From the Department of Biochemistry and Cell Biology, Institute of Gerontology, Nippon Medical School, 1-396, Kosugi-cho, Nakahara-ku, Kawasaki-city, Kanagawa 211-0063, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An amount of human pro-apoptotic Bax as low as 0.01% of total protein was sufficient to cause cell death in Escherichia coli. The bacterial cell death was examined using a viable bacteria-specific fluorescence indicator system and loss of colony formation ability. Co-expression of anti-apoptotic Bcl-xL showed a modest inhibitory effect on the cell death caused by Bax. The trace amount of Bax elongated E. coli and accumulated monounsaturated fatty acids, suggesting an unusual metabolism of redox in the host. In fact, an increase of KCN-dependent O2 consumption accompanied the expression of Bax. At the same time, a fluorescent pH indicator showed the apparent accumulation of protons outside the cell, suggesting that the membrane is intact. Bax increased the level of superoxide anion as measured by the expression of superoxide-dependent promoter. Nicked DNA was significantly generated, and the frequency of mutations resistant to rifampicin was increased by 30-fold, depending upon the expression of Bax. It is proposed that trace amounts of Bax increase oxygen consumption, triggering generation of superoxide, which affects DNA, leading to bacterial death.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis in multicellular organisms is an active cellular self-destruction that is directed by genes. It is not only a physiologically important process in tissue homeostasis and developmental elimination but also a final defense against viral infection and the emergence of cancer (1-5).
Bcl-2 protects cells from apoptosis induced by a wide variety of
stimuli, including radiation, growth factor deprivation, free radicals,
alterations in Ca2+, viral infection, chemotherapeutic
drugs, and axotomy (6). Its related proteins, the Bcl-2 family,
regulate apoptosis by interacting one with each other (7). Members of
the Bcl-2 family are functionally classified into two groups (8, 9).
The first group, including Bcl-2, Bcl-xL, Mcl-1, and A1,
suppresses apoptosis. In contrast, the second group, including Bax,
Bak, Bad, Bik, Bcl-x, and Bcl-xS, promotes
apoptosis.
Bax is a 21-kDa membrane protein with a membrane anchor sequence at the C terminus and promotes apoptosis (10). Bax has three conserved motifs, BH1, BH2 and BH3, which are conserved among several members of the Bcl-2 family (11, 12). Overexpression of Bax countered the death-protecting activity of Bcl-2 and accelerated apoptosis of pro-B cells induced by interleukin-3 withdrawal, whose rates were affected by the ratio of Bax to Bcl-2 (10). Bax forms a heterodimer with Bcl-2 (10). Yeast two-hybrid assays showed that Bax can also bind to the apoptosis-suppressing factors, Bcl-xL, Mcl-1, and A1 (13, 14). Reversely, Bcl-2 and Bcl-xL can bind to the apoptosis-accelerating factors, Bcl-xS (13, 14), Bak (15), Bik (11, 12), and Bad (13, 14). A set of complex and selective interactions among apoptosis-suppressing and apoptosis-accelerating factors appears to dictate the fate of the cell, survival or death following an apoptotic stimulus (14).
Bax expression in the budding yeast Saccharomyces cerevisiae caused cell death (13, 16). Dimerization and targeting to mitochondrial membrane appear to be essential for Bax to exert cytotoxicity in both yeast and mammalian cells (17). Recently Bak as well as Bax induced cell death when expressed in the fission yeast Schizosaccharomyces pombe (18). Co-expression of anti-apoptotic proteins Bcl-2, Bcl-xL, or Mcl-1 abolished the cytotoxicity of Bax or Bak in yeast cells (13, 16, 18, 19). Bax/Bak-induced cell death of yeast partially resembled Bax-induced apoptosis of mammalian cells regarding dead cell phenotype (18). Inducible expression of Bax in mammalian Jurkat T cells initiated apoptosis without an extra death stimulus (20). This process accompanied the generation of reactive oxygen species (ROS),1 and decrease of mitochondrial membrane potential (20). The cell-killing activity of Bax appears to function in biological systems ranging from eukaryotic unicellular organisms to mammalian cells.
