Role of a hisU gene in the control of stable RNA synthesis in Salmonella typhimurium

,J. Marl. Riol. (1982) 157, 237-264

Role of a hisU Gene in the Control of Stable RNA Synthesis in Salmonella typhimurium JOHX P. DAVIDSON.

DONELLA

Department

(Kecriwd

17 Junr

J. WILSON AND 1,. S. WIIAIAMH of Biology

1981. and in revised fwm

4 Ikcernber

1981)

D’e have previously reported a fivefold reduction in expression of the iWED. operon in a hisL’ mutant (hisC!1820) originally isolated as a histidine regulatory mutant that exhibited derepressed (deattenuated) expression of the his operon. More recently, we have reported that a unitary explanation of the effect of this mutant on amino acid control is complicated by the observation of relaxed control of stable RNA synthesis during carbon/energy source downshifts. In the present study, we report the results of an analysis of the relaxation in control of RNA synthesis in relation to the accumulation of the guanosine polyphosphates, ppGpp and pppGpp. Unexpectedly, we observed that, despite the inability to restrict RNA accumulation upon carbon/energy downshifts, this mutant formed ppGpp at the normal rate. Further. the evidence clearly indicates that the defective control of RNA in this hisU mutant is not owing to an alteration in the spoT gene and that the rulil-mediated RNA control is unaltered. However. relaxed RNA synthesis in hisl' is suppressed by hyper-elevated levels of ppGpp : thus, an inverse correlation bet.ween RNA accumulation and ppGpp level during carbon/energy downshifts is still demonstrable in the hisU mutant. These data led us to the observation that the increased accumulation of stable RNA upon a carbon/energy downshift, is apparently the consequence of a I&U-conferred increase in RNA stability.

1. Introduction I)uring transitions in growth rate due to the manipulation of carbon/energy source. bacteria exhibit a rapid and profound change in the rate of RNA accumulation. However. the best understood aspect of RNA control is manifested when protein synthesis is inhibited as a consequence of restricted transfer RNA aminoacylation. Manipulations resulting in restricted tRNA aminoacylation are characterized b? the pronounced reduction in the rate of RR’A accumulation. This “stringent” couple of protein synthetic capacity with RNA accumulation exhibits a rather striking property ; i.e. it requires the binding of an uncharged tRNA to its cognate codon at the acceptor site of the ribosome. This and other properties of the stringent response were determined owing to the existence of strains exhibiting “relaxed” control for RKA synthesis. The mutant class exhibiting the most profound effect is designated relA and is locat’ed at about 60 minutes on both Snlrrtot~ella typhimurium and Escherichia coli genetic maps (Sanderson & Hartman. 00%2836/82/1402S’i-28

so3.00/0

‘CC’1981 Academic Press Inc. (London)

Ltd.

23X

J. 1’. I)AVII)SOS.

I). .I. WILSON

ANI)

I,. S. WlLLIAMS

1978: Bachmann & Low, 1980). The r&l’ gene shows dominance ov-er its rrlAf alleles. The normal product of the reld gene (the stringent factor) is a protein of 77.000 molecular weight found associated with the 50 S ribosomal subunit A fact t)hat was to have enormous impact on subsequent studies was t,he observation that st,ringent factor catalyzes the synthesis of two unusual nucleotides. guanosine-5’. diphosphate 3’.diphosphate (ppGpp) and guanosine-5’.triphosphate 3’.diphosphate (pppGpp) also referred to as MS1 and MSII. respeetirely. The synthesis of MS1 and MS11 by stringent factor is significantly stimulat,ed by the codon-specific binding of uncharged t)RNA to t,he acceptor site of the ribosome. Thus MS1 a,nd MS11 accumulation rapidly ensues upon triggering the stringent response in r~/d ’ but not rplA strains (see Cashel, 1975. for a review). Alternatively. by manipulating the supply of nutrients in a culture. two contrasting growth rate transients can be achieved : namely. a shift up in response to the addition of richer supplements or a shift down transient mediated by- the substitution of poorer carbon/energy source supplement~s. Stable RN.4 accumulation shows a dramatic and rapid increase following a shift up transieiit while protein synthesis is more gradually affected (Maaloe Kr Kjeldgaard. 1966). (‘onversely. following a downshift. stable RNA accumulation undergoes rapid restriction while DNA synthesis continues and protein synthesis exhibits partial restriction (Neidha,rdt & Magasanik, 1960). Neidhardt (1963) further showed that the pat’tern of accumulation of macromolecules was similar in rel=l ’ /rel-4 strains following downshifts. Lazzarini et (11. (1971) showed. interestingly. that E. co/i accumulates substantial quantities of ppGpp following downshifts : and despite the failure of rvlil strains to accumulate ppGpp during amino a,cid deprivation ((‘ashel. 1969). the rr1A mutant was also capable of amassing signiticant~ quantit,ies cjt’ ppGpp following downshifts. Subsequent, studies of carbon/energy downshift~s revealed similar patterns of RNA accumulation in t-d;1 and rrl,-I ’ although t,he relz-l mutant shows a very slow decline relative to rdd + in the instantaneous rate of stable RNA synthesis (Winslow. 1971: Hansen et rrl.. 1975: Molin it ~1.. 1977). These aut,hom. together with Gallant et nl. (1976). showed that the absolute level as well as the kin&es of pp(:pp accumulation could vary widely depending on the allelic state of t,he rrld gene: hence the instantaneous rat)e of synthesis but not the accumulation of stable RNA may show an obligatory eorrrlat~ion with 1ipGpp levels following downshifts. The ra,te of accumulation of ppGpp is a function of its rat,e of synthesis relative to its rate of degradation in E. co/i and both processes are subject to specific: genetic* controls (Fiil et (11.. 1976). The spoT locus encodes a 3-nucleotide pyrophosphatase activity specific for (p)ppCpp (Sy, 1977: Heinemeyer &, Richter. 1!178). The spoT encoded ppGppase shows a strict manganese dependence and exhibits energy activation (de Boer rt (xl.. 1977: Tetu et nl.. 1980). (’ onsequently. rnutants for the spo’f locus exhibit, an elevated basal level of ppGpp during exponential growth Increased st,ability of ppGpp caused by a decrease in act)ivity of the ~~07’ product accounts. at least in part, for ppGpp accumulation during earbon/enrrgy source downshifts. interestingly. no strains particularly defective in restri&ing stattle RNA accumulation following carbon/energy downshifts have been reported for /i:. ~-o/i.

