Journal of General Microbiology (198l), 123, 383-386. Printed in Great Britain
383
SHORT COMMUNICATION
Stringent Control in an Extreme Thermophilic Bacterium. Preliminary
Characterization of a Guanosine 3’,5’-Polyphosphate [(p)ppGpp]
Synthesizing Enzyme from Thermus thermophilus
By I N G R I D L I E N E R T A N D D I E T M A R R I C H T E R *
Inst itu t fur Physiologische Chemie, A bteilung Zellbiochemie, Universitat Hamburg,
2 Hamburg 20, Martinistrasse 52, West Germany
(Received 23 September 1980; revised 17 November 1980)
A guanosine 3’,5‘-bis(diphosphate) (ppGpp) and guanosine 3’-diphosphate 5‘-triphosphate
(pppGpp) synthesizing enzyme is present in the ribosomal fraction of Thermus thermophilus.
The enzyme is comparable to the Escherichia coli (p)ppGpp synthetase I (stringent factor) in
that it requires ribosomes, mRNA and codon-specific, uncharged tRNA. The ribosomal
complex can be replaced by methanol. Ribosomes from Escherichia coli do not function as
effectors of the Thermus thermophilus enzyme.
INTRODUCTION
The regulatory process termed stringent response, governed by the relA gene, is
characterized by the ability of the bacterial cell to coordinate diverse biosynthetic processes in
response to the availability of nutrients or other factors essential for growth (Cashel, 1975).
In Escherichia coli lack of an essential amino acid triggers the stringent response which is
characterized by the curtailment of synthesis of rRNA, tRNA and some mRNA species and
by the simultaneous accumulation of the two nucleotides, guanosine 3’,5‘-bis(diphosphate)
(ppGpp) and guanosine 3’-diphosphate 5’-triphosphate (pppGpp). The relA gene prcduct, a
(p)ppGpp synthetase, transfers pyrophosphate from ATP to the 3’-position of GDP or GTP
(Richter, 1980). This enzyme, originally termed stringent factor, is an ATP :GTP(GDP)
pyrophosphotransferase that catalyses this reaction only when activated by a complex
consisting of ribosomes, mRNA and codon-specifically bound uncharged tRNA. Another
(p)ppGpp-synthesizing enzyme has been found in Bacillus brevis (Sy & Akers, 1976) and
Bacillus stearothermophilus (Fehr et al., 1979); this differs,from the E. coli enzyme in that it
functions independently of the ribosome. mRNA. tRNA complex. The enzyme that depends
on the ribosome complex is designated as (p)ppGpp synthetase I; the other, which functions
independently of the ribosome complex, is designated (p)ppGpp synthetase I1 (Richter,
1980). In the present communication, studies on the regulatory function of (p)ppGpp and its
metabolism have been extended to the extreme thermophile Flavobacterium thermophilum,
now renamed Thermus thermophilus (Oshima & Imahori, 197 1).
METHODS
Materials. [a-32PlGTPand carrier-free [ 32P]orthophosphoricacid were obtained from New England Nuclear
(Boston, U.S.A.)and [3Hluracilfrom Amersham Buchler (Braunschweig, F.R.G.). Pseudomonic acid (Na+ salt)
was kindly provided by Dr E. Cundliffe (Department of Biochemistry, University of Leicester) and purified
stringent factor and ribosomal subunits (E. coli and B. stearothermophilus) by S. Fehr (Physiologisch-Chemisches
Institut, Abteilung Zellbiochemie, Universitat Hamburg).
0022-1287/81/0000-9551
$02.00O 1981 SGM
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384
Short commmica tion
Organism and growth. Thermus thermophilus ATCC 27634 was grown in peptone/yeast extract/salt medium
as outlined below; for storage 1 ml samples of the cell culture (about 1 A 2 6 0 unit ml-') were diluted with 2 g
glycerol, mixed well and kept at -20 "C. For inoculation 0.2 ml of the glycerol stock was added to 50 ml medium.
[For definition of absorbance units see Fehr & Richter (1981).] Thermus thermophilus was grown at 75 OC with
stirring in medium containing, per litre, 8 g peptone, 4 g yeast extract and 2 g NaCl; the pH was adjusted to 7.0 (at
75 "C) with 1 M-NaOH. The generation time was estimated to be 50 min.