We found that an Escherichia coli cell transformed with a prokaryote expression vector carrying mammalian bax or bak cDNAs grew poorly in solid and liquid media. To biochemically investigate the function(s) of Bax in detail, we have chosen this organism as a Bax-expressing host, because there is no report that E. coli has endogenous bcl-2-related genes for interaction with Bax. Here, we present that a trace expression of Bax kills E. coli cells and that this process includes many physiological changes with regard to monounsaturated fatty acid composition, dioxygen consumption, generation of reactive oxygen species, and nicked DNA.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial Cells, Medium, and Culture--
E. coli
strains DH5MCR F
mcrA
(mrr-hsdRMS-mcrBC)
80dlacZ
M15
(lacZYA-argF)U169
endA1 recA1 deoR thi-1
supE44
gyrA96 relA1
(Life Technologies, Inc.), BL21(DE3) F
dcm ompT
hsdS(rB
mB
) gal
(DE3) (Stratagene), and a SOD mutant QC774 F
lac4169 rpsL
(sodA-lacZ)49
(sodB-kan)1-
2 Cmr
Kmr (21), a kind gift from Dr. Yonei of Kyoto University,
Faculty of Science, were used. Media used were as follows: L-broth
containing 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter; L-broth solidified by 1.5% Bacto agar (L-plate);
M9CA medium containing 42 mM
Na2HPO4, 22 mM
KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, and 0.2% glucose, supplemented
with 0.2% casamino acids. Ampicillin (Ap) and chloramphenicol were
added at 50 and 25 µg/ml, respectively.
Cloning of Mammalian bax and bak cDNAs and Expression of
Mammalian Apoptotic Factors in E. coli Cells--
Human bax
cDNA was synthesized and amplified from poly(A)+ RNA
(QuickPrep Micro mRNA purification kit, Amersham Pharmacia Biotech) of human B cell derivatives by PCR with a pair of primers,
5'-NNAGATCTNNCATATGGACGGGTCCGGGGAGCA-3' and
5'-CCGAATTCAGCCCATCTTCTTCCAGAT-3'. Mouse bak cDNA was
synthesized and amplified from poly(A)+ RNA of mouse
(BALB/c, 5 weeks old) thymus by PCR with a pair of primers,
5'-NNNGGATCCATATGGCTTCGGGGCAAGG-3' and
5'-NNNGAATTCATGATTTGAAGAATCTTC-3'. The PCR product of bax
cDNA was digested with a combination of NdeI and
EcoRI, or BglII and EcoRI, and cloned
into the NdeI/EcoRI-digested pProEX-1
(histidine-tagged, Life Technologies, Inc.) or
BamHI/EcoRI-digested pGEX-3X (glutathione
S-transferase fusion, Amersham Pharmacia Biotech),
respectively. For convenience, these vectors, pProEX-1 and pGEX-3X, are
referred to as pHis and pGST, respectively, in this paper. The
resultant constructs for expression of Bax were named pHis-bax and
pGST-bax, respectively. The PCR product of bak cDNA was
also digested with a combination of NdeI and
EcoRI, or BamHI and EcoRI, and then
cloned into NdeI/EcoRI-digested pHis or
BamHI/EcoRI-digested pGST to give pHis-bak or
pGST-bak, respectively. The amino acid sequence deduced from the cloned
bax cDNA has one substitution of Leu59 to
Pro59, compared with the published amino acid sequence
(10). When this cDNA was introduced into mammalian cell FDC-P1,
this version of Bax accelerated apoptosis induced by interleukin-3
withdrawal (22). The DNA sequence of the bak cDNA cloned
was confirmed. A rat bcl-xL cDNA (23) was
inserted into pHis to give pHis-bcl-x, like pHis-bax. The
bcl-x cDNA was also tandemly inserted into the
XhoI site downstream of the bax coding region in
pHis-bax to give pHis-bax/bcl-x. In this construct, the bax
and bcl-x coding regions should be transcribed in a
polycistronic mRNA. To subclone the bax cDNA into
the vector pACYC184 (24), pHis-bax was digested with SphI. A
fragment containing the lacIq gene, the
trc promoter, and the bax cDNA was inserted
at the SphI site of pACYC184, which is located in the
tetracycline resistance gene. The construct was named pACYC-bax. To
express two mutant Bax versions as described by Simonian et
al. (25), pACYC-baxGD67-68 and pACYC-bax63-71 were
constructed by a two-step PCR mutagenesis method (26). Expression of
Bcl-x with the C-terminal hydrophobic region deleted (Bcl-xG196) from a
plasmid pROG196 was directed by the T7 RNA polymerase promoter as
described by Aritomi et al. (27). Induction of the
polymerase with IPTG in BL21(DE3) cells allows the expression of genes
placed downstream of the T7 RNA polymerase promoter.
