(Received for publication, April 24, 1995; and in revised form, June 2, 1995)
From the
Physiological functions of spermidine acetyltransferase in Escherichia coli have been studied using the spermidine acetyltransferase (speG) gene-deficient mutant CAG2242 and the cloned speG gene. The growth of E. coli CAG2242 in the defined M9 medium was normal in the presence and absence of 0.5 mM spermidine. However, cell viability of E. coli CAG2242 at 48 h after the onset of growth decreased greatly by the addition of 0.5 mM spermidine. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. Transformation of the cloned speG gene to E. coli CAG2242 recovered the cell viability. Decrease in cell viability of E. coli CAG2242 was observed even when 0.5 mM spermidine was added at 24 h after the onset of growth. The results indicate that accumulated spermidine functions at the late stationary phase of growth. The accumulation of spermidine caused a decrease in protein synthesis but not in DNA and RNA synthesis at 28 h after the onset of growth. The synthesis of several kinds of proteins was particularly inhibited. They included ribosome modulation factor and OmpC protein. Since the ribosome modulation factor is essential for cell viability at the stationary phase of growth (Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A.(1993) EMBO J. 12, 625-630), the decrease in the protein was thought to be one of the reasons for the decrease in cell viability. The decrease in the ribosome modulation factor mainly occurred at the translational level.
Polyamines, aliphatic cations present in almost all living
organisms, are known to be necessary for normal cell
growth(1, 2, 3) . Thus, it is important to
understand the mechanism by which the cellular polyamine concentration
is regulated. Polyamine content is regulated by biosynthesis,
degradation, uptake, and excretion. Although a rate-limiting enzyme of
polyamine degradation in eukaryotes, spermidine/spermine N-acetyltransferase, has been recently studied in
detail(4) , that in prokaryotes, spermidine acetyltransferase
(SAT), (
)has not. In eukaryotes, spermidine/spermine N
-acetyltransferase is induced strongly in the
presence of polyamines (5) and bis(ethyl)polyamine
derivatives(6) , and it probably plays an important role in the
decrease in polyamine toxicity by its
accumulation(7, 8) . In Escherichia coli,
spermidine acetylation increased in response to high pH (9) and
cold shock(10) , but SAT activity did not change significantly
under these conditions(11) . To help understand the
characteristics of polyamine degradation in E. coli, we
purified SAT and isolated the gene for SAT (speG)(12) . We found that SAT is present in very
small amounts in cells and increases only by 2.5-3.5-fold under
poor nutrient conditions. In this study, the physiological functions of
spermidine acetyltransferase in E. coli were studied with E. coli CAG2242 (speG) and the cloned speG gene. We found that the accumulated spermidine due to the lack of
SAT caused a decrease in cell viability at the stationary phase of
growth through its inhibition of protein synthesis. The synthesis of
several kinds of proteins was particularly inhibited. The decrease in
cell viability paralleled the decrease in ribosome modulation factor
(RMF), which is essential for cell viability(13) . RMF is a
protein associated with 100 S ribosome dimers (14) and is
synthesized during transition from exponential growth to the stationary
phase(13) . We also recently found that RMF is a stationary
phase-specific inhibitor of protein synthesis and that 100 S ribosome
dimers are dormant forms of ribosomes. (
)
Figure 1:
Effects of spermidine and SAT on cell
growth (A) and viability (B) of E. coli C600
and CAG2242. Cell growth (A) and viability (B) were
measured as described under ``Experimental Procedures.''
, C600;
, C600 grown in the presence of 0.5 mM spermidine;
, CAG2242;
, CAG2242 grown in the
presence of 0.5 mM spermidine;
, CAG2242/pMWSAT;
, CAG2242/pMWSAT grown in the presence of 0.5 mM spermidine. Each value is the average of duplicate
determinations.
Next, polyamine contents in E. coli cells were measured (Fig.2). When E. coli C600 cells were cultured in the presence of spermidine, spermidine accumulation was not observed. However, when E. coli CAG2242 cells were cultured in the presence of spermidine, spermidine accumulation was observed. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. The transformant of E. coli CAG2242 with pMWSAT did not cause the spermidine accumulation. The results strongly suggest that the accumulation of spermidine decreased the cell viability of E. coli cells.