Preparation of the ribosomalfraction. Cells grown in peptone/yeast extract/salt medium were harvested during
the exponential growth phase at an A,,, of 1 to 1.5 and homogenized in 2.5 vol. buffer (10 mM-Tris/HCl pH 7.8,
10 mM-magnesium acetate, 2 mM-MnCl,, 2 mM-dithiothreitol, 2 pg DNAase ml-') using a French laboratory press
(Heinemeyer & Richter, 1978). The extract was differentially centrifuged at 18000 rev. min-' (39000 g) for 30
min (Sorvall rotor SS34) and at 40000 rev. min-' (1 10000g) for 3.5 h (Beckman rotor Ti60). The pellet from the
last centrifugation was suspended in a small volume of distilled water (about 600 to 700 Az6, units ml-') and stored
in liquid nitrogen; it is referred to as the ribosomal fraction.
Exfracfionof ribosomes with ammonium chloride. One ml (620 A260units) of the ribosomal fraction was diluted
with 3.5 ml buffer A [ l o mM-Tris/HCl pH 7.8, 1 mM-dithiothreitol, 14 mwmagnesium acetate, 60 mM-potassium
acetate, 5 % (w/v) glycerol, 2 mM-EDTA containing 0.5 M-NH,C~and 0.13 mg phenylmethylsulphonyl fluoride
ml-'I. After stirring for 5 h at 4 "C the suspension was centrifuged at 45000 rev. min-' for 2 h (Beckman Ti
SW65 rotor). The ribosomal pellet, referred to as washed 70s ribosomes, was suspended in 2 ml buffer A (without
glycerol and EDTA) and stored in liquid nitrogen. The supernatant fraction, containing the (p)ppGpp-synthesizing
activity, was recentrifuged as indicated above to remove residual ribosomes. To 100 ml of the resulting supernatant
fraction 49.5 g (NH,),SO, was added and the pH was adjusted to 7.8 with 1 M-NaOH. The suspension was stirred
for 1 h at 4 "C and the precipitate was collected by centrifugation at 15 000 rev. min-' for 30 min (Sorvall rotor
SS34). The pellet was dissolved in 500 pl buffer A and dialysed against the same buffer. As with the E. coli
enzyme, the (p)ppGpp synthetase I from T. thermophilus formed aggregates at low ionic conditions. The
aggregated material was centrifuged, dissolved in 100 pl buffer A containing 1 M-NH,CI and stored in liquid
nitrogen. The protein fraction is referred to as (p)ppGpp synthetase I.
Assayfor synthesis of (p)ppGpp in vitro. (1) Ribosome system. The standard assay system (50 pl) contained
20 mM-Tris/HCl pH 7.8, 20 mM-magnesium acetate, 40 m ~ - N H , c l , 4 mM-dithiothreitol, 4 mM-ATP, 0.4
~ M - [ ~ - ~ ~ P (specific
I G T P activity 25 Ci mol-', 925 GBq mol-'), 6.25 pg poly(U), 1 pg tRNA$is,, 11 pmol 70s
washed ribosomes and 2 to 5 pg (p)ppGpp synthetase I. Unless otherwise stated, incubation was carried out at
50 "C for 1 to 2 h. The reaction was stopped by adding 1 pl 88% formic acid. The products were analysed as
reported by Heinemeyer & Richter (1978). (2) Methanol system. The assay mixture (50 pl) contained 20
mM-Tris/HCI pH 7.8, 20 mM-magnesium acetate, 4 mwdithiothreitol, 20 % (v/v) methanol, 200 mM-NH,Cl, 4
mM-ATP, 0.4 mM ~ C X - ~ ~ P(specific
~ G T P activity 25 Ci mol-', 925 GBq mol-') and 4 pg (p)ppGpp synthetase I.
Incubation was carried out at 21 "C for the time indicated.