COSTROI,

OF RNA

SYKTHEGIS

IN

A hisl’

MI’TANT

“39

We (Davidson & Williams, 1979a) observed such a mutant in S. typhimurium. This mutant is one of the class of hisU mutants originally isolated as histidine regulatory mutants (see Brenner & Ames, 1971, for a review). In this report we show that in one of these mutants (hisC72820) the restriction of stable RNA accumulation is suspended following shift down transitions even though normal levels of ppGpp are amassed with normal kinetics. We provide evidence that RNA accumulation is the result of increased RNA stability in the his17 mutant. We propose that a moderate manifestation of reL4-mediated synthesis of ppGpp is essential for the normal pattern of ppGpp accumulation following growth rate downshifts and that postdownshift differences in the kinetics and levels of ppGpp accumulation can occur as a function of the extent of the &A-mediated contribution. We suggest that while these differences may show no obligatory correlation with the rate of post-shift RX.4 accumulation, we propose that they may have enormous consequences on the facility with which post-shift cultures approach a new steady-state. Hence, the abrupt cessation of RNA accumulation occurs as a consequence of either blocked RSA synthesis or increased RNA degradation or a combinat’ion of the two. During amino acid deprivation the rapidly achieved, highly elevated level of ppGpp is sufficient to completely halt RNA accumulation without significant degradation. (‘onversely. the carbon/energy downshifts employ both ppGpp-mediated blockage of RNA synthesis and increased RNA degradation to curb RNA accumulation in ~Wmonella typhimurium. 2. Materials (a) Hacterial

and Methods

and growth

con,ditiorw

The strains used in this study are listed in Table 1. Bacteria were grown in the basal salts solution described by Fraenkel & Neidhardt (1961) except for (p)ppGpp measurements where the low phosphate medium of Cohen and Rickrnberg as modified by Simon 8r Van Pragg (1966) was used. Glucose to succinate downshifts were performed as previously described (Davidson & Williams, 1979a). When the downshifts were imposed by the addition of a-methylglucoside (a-MG). the ratio was 20/l, a-MG/glucose. The deprivation of charged tRNAS”’ was effected by the addition of r)L-serine hydroxamate at a concentration of 500 &ml. Growth was monitored turbidimetrically in a Klett coloritieter using a 660 filter. 1

TABLE Strain

Strain S. typhimuriunc TA799 .JClOO TA471 TA7O.i Ir:. coli XF161 XF1035 SF859

list

Genotype

his IT hisl” WlA + 1 his 0.H225.1. h&T1504 relA his O-82253. hisT1504 spoT. r&4 + &A. spoT + spoT + rrlA +

Source P. K. Hartman

This laborator,v M. Levinthal M. Levinthal .J. Gallant J. Gallant J. Gallant

240

J. P. DAVIDSON,

D. J. WILSON

AND

l,lO-phenanthroline was added to a concentration of 620 the specificity of l,lO-phenanthroline for the spoT concentration. Levallorphan was added to a concentration from Hoffman & Roche). Chloramphenicol and rifampicin of 100 pg/ml. (b) Measurement

of ppGpp

I,. S. WILLIAMS

m&r. Divalent cations used to test product were added to 61 mM of 2.0 mM (levallorphan was a gift were both used at a concentration

an,d pppGpp

Exponential cultures were labeled with 50 &i of [ 32P]orthophosphate/mI for 1 h prior to sampling. Samples (01 ml) were pipetted into equivolumes of 2 M-formate. Chromatograph> was performed on poly(ethylene)imine cellulose thin-layer plates (Rrinkman Instruments) by ascending development with 15 M-KH,PO, according to Cashel (1969). (c) In vivo degradation

of ppGpp

Elevated levels of ppGpp were induced with serinr hydroxamatr in cultures labeled with [32P]orthophosphate. Chloramphenicol was added to 100 pg/ml and @I ml samples were pipetted into equivolumes of 2 M-formate. The radioactivity in ppQpp was plotted as ‘!. remaining activity r1ersu.Etime. (d) RAVE degradation Exponential minimal medium cultures growing with glucose were labeled for 2 or more doublings with [3H]uridine. Cultures were harvested and washed at room temperature in starvation medium containing excess amounts of cold uridine. Cells were resuspended at 37°C in starvation medium containing cold uridine. Samples (1 ml) were pipetted irno equivolumes of lo?/, trichloroacetic acid. filtered and the radioactivity determined. The radioactivity was plotted as o/o remaining activity versus time. (e) Radioactivity

labeling

The accumulation of RNA and protein was measured by the incorporation of [ 3H]uracil and [ 14C]leucine. respectively, into trichloroacetic acid-precipitable material. Samples (1 ml) were brought to 5% trichloroacetic acid. Samples were filtered through glass filter fibers. dried and counted in toluene-based scintillation liquid. The radioactivity was plotted as relative disints/min per klett660 versus time. (f’) RL’VA4marker

puri$cation

Preswollen Sephadex Cl00 (Pharmacia) was packed into a 1.5 cm x 90 cm column and equilibrated with 0.01 M-Tris buffer. A sample of [14C]uridine-labeled RNA was applied in the same buffer, and the column was eluted with the Tris. HCl buffer at a flow rate of 15 to 20 ml/h. Fractions of approximately 2.0 ml were collected. and 61 ml portions from alternate fractions were placed in 5 ml of Aquasol (New England Nuclear) and the radioactivity determined. The RNA sample was separated into 3 peaks (16 S, 23 S and 4 S RNAs). Fractions corresponding to each peak were pooled and precipitated in 2.5 vol. ethanol. (g) Polyacrylamide

gel electrophoresis

Cells from overnight cultures were inoculated into 2 ml of the indicated medium grown at 37°C. At d420nm of 68, 1.0 ml of these cells from each experimental culture transferred to 2.0 ml of fresh, prewarmed medium with the same or different nutrients (66 Ci/mmol) nutrient broth + glucose media shift) and 20 &i of [5.6.5’-3H]uridine

and was (e.g. were

CONTROL

OF RNA

SYNTHESIS

IN

A hislJ

MITTANT

“11

added to the cultures. Labeling was terminated after 15 min by adding an equal volume of water-saturated phenol containing 91 ml of 2 iv-Tris .HCI at pH 755 ~1 of 96 M-potassium phosphate buffer (pH 7.0) and 1 mg of carrier RNA (extracted from the wild-type strain). This mixture was placed on a rotary shaker and shaken immediately at 37°C for 30 min followed by centrifugation at 10,000 revs/min for 10 min. The supernatant was collected, and the phenol phase was extracted with 0.5 ml of 100 mw-Tris. HCl (pH 75) for 5 min. At the 2 supernatant fractions were combined and the end of the 2nd centrifugation, precipitated in 2.5 vol. 95% ethanol containing 0.5 ml of 20% sodium acetate at -29°C overnight. The precipitated RNA was collected by centrifugation at 12,500 revs/min foi 15 min and then analyzed using a tube gel system (Buchler Instruments, Fort Lee, New Jersey) according to the procedure of Peacock & Dingman (1967). The electrophoresis buffer (pH 8.3) consisted of 108 g of Tris. 993 g of disodium-EDTA and 5.5 g of boric acid/l. The electrophoresis buffer system was discontinuous. The 10% (w/v) polyacrylamide gels that were used to fractionate small RNA species contained 95% (w/v) acrylamide. 05O/; (w/v) S,S’-methylenebisacrylamide, 04% (w/v) 3-dimethylaminopropionitrile, and 95% (w/v) ammonium persulfate. The gels were equilibrated by prerunning them at 3 mA/gel for 1 h before using them for electrophoresis. RNA samples were dissolved in the Tris/borate/EDTA running buffer containing 50% (w/ v ) sucrose and 0005'$/, (w/v) bromophenol blue as a tracking dye. A 200 ~1 sample containing 500,000 to 700,000 cts/min of [3H]RNA was divided equally between duplicate gels. Experiments involving wild type-mutant comparisons were normally done with equal amounts of labeled RNA and, in many instances. 14C-labeled marker RNA species were electrophoresed along with the 3H-labeled experimental samples. The gels were electrophoresed at 2 mA/gel at room temperature. At the end of electrophoresis, the gels were sliced into 1-mm slices and solubilized in a mixture (Amersham) and 10% NCS solubilizer at room of 9o:o organic counting scintillant temperature for 24 h. Radioactivity was determined with a liquid scintillation spectrometer. The 3% (w/v) polyacrylamide gels were used to fractionate the larger RNA species (23 S and 16 S). The gels contained 3.0% (w/v) acrylamide with 915% (w/v) methylenebisacrylamide. The running buffer, that of Peacock & Dingman (1967), contained loo/, (w/v) glycerol and 62’j/,, (w/v) sodium dodecyl sulfate. The RNA sample, dissolved in 100 ~1 of the running buffer, 5O”/b (w/v) sucrose and 0605~o (w/v) bromophenol blue, was heated for 5 min at 65°C. 50.000 to 100,000 cts/min of [3H]RNA were used per gel. All 3% gels were prerun prior to use in an electrophoresis experiment. RNA samples were electrophoresed at 3 mA/gel at room temperature for approx. 3 h. The gels were then sliced, solubilized and counted as described above. 3. Results (a) Protein

