o w t h m e d i u m ( # m o l • m i n - l • g - l dry w t . ) -- glucose + glucose
Percentage of non-repressed transport r e m a i n i n g after glucose addition
not shown in Table IV). It may be concluded that the adenosine highly phosphorylated nucleotides are not required for the expression of catabolite repression. B. Megaterium mutants defective in purine metabolism. A lower level of D-gluconate transport activity was observed in B. megaterium than in B. subtilis using the same conditions for cell growth, induction and transport assay. Nonetheless, this D-gluconate transport activity was subject to D-glucose catabolite repression (Table V). Despite the addition of sufficient adenine {0.02 g/l) to facilitate growth, the pur 562 mutant is clearly much less sensitive to catabolite repression than the wild type, gin- 26R1. The gin- 26 mutant was used in order to test whether repression is mediated at one of the control steps in purine synthesis where glutamine donates an amino group to the base. The D-glucose catabolite repression may be dependent to a certain extent on the presence of glutamine synthetase; the repression of D-gluconate transport activity is lower in the gln- mutant, grown in the presence of glutamine (0.5 g/l) than in its revertant, gin- 25R1. On the other hand, a mutant defective in glutamate synthase obtained from Dr. C. Elmerich [36] {results not shown) exhibited the same degree of repression as its wild type revertant strain of B. megaterium. These results imply that the early stages in inosinic acid synthesis (conversion of 5-phosphoribosyl-l-pyrophosphate to 5-aminoimidazole ribonucleotide) are necessary for catabolite repression, as they are for sporulation [36]. The possible requirement for glutamine though not glutamate is contentious
32 TABLE V GLUCOSE REPRESSSION OF D-GLUCONATE DEFECTIVE IN PURINE METABOLISM
TRANSPORT
IN MUTANTS
O F B. M E G A T E R I U M
All m u t a n t s w e r e o b t a i n e d f r o m Dr. C.E. E l m e r i c h a n d g r o w n in m e d i u m s u p p l e m e n t e d w i t h u r a c i l a n d adenine or glutamine [36].
Mutant: Genotype:
G i n - 26
G i n - 26 R I
Put 562
Sporulation only repressed in p r e s e n c e o f g l u t a m i n e . U- gin- (glutamine synthetase)
spo + U-
Sporulation repressed by adenine but not by uracil U - P u r - (class l a ) *
20.7 2.8
20.8 17.4
13.5
83.7
V 0 in i n d u c e d g r o w t h m e d i u m ( # m o l • rain - l • g - I dry wt.) -- glucose 15 + glucose 3.3
Percentage o f non-repressed transport remaining after g l u c o s e addition
22
* Class l a m u t a n t s a r e d e f i c i e n t in t h e c o n v e r s i o n o f 5 - p h o s p h o r i b o s y l - l - p y r o p h o s p h a t e imidazole ribonucleotide.
to 5-amino-
since the appropriate amino acid supplements had to be added in order to allow culture growth. Ideally the supplements should provide sufficient amino acid to facilitate growth while not allowing catabolite repression in the mutant defective in the production of amino acids involved in repression, but in practire this balance is difficult to achieve. Discussion
The only qualitative or quantitative difference between the formic acid extracts of repressed and non-repressed cells lies in the greater amount of label at the origin, of the extracellular material from the latter cells. If this represents a highly phosphorylated nucleotide, then its most likely identity would be pppGppp. Rhaese et al. [31], report that adenosine and guanosine highly phosphorylated nucleotides complement each other in their presence within the cell so that when one is present at a particular stage of growth or alternatively in a given m u t a n t strain, the other is absent. If this is so, and if the sporulation process is regarded as a removal of the repression exerted on the expression of certain genes, then it may be postulated that the guanosine highly phosphorylated nucleotides not present during sporulation are in fact necessary for the mediation of catabolite repression. The fact that ppGpp is known to alter transcription of selected operons o f E . coli [29] and S. typhimurium [28] supports this view. The complementary highly phosphorylated nucleotides, that is, those containing adenine, may then serve to alleviate the effects of repressing sugars and their metabolites. In the case of B. subtilis the guanosine highly phosphorylated nucleotides
33 may mediate catabolite repression either directly in the role of a negative effector or indirectly by draining the cell of a positive effector or substrate required for transcription. In the latter case it is possible that ATP effluxes from the cell and a decreased rate of synthesis of highly phosphorylated nucleotides occurs since it has been shown that both adenosine [9] and guanosine [34] highly phosphorylated nucleotides require ATP for their formation, ppGpp is synthesised in membrane vesicles of B. subtilis [9] and hence would constitute a plausible initiating signal from the external "rich" environment. Similarly, the adenosine tetra- and pentaphosphates are eligible signals of the external " p o o r " medium [9] and could act by antagonising the synthesis or activity of the guanosine nucleotide(s), pppGppp may then accumulate extracellularly, presumably to prevent a replenishment of the ppGpp and/or pppGpp pools. The mechanism whereby the concentrations of the highly phosphorylated nucleotides are controlled is likely to be extremely complicated. The results with the mutants defective in purine metabolism imply that the early stage in inosinic acid synthesis is important, independent of its role in the synthesis of ATP. In E. coli, ppGpp is known to inhibit IMP dehydrogenase and adenylosuccinate synthetase [34] which are involved in the formation of GMP and AMP from inosinic acid. Finally, ATP and GTP regulate not only their own synthesis but also that of the highly phosphorylated nucleotides. In summary, the sequence of events provoking a change in transcription of catabolite repression sensitive operons in response to transient changes in the nutrient status of the cell, appears to rely on the build-up of a sufficient pool of phosphorylated sugars [7--9,15] from which information involving a phosphate or phosphoryl group for example can be relayed to ATP and GTP and from thence to highly phosphorylated nucleotides. The nature of the final event which alters transcription is not yet known though some suggestions have been made above. Rhaese et al. postulate HPN III, a highly phosphorylated nucleotide containing uridine with a base to phosphate ratio of 1 : 4 as the nuclear signal, for intiation of sporulation, since it requires membrane and cytoplasmic factors for its synthesis and its appearance is also correlated with sporulation [9]. The identity of the final signal will only be elucidated by using an in vitro system of transcription. To demonstrate unequivocally whether or not ppGpp, pppGpp and/or pppGppp accumulation give rise to catabolite repression that is relieved by pppAppp would be a relatively simple matter since a m u t a n t of B. subtilis deficient in the guanosine highly phosphorylated nucleotides has been isolated [38] and the latter c o m p o u n d can now be synthesised and purified [9].
Acknowledgements We thank the National Science Council (Ireland) for partial funding of this work and also the Department of Education of Ireland for research maintenance awards to L.B. and B.D. We are indebted to Miss Mairin O'Sullivan for most of the results shown in Table I.
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