Figure 2:
Effects of spermidine and SAT on polyamine
contents. E. coli cells grown in the presence and absence of
0.5 mM spermidine were harvested at the times shown in the
figure, and polyamines were analyzed by HPLC. &cjs2090;, putrescine;
, spermidine. Each value is the average of duplicate
determinations.
Figure 3:
Effect of spermidine on macromolecular
synthesis at the stationary phase of growth of E. coli CAG2242. DNA (A), RNA (B), and protein (C) syntheses of E. coli CAG2242 grown in the
presence () and absence (
) of 0.5 mM spermidine
were measured by labeling of cells with
[
H]thymidine, [
H]uridine,
and [
S]methionine, respectively, and by counting
cold (A, B) and hot (C) trichloroacetic
acid-insoluble radioactivity as described under ``Experimental
Procedures.'' Each value is the average of duplicate
determinations.
Figure 4:
Two-dimensional analysis of proteins
synthesized in E. coli CAG2242 grown in the presence and
absence of 0.5 mM spermidine. A, without spermidine; B, with 0.5 mM spermidine. , proteins of which
syntheses were particularly inhibited by 0.5 mM spermidine.
Next, the ribosomal proteins and their associated proteins were
analyzed by Western blotting because of the strong inhibition of
protein synthesis by spermidine (Fig.5). The amounts of
ribosomal proteins S1, L20, and L35 (21, 29) in E.
coli CAG2242 grown in the presence of 0.5 mM spermidine
were nearly equal to those in E. coli CAG2242 grown in its
absence. There were also no significant differences in the amounts of
initiation factors 2 and 3. However, the amount of RMF, which is
essential for cell viability(13) , decreased greatly in the
cells grown in the presence of spermidine. When Western blot analysis
was performed using the 30,000 g supernatant instead
of ribosome-associated proteins, essentially the same results were
obtained (data not shown). The results indicate that the amounts of
ribosomal proteins and initiation factors in E. coli CAG2242
grown in the presence and absence of spermidine are nearly equal and
the amount of RMF decreased greatly in the cells cultured with
spermidine.
Figure 5: Western blot analysis of ribosomal proteins and their associated proteins. E. coli CAG2242 grown in the presence and absence of 0.5 mM spermidine was harvested at the times shown in the figure, and Western blot analysis was performed as described under ``Experimental Procedures.''
Since RMF is a stationary phase-specific inhibitor of
protein synthesis and forms temporarily inactive 100 S ribosome dimers (14) , ribosome patterns were analyzed by sucrose
gradient centrifugation. As shown in Fig.6, the relative amount
of 100 S ribosome dimers decreased greatly in E. coli CAG2242
cells grown in the presence of spermidine. At 44 h after the onset of
cell growth, there were no 100 S dimers in the spermidine- accumulated
cells, and the amount of ribosomes (30, 50, and 70 S) decreased
greatly. These results suggest that RMF is not only a stationary
phase-specific inhibitor of protein synthesis but also an
anti-degradation factor of ribosomes.
Figure 6:
Sucrose density gradient centrifugation of
ribosomes. E. coli CAG2242 was harvested at the times shown in
the figure, and sucrose density gradient centrifugation was performed
using 5 A units of the 30,000
g supernatant.
Figure 7:
Effect of spermidine added at the late
stationary phase on cell viability of E. coli CAG2242.
Spermidine was added at 24 h after the onset of cell growth, and cell
viability was measured. , without spermidine;
, with 0.5
mM spermidine. Each value is the average of duplicate
determinations.
Figure 8: Effects of spermidine added at the late stationary phase on ribosomal patterns (A) and the amount of RMF (B). Sucrose density gradient centrifugation and Western blot analysis of RMF were performed as described in the legends of Fig.5and Fig. 6.
Figure 9:
Effect of spermidine on the amount of RMF
mRNA (A) and RMF synthesis (B). A, the
amount of mRNA was measured by primer extension. Primers 3 and 4 in (13) were used in the experiments of a and b,
respectively. 1 and 3, E. coli CAG2242 was
grown in the absence of spermidine, and RNA was prepared from the cells
harvested at 20 and 28 h, respectively; 2 and 4, E. coli CAG2242 was grown in the presence of 0.5 mM spermidine, and RNA was prepared from the cells harvested at 20
and 28 h, respectively. Arrows indicate the initiation sites
of transcription. B, RMF synthesis was measured as described
under ``Experimental Procedures'' using E. coli CAG2242 labeled with [S]methionine. Numbers on the right represent molecular mass in
kDa.