synthesis

and RNA

accumulation

follou!ing

a shift doum transition

Growth rate shift down transitions were imposed by the addition of amethylglucoside or by growth in limiting glucose and excess succinate. a-MG has been shown to be a competitive inhibitor of glucose uptake in E. coli (Hansen et al.. 1975) and in both 8. typhimurium and E. coli, the percentage of growth restriction is proportional to the ratio of n-MG/glucose. In the typical experiment. the glucose concentration was 0.05% and a-MC: was supplemented to l.O”/e. A representative growth curve is shown as an inset to Figure l(a). Figure l(a) compares the incorporation of [3H]uridine and [14C]leucine in strain TA799 (hisU1820) and JCloO, an isogenic normal strain. Zero time is immediately post a-MG addition. The relative incorporation into protein and RNA is significant in hisU while virtually no incorporation is observed in the normal strain. There is also no RNA accumulation observed during downshifts in relA strains of either E. coli or S. typhimurium (data not shown)

242

J. P. I~.~VII)SON.

1). J. WILSO?i

AS11 I,. S. WILLIAMs

Time (min) (b)

Fro. 1. [3H]uridine and [“‘CJleucine incorporation. respectively, [14(l]leucine in hid' (A) and hisV* (A): [3H]uridine in hislr (a) and growth curves (a) hisU: (0) his!r+. Th e arrow in inset represents 114C]leucine in spa!? (A) : ,sy)‘l’ ’ (Q) ; L3H] un ‘d‘me in spoT (0): spo’Pt coli strains NF161 and NF859 as listed in Table 1.

following &-MC: downshift. (a) hisI’+ (0). Inset shows typical time of addition of r-M&. (b) (0). The cells used were the E.

The SJNT gene of Is;. coli encodes an energy dependent ppclpp degrading activity and the s?oT-dependent degradation of ppGpp is antagonized by carbon/energJ source downshifts or treat,ments that mimic carbon/energ,y downshifts such as energ poisons, certain chelators and agents that perturb membrane integrity. The consequence of either manipula,tion in which ppGpp degradation is blocked is the rapid accumulation to significant levels of ppGpp without an apparent. change in the basal level of pp(;pp s?nt3hesis. Events surrounding a number of manipulations resulting in pp(:pp accumulation comprise t,he shift down tmnsit ion. -4s discussed in the Tntroduction, manipulations that result in the restriction of tRMA aminoacylation also result in ppGpp accumulation. The rd.4 gene and genes directly affectZing the re!&+l-mediated response control t’he events triggered by amino acid deprivation. KY contrast, virtually all perturbat,ions known t’hat trigger the downshift, seem to affect’ the ,~poT product. Nevertheless. Figure I(b) shows no RNA accumulation on a spoT mutant of E. coli following an %-MC triggered downshift. We sought, to ascertain whet,her post-shift uridine incorporat’ion rrported in Figure I (a) for S. typhimurimr could be attributed to a differential uridine uptake effcienc~ between his V and his 1” Table 2 shows the result of such an experiment in which cells growing exponential1.v in glucose were collected and resuspended in media containing 13H]uridine and supplemented with succinate as carbon so~ww. Duplicate samples were taken immediately and at subsequent, regular intervals. One sample was filtered immediately and the second sample was brought to GO;, cold trichloroacrtic acid and filtered after an incubation period of’ at least 30

(‘OSTROL

OF RNA

SYSTHESIS

IX

minutes.

The radioactivity determinations patterns of hisU and h&C r+ in marked of RNA incorporation.

showed contrast

uptake

(1)) The effect

A hisI:

of selected agenks 011 post-shift

243

MI’TANT

virtually identical to t)heir respective

RXA

overall patterns

accumulation

In an effort to clarify the basis for post-shift RNA accumulation in hisl’. the effects of a number of chemical agents were tested. As expected, rifampicin completely suppressed post-shift RKA accumulation. Also. not unexpected. chloramphenicol stimulated RKA accumulation in both strains: the effect, being significantly greater in the hisU mutant (the data on the effects of these drugs are also shown in Table 2). Chloramphenicol stimulation of RNA accumulation following shift’ down transitions is apparently the consequence of the inhibition of pp(:pp synthesis (Gallant et al., 1972). Two other drugs suppressed post-shift RN-4 accumulation ; namely. levallorphan and 1 ,lO-phenanthroline. Levallorphan exhibits a moderate suppression of the hisG mutation (Fig. 2). Levallorphan is a morphine analog that inhibits spoT-dependent degradation of ppGpp both in V~PO and in z’itro (Simon & Van Pragg, 1966: Boquet et al.. 1973: SF, 1977). As recently reported by Sy (1980), levallorphan seems to perturb the cell membrane, thereby int’erfering, in a manner yet to be determined. with the energy generating potential of the cell. The product’ of the spoT gene shows a requirement’ for manganese in ciao and itt vi&o. A number of chelating agents show manganese reversible inhibition of sE)o7’-dependent ppGpp degradation (Johnson et al., 1979). Figure 2 shows that inhibit,ion of post-shift RNA accumulation by one such chelator. 1 .I Ophenanthroline. is reversed by manganese while other divalent cations were ineffective (data not shown). TABLE 2 [“Hjuridinr

uptake versus incorporation

following

carbon

source

downshift

Incorporation l-ptake

No addition

Chloramphenicol

Rifampicin

Time (min)

JCIOO

TA799

JClOO

TA799

JClOO

TA799

JClOO

TA799

%UY) 10 20 30 40 60 60

39401 76542 77932 73242 66512 67764 66453

38957 77926 74295 69215 69987 67526 73094

1092 3296 3780 3411 3225 2995 2882

1023 9485 12479 13720 14444 16592 17585

4390 15996 18345 16554 15651 14535 13987

12827 45964 50192 51540 53466 49152 51551

117 54 105 42 112 143 175

li5 < 321 364 163 186 13X 132

I)ata represent cts/min per A,,, unit for the wild-type (JClOO) and the h&U mutant (TA799) strains of S. typhimurium. Chloramphenicol and rifampicin were added to the cultures at the indicated time (min) as described in Materials and Methods.