RESULTS AND DISCUSSION
In order to find out how T. thermophilus responds to starvation conditions known to
induce stringency in other bacteria, synthesis of RNA and guanosine polyphosphates was
measured under various conditions. Stringency was observed on addition to the medium of
hydroxylamine, which is known to block amino acylation of tRNA (Lund & Kjeldgaard,
1972), and on shifting the temperature from 75 "C to 37 OC. In both cases growth and RNA
synthesis were restricted and (p)ppGpp production increased above the basal level. Attempts
to trigger stringency with pseudomonic acid, known to inhibit isoleucyl-tRNA synthetase
(Hughes & Mellows, 1978), gave mixed results. In some, but not all, experiments
pseudomonic acid showed inhibitory effects similar to those in E. coli. It was not clear why
the pseudomonic acid experiments were not reproducible; heat instability of the antibiotic can
be excluded since at 75 "C preincubated pseudomonic acid induced stringency well in E . coli.
Fractionation of the cell extract by differential centrifugation and subsequent analysis for
(p)ppGpp-synthesizing activity revealed that most of the activity remained with the
ribosomes. As with E . coli, the (p)ppGpp synthetase I could be extracted from the ribosomes by high salt treatment (0.5 to 1.0 M-NHJI). The enzyme required the
ribosome. mRNA .tRNA complex for its activation. In this reaction mainly pppGpp was
formed; less than 10% was found to be ppGpp as analysed by two-dimensional thin-layer
was replaced by
chromatography (Heinemeyer & Richter, 1978). When [cc~~PIGTP
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Short communication
385
2
Time of incubation (h)
Fig. 1. Non-ribosomal synthesis of (p)ppGpp in the presence of 20% (v/v) methanol and 200
mM-NH,CI. Incubation was carried out at 21 "C with methanol (O),
at 21 "C without methanol (0)
and at 37 OC with methanol (A).
Table 1. Interchangeability of (p)ppGpp synthetase I and ribosomes from T. thermophilus,
E. coli and B. stearothermophilus
Assay mixtures were as described in Methods, except that when E. coli or 3. stearotherrnophilus
ribosomes were used the 50 p1 assay contained 8 pmol 50s and 9 pmol 30s ribosomal subunits.
Incubation was carried out at 37 "C for 120 min; under these conditions the (p)ppGpp synthetase I
from either E. coli or T. therrnophilus synthesized less than 0.5 nmol (p)ppGpp in the absence of the
ribosome. mRNA .poly(U) complex.
(p)ppGpp synthesized (nmol)
7
-
Source of ribosomes
T. thermophilus
E. coli
B . stearothermophilus
Source of (p)ppGpp synthetase I
T. therrnophilus
E. coli
3-30
0.46
2-50
14.6
13.4
12.6
[3HlGDP as substrate, again mainly pppGpp was synthesized, apparently due to the rapid
conversion of GDP into GTP by a kinase contaminating the ribosomal fraction and/or the
(p)ppGpp synthetase. Another property significant for the (p)ppGpp synthetase I enzyme
from other bacterial species (Richter, 1980) is its reactivity to organic solvents. In the absence
of ribosomes E. coli (p)ppGpp synthetase I can be activated by 20% methanol. Figure 1
shows that, as for E. coli, the T. thermophilus (p)ppGpp synthetase I is activated by
methanol. While in the ribosomal system the optimal incubation temperature was 50 O C , the
methanol system required 2 1 O C .
To test whether the (p)ppGpp synthetase I from T. thermophilus could be activated with
ribosomes from other bacterial species and vice versa, the enzyme was complemented with E .
coli or B. stearothermophilus ribosomes. Table 1 shows that the T. thermophilus enzyme was
active with B. stearothermophilus ribosomes and, to a significantly lower degree, with E . coli
ribosomes. On the other hand, the E. coli (p)ppGpp synthetase I reacted perfectly well with
ribosomes from either T. thermophilus or B . stearothermophilus. This result is in agreement
with recent findings (Fehr & Richter, 198 1) indicating that the (p)ppGpp synthetase I from B.
stearothermophilus was less active with E. coli ribosomes than with the homologous system.
In conclusion, the (p)ppGpp synthetase I from T. thermophilus has properties similar to those
of the enzyme from E. coli or B. stearothermophilus.
We thank Deutsche Forschungsgemeinschaft for financial support.
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