The physiological functions of SAT were examined in this study. We found that SAT is important for the maintenance of cell viability at the stationary phase of growth through the inhibition of spermidine accumulation. When SAT was deficient in E. coli cells, spermidine accumulating in the cells grown in the presence of 0.5 mM spermidine was approximately three times more than that in the cells grown in the absence of spermidine. In eukaryotic cells, accumulated polyamines and bis(ethyl)polyamines inhibited cell growth(7, 30) . The inhibition of cell growth paralleled the inhibition of protein synthesis, especially of mitochondrial protein synthesis due to the existence of unstable tRNAs in mitochondria. In E. coli, however, the accumulated spermidine neither decreased the cell growth nor inhibited protein synthesis at the logarithmic phase of cell growth. At the stationary phase, the accumulated spermidine due to the lack of SAT started to inhibit protein synthesis and caused a decrease in cell viability. Since almost all kinds of protein synthesis were inhibited (Fig.4), the amount of spermidine bound to ribosomes may increase at the stationary phase of growth and cause the inhibition of protein synthesis, which was observed in eukaryotic cells. Actually, the amount of ribosomes decreased at the stationary phase of growth. Accordingly, the relative amount of spermidine to ribosomes increased at the stationary phase of growth. These phenomena were recovered by the transformation of speG gene to SAT-deficient cells. The results clearly show that deficiency of SAT caused the accumulation of spermidine followed by the inhibition of protein synthesis and the decrease in cell viability.
Among the proteins synthesized at the stationary phase, synthesis of OmpC protein and RMF was strongly inhibited by the accumulated spermidine. As expected, the inhibition of RMF synthesis mainly occurred at the translational level. We recently reported that most polyamines exist as a polyamine-RNA complex in cells and contribute to the stimulation of some kinds of protein synthesis under normal conditions(31, 32) . In other words, the degree of stimulation of protein synthesis by polyamines differs in each protein. In a eukaryotic cell-free system, we also reported that low concentrations of spermidine strongly stimulate protein synthesis directed by the mRNA having a GC-rich 5`-untranslated region and that high concentrations of spermidine inhibit the protein synthesis greatly (33) . Thus, it is presumed that the degree of inhibition of protein synthesis by the accumulated spermidine also differs in each protein. Our present results may be in accordance with the concept that the polyamine regulation of protein synthesis depends on the base composition of mRNA, that is, the tertiary structure of mRNA. Further studies will be necessary to provide a clear explanation for the late disappearance of RMF in cells accumulating spermidine.
RMF is known
to be essential for cell viability (13) and is synthesized only
at the stationary phase of growth. Accumulated spermidine also
functions at the stationary phase (Fig.7). A decrease in OmpC
protein did not influence cell viability. It has been reported that the
stationary phase sigma factor is also involved in
cell viability(34) , and its synthesis is positively regulated
by guanosine tetrasphosphate (ppGpp)(35) . The amounts of
measured by Western blotting and of ppGpp measured
by thin layer chromatography did not change significantly in the cells
grown in the presence and absence of spermidine (data not shown). Thus,
the decrease in RMF by the accumulated spermidine is probably the major
reason for the decrease in cell viability.
Although the rates of RMF synthesis were different in cells grown in the presence and absence of spermidine (Fig.9), the amounts of RMF existing in the cells were nearly equal until 28 h after the onset of cell growth (Fig.5). The reason for this remains to be clarified. It may be that excess amounts of RMF that cannot be bound to 100 S ribosome dimers are degraded rapidly.
RMF is essential for cell viability at the stationary phase of growth (13) , but we do not know yet how RMF is involved in cell viability. We suggest that RMF may function as an anti-degradation factor of ribosomes since the amount of ribosomes decreased greatly in the spermidine-accumulated cells harvested at 44 h after the onset of cell growth. However, the possibility that degradation of ribosomes may be the result of cell death can also not be ruled out at present.