244

J. P. 1)AVIDSON.

I). J. WILSON

AND

I

1,. S. WILLIAMS

A

IO

20

30 40 Time (mln)

50

60

FIG. 2. [3H]uridine incorporation in hisi of 8. typhimuriurrr during glucose/succinate transition following drug treatments. The treatments were imposed as described in Materials and Methods: untreated (0); 1.10.phenanthroline (a); phenanthroline and Mn*+ (0): levallorphan (A).

6-

-4

0

8

4 Time

12

16

20

(mln)

(a) FIG;. 3. The accumulation of ppGpp following a-MG. (a) ppGpp is plotted as relative change in ppGpp level; hisU (0); hisC’ (0). (b) and (c ) are autoradiograms of thin-layer chromatography from which plot in (a) is taken. Time of sampling is indicated at bottom of Figs: (b) hisU+ ; (c) h&U. The arrows represent the addition of a-MG. The thin-layer chromatography was exposed to Kodak XR-5 X-ray film for 24 h and developed.

CONTROL

OF Ri\‘A

SYNTHESIS

IN

.4 hisl

MUTANT

-ATP -GTP

-PPGPP

-PPPGPP

4.0

8.0

12.0

16.0

(b)

ATP GTP

PPGPP

PPPGPP

-4.0

+ 1-o

8.0

40

(cl FIG. 3.

12.0

16.0

J. f’. I)ACIDSON.

146

(c) Measurement

1). J.

WILSON

of unusual

AND

I,.

guanosine

H. WILLIAMS

polyphosphates

We then examined the intracellular levels of the unusual guanosine polyphosphates in hisll and hisU+ during balanced growth and during growth rate transients achieved by a variety of manipulations. In normal E. coli there seems to be an inverse correlation between ppGpp and the rate of post-shift RNA accumulation. Figure 3 shows the kinetics of ppGpp accumulation in hisTl/hisl’+ following an %-MC; triggered shift down transition. The two strains show Vera similar patterns of accumulation of ppGpp and little or no detectable increase in pppGpp, The pattern is characteristic for shift down transitions. The ppGpp pool undergoes rapid expansion, peaking rather sharply at an approximate sixfold elevation then receding to a level only moderately higher than found in the preshift culture. We previously reported (Davidson & Williams, 1979a) that the alteration in control of RNA synthesis in hislJ is independent of the relA gene product. In that report. using a leucine-requiring derivative of hisC. we observed stringent cont,rol of RNA synthesis during leucine deprivation. In this report we examined the accumulation of ppGpp and pppGpp following the deprivation of charged tRNA”” in S. typhimurium. The diminution of charged tRNAS”’ was effected by thca addition of serine hydroxamate and the pattern of accumulation of ppGpp and pppGpp is shown in Figure 4. Autoradiograms depicting the pattern and kinetics of following the addition of serine hydroxarnate is MS accumulation in his~~/hial’+ shown in Figure 11. As shown in Figure 4, both hisc! and hisC+ show rapid accumulation of ppGpp. However, the level achieved is twice that obtained for thr downshift and this high level of ppGpp is maintained throughout the period of

I

-8 -4

0

4

8

12 16 20 24 28 32 Time (a)

I

I

1

L

-8 -4 (mill)

I

0

4

8

,

I

,

,

,

,

12 16 20 24 28 32 (b)

FIG. 4. MS accumulation in S. typhimurium following deprivation of charged tRNA”“. of charged tRNAsPr was effected by the addition of serine hydroxamate as described Methods. (a) Depicts MS accumulation in hisl:‘: ppGpp (0): pppGpp (a). accumulation in hislT: ppGpp (0): ppp(:pp (0).

The diminution in Materials and (b) Ijepirts MS

(‘OSTROL

deprivation. Roth strains latter nucleotide reaching

OF RN.4

SYNTHESIS

accumulate a maximum

IX

A hisl’

MVTANT

significant quantities fold elevation equal

217

of pppGpp with the to about half that of

I’P(:PP. (d) ,4 comparison

of the his11 and spoT mutants

In the Introduction it was noted that relA mutants restrict RISA accumulation following downshifts. Neither the reL4 nor any other gene product has been shown to be indispensable to growth rate control of RNA in E. coli. For reasons already in growth rate control stated. the spoT gene product has been strongly implicated of stable RNA. However, as seen in Figure l(b), spoT mutants also restrict RXA accumulation following downshifts. Thus the role of ppGpp in growth rate control of RNA accumulation remains unclear. The spoT gene ptoduct catalyzes the energyof energy flow results in dependent degradation of ppGpp, and a restriction decreased turnover and therefore the accumulation of ppGpp. Consequently, mutants altered at the spoT locus in a manner such as to reduce ppGpp degrading activity need not be defective in ppGpp accumulation upon downshifts. lt’ therefore seemed possible that the array of spoT mutants extant may not have permitted a complete assessment’ of the function of the SPOT gene. Furthermore. the relaxed RNA accumulation in the hist: mutant during shift down transients is suppressed by agents known to inhibit the spoT product. The spoT gene has not been identified in S. typhimurium: however. SPOT of E. coli and his{’ of S. typhimwium are located at, analogous positions on their respective chromosomes. We therefore a,ttempt,ed to determine if normal hisl: encodes the spoT-like function. spoT mutants exhibit reduced levels of pppC:pp and are unable to acclnnulate pppGpp during amino acid derivation. In addition. ppGpp shows increased in GPO stabilit,y in spoT mutants. As seen in Figure 1, hisP retains t’he capacity to accumulate ppp(:pp, Figure 5 compares t’he in ??i~ostabilit’y of ppGpp

I

0

IO 20 30 40 50 60 70 00 90 Time (s)

Fit:. 5. In viva degradation of ppGpp. ppGpp degradation was determined as described in Materials and Methods. ppGpp is plotted as the o/o remaining ~WS%LS time. hiss’+. no addition (0): hisl:. no addition (A): hisV+ with phennnthrolinc (0); hisli with phenanthroline (A).

248

J. 1’. DAVIDSON.

I). J. WILSON

ASI)

L. S. WILLIAMS

in hisc’ and hial;' following chloramphenicol treatment. The ppGpp decay is similar in the two strains (i.e. a half-life of about 25 s). Also shown in this Figure is the effect of l,lO-phenanthroline on the decay of ppGpp. Whereas ppGpp in untreated cultures is degraded to background in 50 seconds, in the presence of 1.1 Ophenanthroline ppGpp is reduced by only 20% after 90 seconds. Johnson et nl. (1979). using picolinic acid, a chelator wit,h properties similar to 1 .10-phenanthroline. observed the accumulation of ppGpp. Intriguingly. picolinate triggered ppGpp accumulation in SPOT+ but not spoT strains. They further showed that picolinate greatly retarded degradation in both spoT and SPOT’ , thus posing the question of whether retarding the turnover of ppGpp is sufficient to account for the spoT-dependent accumulation of ppGpp. Hence. anot,her property of’ spo7’ mutants appears to be the inability to accumulate ppGpp when challenged with a manganese reversible ppGpp-ase inhibitor. Analogous observations involving other inhibitors of ppGpp-ase can be found in earlier literature (E:dlin & 1)onini. 1971 : Harshman & Yamazaki. 1976: Roquet, et 01.. 1973: Haue & (+ruber, 1974). Possible implications of these observations are considered in the next sect,ion. Wr t,reated exponential cultures with 1.10.phenanthroline and monitored the accumulation of ppGpp. The results are shown in Figure 6. There is substantial accumulat,ion of The elevation is nearly threefold that, found ppGpp in both hisl’ and his{“. following an vMG downshift and even greater than found with serine hvdroxamate. It seems from the above observations that, hist’ is not a spoT mutation of S. typhinruri7crrc 18 16 -

ke----4

8

12 16 20 24 28 32

Time

(mln) (a)

FIG. 6. The effect of l,lO-phenanthroline on ppGpp accumulation. (a) hisV+ (0); hisl: (a). (b) and (c) are autoradiograms of thin-laver chromatography from which plot is taken. (b) is hisL’+ and (c) is h&F. The arrow represents the time of addition of phenanthroline.

(“ONTROL

OF RNA

SYNTHESIS

IS

A hisl’

24!)

MVTANT

-ATP .GTP

-PPGPP

-8.0

4 2.0 4.0

200

12.0 (b)

28.0

-ATP -GTP

-PPGPP

-8-O

+ 2.0 4.0

12.0 (cl

20.0 Rc:. 6.

28-o

250

J.

P.

DAVII)SON.

(e) The effect

I).

J.

of phenanthroline

WILSOS

ANI)

on guanosine

L. S. WILLIAMS

metabolism

polyphosphate

The metabolism of the guanosine polyphosphates appears to be normal in ever! respect in the hisli mutant. The h&U gene must therefore code for another requisite function in RNA control. Understanding how 1.10.phenanthrolinr phenotype could provide insight into understanding the suppresses the h&U normal function of the hisU gene. Figure 7(a) shows the accumulation of ppGpp following treatment’ with r-MG and l.lO-phenanthroline. The pattern of accumulation is identical to that obtained with ,I-MG. alone: however. the magnitude of the change is nearly doubled when phenanthroline is also added. Manganese reverses the effect of phenanthroline on RKA accumulation in l-M(:treated cultures of the hisU mutant. The effect of the latter combination on ppGpp accumulation is shown in Figure 7(b). The pattern and magnitude of accumulation is identical to that observed with a-MG, alone. A simple interpretation of these results is that the normal post-shift level of ppGpp is inadequate to restrict, RSA accumulation in hisU: the implication being that the normal hisr: product somehow mediates ppGpp-dependent RNA restriction. Alternatively. the intrinsic. sensitivity to ppGpp may be unchanged: rather. hisP may encode a ppGppindependent function that normally participates in growth rate control of RSA. Adjustments in the rate of RNA accumulation can occur at the level of synthesis or degradation. The level of pp(:pp obtained during amino acid derivation appears to be sufficient to restrict, RN4 accumulation without appreciable RNA degradation (Gausing. 1977). As the level of pp(:pp obtained following carbon/rnergJ downshifts is about half that obtained during amino acid derivation. we examined

1

0’

-4

n ’ -2

I

2

4

6

8

IO

12

14

16

I8 Time

(a)

I

-4

I

-2

2

4

6

8

IO

I2

14 I6

18

(m(n) (b)

FIG:. 5. The effect of I,lO-phenanthroline on ppGpp accumulation following a-MG-elicited downshift. (a) hiaCT+ with phenanthroline (0): h&U with phenanthroline (a). (b) hisU+ with phenanthroline and from which these plots are taken Mn2+ (0): his17 with phenanthroline and Mn’+ (0). Autmadiograms are shown in (c) to (f), representing hisl'+ with phenanthroline. hisl’ with phenanthroline. hisf'+ with respectively. The time of addition of 1phenanthroline and Mn*+. hisC with phenanthroline and Mn’+. was added immediately following T\MG is indicated with an arrow. Phenanthroline alone or with Mn” MG.

CONTROL

OF RNA

SYNTHESIS

IS

B hisC’

2.51

MVTANT

,ATP ,GTP

-PP%~

-40

+1-O 2.0

6.0 (cl

100

14.0

18.0

TP TP

,GPP

-4.0

6.0

100 (d) FIG. 7.

14.0

18.0

J. I’.

DAVIDSON.

I). J. WILSON

AND

I,. S. WILI~IAMS

ATP GTP

,PPGPP

-4.0

+ 1.0 2.0

60 (e)

100

14.0

18.0

-ATP .GTP

-PPGPP

- 4.0

f 1.0 2.0

6.0 (f)

10.0 FIG.7.

14.0

180

COSTROL

OF RNA

IO

SYNTHESIS

20

IN

30 Time

40

A hisl’

50

MUTANT

153

60

(mln)

FIG. 8. RNA degradation following carbon source downshift. RN4 degradation was determined as described in Materials and Methods. RNA is determined as trichloroacetic acid-precipitable counts following [3H]uridine incorporation and plotted as y0 precipitablr material remaining versus time. hid:+

(0):

hinl!

(a).

t’hr possibility that the normal adjustment to carbon/energy downshifts might reflect an increase in RNA degradation. Strains TA799 and JClOO were cultivated in glucose and submitted to long-term labeling with r3H]uridine. Following the labeling period. cultures were washed and resuspended in medium containing no glucose and cold uridine. Samples were taken at regular intervals and precipitated in trichloroacetic acid. The radioactivity in the samples was determined and plotted as the percentage remaining activity. The result of that experiment is shown in Figure 8. The results show that 60% of the preformed RNA is degraded in the normal strain in ten minutes while only loo/& of the preformed RNA is degraded during the same period in the hisU mutant. If RNA. newly made during the carbon/energy downshift. exhibits a similar disparity between hisC and h&l'+. then the failure to restrict RNA accumulation in hisU is explained. It seems likely that’ the hisI; gene encodes a function affecting the stability of ribosomal RNA during shift down transients. The possibility of a defective nuclease is being explored although other explanations are plausible (see Maaloe. 1979). To further examine the question of RN4 synthesis and in viva stability. we conducted electrophoretic analyses of RNAs radioactively labeled during growth under specific conditions as described in Materials and Methods. As compared to the Sephadex G-100 chromatographic profile of RNA species of t’he hial'+ strain grown in nutrient broth (Fig. 9(a)). both the hisU+ and hisc’ mutant synthesized the small stable RNA species (5 S and 4 S) during steady-state growth in minimalglucose medium (Fig. 9(b) and (c)). A similar polyacrylamide gel profile was obtained for the labeled RNAs extracted from both strains grown in nutrient broth (result,s not shown). As expected, the hisU mutant exhibited significantly greater amount,s of in GVO formed small RNAs than the hisU+ strain (compare Fig. 9(b) and (c)). Consistent with the results of the uridine incorporation studies. the hiat'+ strain exhibited a stringent response upon a nutrient brot’h to glucose transition

J. P. L,AVII~GON.

I). J. WILSON

ASI)

I,. S. WILLIAMS

700 600 500 400 300

9oL 80’ 7Ob 60+

Tube number (a)

!

-I

132 15 7

14 13 I2 II IO ‘9

6 5 4

rl

2

E

a 7 6 5 4

3

I&h Y

15

1

‘1 II IO 9 8 7 6 5 14

1 43

1-i

/

5

Y@

I

15

I

25

-

I

35 Slice

1

45

number

55

65

75

(I mm/shce

85 )

95

105

CONTROL

OF RNA

SYNTHESIS

IS

A hial'

MI’TANT

25.i

22 20 18 16 14

8 6

1 8

,6

14

0

5

15

25

75 85 35 45 55 65 Slice number ( I mm /slice)

95

10;

gel electrophoretic profiles of 3H-labeled small RNA species following a Frc:. 10. Polyacrylamide nutrient broth to glucose nutritional transition. RNA was labeled with [jH]uridine. extracted and subjected to electrophoresis as described in Materials and Methods for the hislJ+ strain (a) and the hial' mutant (b).

(Fig. 10(a)). Conversely. as shown in Figure 10(b), upon the same nutrient broth to glucose transition the hisT.:’ mutant continued to synthesize these small RNAs at a substjantial level (i.e. fourfold increase over that of the wild-type strain experiencing the same growth media transition). These data (Figs 9(a), (b) and (c) and 10(a) and (b)) clearly indicate that’ this hisC mutant (1) overproduces stable

FIG;. 9. Separation of stable RNAs by Sephadex chromatography and polgacrylamide gel electrophoresis. Labeled RNA samples were prepared as described in Materials and Methods and fractionated on a G-100 Sephadex column (marker RNA) and on 10% (w/v) polyacrglamide gels. (a) GI00 Sephadex column profile of RNA samples from his Cl+. The fractions from each peak were pooled and used as purified RNA markers in the polyacrylamide gel electrophoresis analyses of the RNAs shown in (b) and (c). (b) 10% polyacrvlamide gel electrophoresis of RNA of the hislJ+ strain grown in glucose medium after a 15min labeling period. The open svmbols represent the l’%]marker RNA. (c) IO:/, polyacrylamide gel electrophoresis of RNA of the hisI’ strain grown in glucose medium after a 15. min labeling period. The open circles represent the [‘4C]marker.

tJ. P. D4VIDSON.

11. J. WILSON

ASI)

1,. S. WILLIAMS

.ATP -GTP

-DOGPP -PPP( ZPP

-8.0 -4-O+ 2.0 4.0

12.0 f+ 20.0

280

(a)

,ATP GTP

,PPG PP .PPP( SPP

-8.0 -4.0 f 2.0 4.0

12.0 $$ 20.0 (b) FIG. 11.

28.0

CONTROL

OF RNA

SYNTHESIS

I 0

60

120 180 240 x)0 (a)

FIG:. 12. Relative change in induced levels of MS were induced aa in Fig. versus time. (a) Phenanthrolme alone; (0): pppGpp (0). (c) Phenanthroline

0

60

I

IN

I

I

120 180240300 Time (s) (b)

A hisU

I

tl 0

Lii

MUTANT

60

I I I 120 180240

I xx)

(c)

MS level following treatment with 1.lO-phenanthroline. Elevated 11 and the effect of phenanthroline is plotted aa relative change ppGpp (0); pppGpp (a). (b) Phenanthroline and Mn’+ : ppGpp and Mg’+: ppGpp (0): pppGpp (a).

RNAs during growth in steady-state culture, (2) exhibits relaxed control of RNA synthesis under growth conditions that evoke the production of ppGpp and (3) the degradation of newly formed RNAs appears to be insignificant as a control response. The focus thus far has been on the dramatic accumulation of ppGpp following downshifts. Equally dramatic is the dearth of pppGpp following downshifts. Already noted is the fact that carbon/energy downshifts are generally characterized by the accumulation of only ppGpp. Johnson et al. (1979) noted that SPOT mutants of E. coli fail to accumulate ppGpp when treated with picolinate and drew attention to the fact that these spoT mutants lack pppGpp. We examined the possibility that SPOT-dependent ppGpp accumulation in S. typhimukm also reflects. in addition to the decreased turnover of ppGpp, a preferential channeling to ppGpp at the expense of other guanosine polyphosphates. Cultures were treated with serine hydroxamate. inducing the accumulation of both ppGpp and pppGpp. These cultures were then treated with l,lO-phenanthroline and the results are shown in Figure 11. Phenanthroline addition precipitates the rapid disappearance of pppGpp and an increase in ppGpp over its already elevated level. The has no significance for the disappearance of pppGpp. per se. probably phenanthroline-elicited suppression of the hisU mutation, since little pppGpp is formed in either his&’ or hisV+ when treated with a-MG (see Fig. 3). In order to ascertain that the adjustment in the level of MS is spoT-dependent. high levels of MS were induced with serine hydroxamate, the effect of l,lO-phenanthroline

FIG:. 11. The effect of l.lO-phenanthroline on induced MS. Elevated levels of MS were induced as described for Fig. 4. The single arrow represents time of addition of serine hydroxamate and the double arrows represent time of addition of phenanthroline: (a) hisU+ : (b) hi$U.

258

J. P. l)A\‘IDSOiK.

I). J. WILSOX

ANI)

1,. S. WILLIAMS

was monitored at short intervals with or without the addition of manganese or magnesium. Figure 12 shows an increase in MS1 and a concomitant disappearance of MSII; the addition of manganese suppresses the effect of phenanthroline while magnesium addition resembles the effect, of treatment w&h phenanthroline alone. The spoT product appears to function, through a mechanism that, has not been determined. in establishing the ratio of MSI/MSII: when the ppGpp-ase is most act,ive the ratio of MU/MS11 is small but when ppGpp-ase is inhibited MSI/MSTI increases dramatically.

4. Discussion We had suspected that an alteration in the spoT gene might explain defective control of RNA in the hisC: mutant. However, hisl: appears normal in exhaustive examinations of known spoT-associated phenotypes, including : (1) normal accumulation of ppGpp upon carbon/energy downshifts, (2) normal accumulation of pppGpp and ppGpp upon amino acid derivation, (3) no alteration in ppGpp in vivo stability, and (4) retention of the capacity to accumulate ppGpp when challenged with agents that mimic the downshift (see Table 3). Suggestive evidence for the possible function of the hisr’ gene was obtained 1,~ examining chemical agents that tended to modulate the hisc’ phenotype. Lavallorphan and 1, IO-phenanthroline each suppressed post-shift RNA accumulation in hisli. i2s shown in Table 3. these ‘agents are known to trigger an avalanche of events characteristic of the carbon/energy downshift. Furthermore. this mimicry of carbon/energy downshift is exerted. at least in part. through the SPOT gene product. The manganese-specific reversal of the 1, lo-phenanthroline suppression of post-shift RNA accumulation (i.e. zinc. magnesium. cobalt’. molybdenum and iron are ineffective) indicate this response is independent of chelator-mediated inhibition of RNA polymerase. Moreover. chloramphenicol addition results in post-downshift RNA accumulation in excess of the level caused TABLE Mechanisms

Types of downshifts A.

Nutrient

deprivation

H. C”.

Disruptor of the protonforce Membrane perturbations

D.

Chelation

for

rliciti~ng

Putative

thu downshift

modes of action

I)ecreased efficiency energy utilization Inhibition

3

of

of ATPase

motive

Disruption of protonmotive gradient Decreased Mn*+ concentration (chelator must enter cell)

These operations tend to elicit the accumulation

Diminution of carbon or nitrogen source Cyanides. 2.1. dinitrophenol I,evallorphan. hypertonic salts l.lO-phenoanthroline. picolinate. tetracycline

of the single pp(Zpp by inhibiting

the qw7 product.

CONTROL

OF RNA

SYNTHESIS

IN

A his17 MUTANT

zxi

by the hisU mutation. Levallorphan and 1.10.phenanthroline each enhance the level of ppGpp accumulation while chloramphenicol restricts the level of ppGpp accumulation. Thus, despite the inability of normal levels of ppGpp to totally restrict RNA accumulation in hisU, the his/Y mutant continues to show an inverse correlation between RNA accumulation and ppGpp level. Suppression of hisU is accomplished by hyper-elevated levels of ppGpp. These observations permit two alternative interpretations ; the first is that the hisli mutant has acquired a resistance to ppGpp. Resistance to ppGpp inhibition of RNA synthesis could result if the normal hisU product is a factor that mediates ppGpp inhibition. The second alternative assumes that the hisU product and ppGpp are independent participants in RNA restriction following downshifts. Realizing that differences in the rate of RNA accumulation might represent differences in synthesis and/or stability. we compared the stability of preformed RNA in hisU and hisU+ during the shift down transient. Figure 8 shows that the RNA in the hisU mutant is more stable than in hisIJ+ following carbon/energy downshifts. We therefore conclude that the increased accumulation of RNA in hisi7 is the consequence of increased RNA stability. Increased turnover of rRNA is a normal characteristic of E. co/i during shift down transients (Nierlich. 1978). Ribosomal subunits appear to be more sensitive to nuclease digestion and an increase in monosomes (a probable prerequisite for subunit dissociation) appears to occur as a consequence of an excess of ribosomes to mRNA following the downshift (Maaloe. 1979). The hisU mutation may have resulted in a defective nuclease, or alternatively, the his.51 mutation may have caused an alteration in the infrastructure of the ribosome; thereby rendering bhe ribosome resistant to nuclease digestion. Discriminating between these alternatives must await further studies. Interestingly, a recent report indicates that another of the histidine regulatory mutants, hisT1504, is unable to accumulate ppGpp upon deprivation of histidine, while arginine and threonine starvation resulted in substantial production of both ppGpp and pppGpp (Spadaro et al.. 1981). In comparing these two regulatory mutants of 8. typhimurium, it is evident that the hisu mutant exhibits relaxed control of RNA synthesis upon carbon/energy source transitions despite the accumulation of ppGpp; while the hisT mutant evokes the stringent control response without the histidine starvation-mediated re2il -dependent formation of ppGpp. Moreover, we have recently shown that a cold-sensitive hisW regulatory mutant exhibits stringent control of stable RNAs during carbon/energy source transitions. but overproduces stable RNAs during incubation at the non permissive growth temperature (Davis & Williams, unpublished results). The role of ppGpp as well as the precise mechanism of ppGpp accumulation during carbon/energy downshifts continue to be a source of some controversy. Examples of RNA restriction in the absence of ppGpp accumulation can be found in the literature (Gallant et al., 1976 ; Yao & Dyess, 1981). Furthermore, at least one previous example of RNA accumulation despite significant ppGpp accumulation has been reported (Gallant et al., 1977). Indeed. the observations presented in this paper suggest that ppGpp accumulation during the shift down transient is somewhat gratuitous. Figure 13 illustrates the metabolism of the predominant unusual guanosine polyphosphates in E. coli. The gpp locus was recently described

260

J. P. 1)AVIDSON.

D. ,J. WILSON

AND

L. S. WILLIAMS

PPPG

k!?!a PPG

FIG. 13. The metabolism of MS. Biochemical steps are indicated by continuous lines with the arrow showing the prevailing direction of the reaction. Putative regulatory effects are indicated with broken lines; inhibition and stimulation are indicated with ( - ) and ( + ). respectively. Genes coding for a number of the enzymatic activities are also indicated: the parentheses denote those genes for which there is no direct evidence in 5’. typhimurium. Two classes of effects are indicated ; primarv effects (1 ‘) as a direct consequence of the downshift and secondary effects (2”) ensuant upon the inhibition of the spoT product.

for E. coli by Somerville & Ahmed (1979). The gpp gene has not been described for S. typhimurium: a point of interest since the presence of this activity would be expected to affect the relative levels of MS1 and MSII. The rel8 gene was also recently discovered in E. coli (Engel et al., 1979) and appears to encode a ribosomeindependent ppGpp synthetase. The latter activity is believed to account for basal level ppGpp in lieu of relA. Richter (1979) has noted the presence of a ribosomeindependent ppGpp synthetase in bacteria other than E. coli and, no doubt, such an activity exists in S. typhimurium as well. This paper presents physiological and biochemical evidence for the .spoT gene in S. typhimuriwm. Genetical evidence for the spoT gene in A’. typhimurium has not been reported : however, a spoT mutation has been discovered in the laboratory of Bruce Ames (personal communication). The relA locus was described for S. typhimurium by Martin (1968). We examined this relA mutant during histidine starvation and found it unable to accumulate ppGpp in contrast to an isogenic rel.4+ (Fig. 14). Further evidence that the unusual guanosine polyphosphates are metabolized in the same manner in A’. typhimuriuwL as in E. coli is provided in Figures 11 and 12. The addition of 1 ,lO-phenanthroline causes increased accumulation of ppGpp and the concomitant disappearance of pppGpp in S. typhimurium. Similar observations in E. coli were initially interpreted as evidence that ppGpp is a precursor of pppGpp (Lazzarini & Stamminger, 1976). We now believe. in light of subsequent observations, that the phenomenon observed in Figure 12 is compatible with the pathway shown in Figure 13. Compelling evidence for this interpretation is derived from a number of sources. Molin et al. (1977) showed that the pattern of ppGpp accumulation is remarkably different in relA/relA ‘. The data from Table 2 of Molin et al. (1977) for ppGpp accumulation are plotted in Figure 15. The level of ppGpp in re1.4 ’ is fivefold that in relA at two minutes post-downshift. We interpret the relil dependence on the pattern of ppGpp accumulation as evidence that t,hr carbon/energy downshift normally elicits re2A-mediated synthesis of ppGpp. An

CONTROL

OF RNA

(b)

SYNTHESIS

IN

A hisU

MUTANT

(d)

FIG. 14. The absence of MS accumulation in relA of S. typhimurium. Isogenic re/A/relA+ were grown in growth rate-limiting and excess histidine. respectively. and the capacity of these strains to accumulate MS was determined as described in Materials and Methods. Lane (a) relA unrestricted: (1)) rel.4 limited for histidine; (c) rd=l + unrestricted; (d) r~l.4 + limited for histidine.

addit’ional observation of particular interest from Molin et al. (1977) is that the instantaneous rate of total RNA synthesis in relA+ is reduced by 55% in the first two minutes of downshift while total RNA accumulation is reduced by 900/,; alternatively. while the instantaneous rate of total RNA synthesis is reduced by onlv 50/ during this same two minute period in relA. RNA accumulation is st~ill reduced by 75%. Direct evidence for the pathway shown in Figure 13 was provided by Fiil et al. (1976.1977) for E. coli. These authors compared the rate of accumulation of MS1 and MS11 and found that the expansion of the MS11 pool precedes the increase in the pool of MS1 during isoleucine starvation. They further showed that the MS11 pool exhibits only a burst upon isoleucine starvation in spoT mutants. We believe that the behavior of MS upon carbon/energy downshifts is qualitatively analogous to the behaviour of MS during amino acid starvation in spoT mutants. Figure 13 shows two primary effects of the downshift: a stimulation of relA and inhibition of

iw

J. P. DBVIDSOP;.

D. J. WILSON

-2

2 5

IO Time

AND

20

1,. 8. WILLIAMS

3c

(min)

of K. coli following a-MC&elicited FIG. 15. Comparison of ppGpp accumulation in relA/relA’ downshift. The data plotted in Fig. 15 are taken from Table 2 of Molin et nl. (1977); relA+ (0); relA (0).

spoT activity.

The inhibition of spoT is depicted as subsequently resulting in the leading to the disappearance of MS11 while MS1 retrenchment of relA activity persists due to the absence of turnover. Figure 13 also depicts a stimulation of the reaction converting MS11 to MS1 also owing to the inhibition of spoT. We believe the latter effect to be the result of a channeling of MSII breakdown to MSI. Somerville & Ahmed (1979) noted that gpp mutants accumulate higher levels of accumulate MS11 and interestingly, that. gyp and leaky spoT double mutants higher levels of MS11 than gpp mutants alone. In vitro evidence that the SPOT product degrades MS11 has been reported by Richter (1979). The product of SPOTdependent degradation of MS11 would presumably be pppG not ppGpp. A compensatory relationship has evolved between r~l=2 and spoT such that ppGpp synthesis is curtailed as ppGpp degradation decreases (Fiil et ~1.. 1976). We believe that the relative contribution of reL4 and spoT to ppGpp accumulation may vary considerably depending on the manner by which the downshift is invoked. and that the greater the rdA contribution relative to spoT. the higher the level of MS11 achieved. In assessing the importance of ppGpp accumulation during carbon/energy downshifts it is perhaps informa.tive to compare RNA control during the downshift with control during amino acid deprivation. We have previously noted that the elevated level of ppGpp formed during amino acid deprivation is apparently sufficient to restrict RNA accumulation in the absence of appreciable degradation. These differences in control mechanisms employed for the two types of transitions may serve to facilitate the resumption of balanced growth when transient is complete. Restoration of depleted amino acid enables cultures to resume pretransient growth rates while resumption of growth following the downshift is at an obviously slower rate. While it would be futile not to restrict RNA synthesis

(IONTROL

OF RNA

SYNTHESIS

IN

A hisC

MITTANT

263

during downshift, restriction of synthesis alone would appear to be relatively ineffective in the requisite downward adjustment of ribosome levels. Finally, we have previously reported a pleiotropic effect of the hisU mutation on isoleucine and valine biosynthesis. Davidson & Williams (1979b) reported a fivefold reduction in expression of the ilvGEDA operon in the hisU mutant, from which we proposed that the hisU mutant could have reduced levels of a metabolic factor such as ppQpp. which acts as a positive effector on amino acid operons and a negat,ive effector on stable RNA operons (Stephens et nl., 1975). The notion may now need revision in the light of the present observations. It is tempting to speculate that the retrenchment of specific activity of the ilv gene products is the consequence of increased accumulation of ribosomal proteins (e.g. see Wirth & Bock. 1980). However, the latter supposition is complicated by the observation that the his{: mutation is epistatic to $r mutations that cause significant derepression of ilvGEL)rl in hisI]+ (Davidson & Williams. unpublished observations). It should be recalled that this hisU mutant was identified in a search for histidine regulatory and exhibits derepressed (de-attenuated) levels of the mutants of S. typhimurium, Thus, the singular consequence of the hisl’ histidine biosynthetic enzymes. conferred regulatory factor on RNA accumulation during growth rate transitions notwithstanding, the precise role of the hiall gene product in the control of the his and ilv (and perhaps others) biosynthetic operons remains an intriguing question. We thank Myrna Sales and Joseph Whittaker for helpful discussions throughout most of these studies. Some of the results reported herein are from a thesis submitted by one of t,hr authors (D.J.W.) to Purdue University in partial fulfillment of the requirements for t,he Doctor of Philosophy degree. This research was supported by National Institutes of Health grants numbers GM 21878 and GM 29200 (to L.S.W.).

REFERENCES Bachmann, B. J. & Low. K. B. (1980). Microhiol. RPV. 44, l--56. Boquet, P.-L., Devynck, M. A., Monnier, C. & Fromageot, P. (1973). Eur. J. Biochem. 40.31l 42. Brenner, M. 8r Ames, B. N. (1971). In Metabolic Pathways (Vogel, H. J., ed.). vol. 5. pp. 349387, Academic Press, New York. Cashel, M. (1969). J. Biol. Chem. 244, 313333141. Cashel, M. (1975). Annu. Rev. Microbial. 29, 301-318. Davidson, J. P. & Williams, L. S. (1979u). Biochem. Biophys. Res. Commun. 88. 682-687. Davidson, .J. P. & Williams, L. S. (1979b). J. Mol. Biol. 127, 2299235. de Boer, H. A., Bakker, A. J. & Gruber, M. (1977). FEBS Letters. 79, 19.-24. Edlin, G. & Donini, P. (1971). J. Biol. Chem. 246, 4371-4373. Engel, ?J. A.. Sylvester, J. & Cashel, M. (1979). In Regulation of Macromolecular Synthesis by Lou: Molecular Weight Mediators (Koch. f$. & Richter. D.. eds), pp. 25-38. Plenum Press, New York. Fiil. N. l’.. Mortensen, U. & Friesen. ?J. D. (1976). In Control of Rihosome Synthe.sis-A41fred Ben-on Symposium IX (Kjeldgaard, N. C. & Maaloe, 0.. eds), pp. 4377444, Academic Press, New York. Fiil. PI’. I’.. Willumsen, B. M., Friesen. J. D. & von Meyenburg. K. (1977). Mol. Oen. &wt. 159, 87-101. Fraenkel. D. G. & Neidhardt, G. C. (1961). Biochim. Biophys. Acta. 53, 96110. (iallant. J.. Margason. G. & Finch, B. (1972). J. Biol. Chem. 247, 60X-6058.

264

J. P. DAVIDSON,

D. J. WILSON

AND

L. 6. WILLIAMS

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