Plasmid Preparation-- Since the plasmid containing cDNA of bax was easily mutated after overgrowth of the host, the transformant was picked from a small colony, grown in a rich L-broth medium containing 20 mM glucose and harvested before a stationary state to prepare the plasmid.
Viability Assay--
DH5MCR cells were transformed with the
indicated plasmids and incubated on L-plates containing Ap for 16 h at 37 °C. After enumerating the colonies, Ap-resistant
transformants on the plate were harvested and pooled in L-broth. To
asses colony-forming ability, a number of cells was microscopically
examined with a hemacytometer for bacteria. The cell suspensions were
adequately diluted and spread on fresh L-plates with Ap and the number
of colonies were counted on the following day. For the fluorescence analysis of viability, the cells in the pooled cell suspension were
counted and treated with LIVE/DEAD BacLightTM bacterial viability kits
(L-7012) according to the protocol provided by the supplier (Molecular
Probes).
Detection of the Bax Protein--
DH5MCR cells carrying the
vector or pHis-bax were cultured in L-broth. The cells were harvested,
washed, and disrupted by sonication in a solution containing 6 M urea and 2% SDS. Solubilized proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene difluoride membrane. The membrane was treated with
anti-Bax polyclonal antibody N-20 (Santa Cruz Biotechnology), then
incubated with a peroxidase-conjugated anti-rabbit IgG (Cappel). Bax
was visualized by a chemiluminescence method using RENAISSANCETM (NEN
Life Science Products). To obtain the purified Bax protein as a
standard, Bax with (His)6 tag was expressed in E. coli for 30 min by adding an inducer, IPTG, to 1 mM.
The expressed Bax was purified by nickel column chromatography, followed by gel filtration. The purified protein was identified by
Western blotting using the anti-Bax antibody. The Bax was eluated in
the fractions corresponding to 50 kDa by gel filtration chromatography.
Scanning Electron Microscopy--
Scanning electron microscopy
was performed by a conventional method. E. coli cell
DH5MCR cells harboring the vector or pHis-bax was cultured in
L-broth. The cells were applied on isopore track-etched membrane
filters (Millipore) at A600 = 0.3 (1.4 × 108 cells/ml). After washing with phosphate-buffered
saline, the filters with cells were immersed in 2.5% glutaraldehyde.
The cells were dried by a critical point drying method after
dehydration with ethanol. The cells were coated with gold and observed
with JSM-T20 (JEOL, Tokyo).
Fatty Acid Composition--
DH5MCR cells carrying pHis-bax or
the vector were cultured in L-broth for 2.5 h at 37 °C after
cessation of growth. The washed cells were freeze-dried. According to a
conventional method, fatty acid methyl esters were extracted by hexane
following methanolysis (28). The extracts were analyzed with a gas-mass
spectrometer GCMS QP5000 (Shimazu, Kyoto).
Oxygen Consumption--
DH5MCR cells harboring the vector or
pHis-bax were inoculated in L-broth containing Ap at
A600 of 0.01 and grown with vigorous shaking at
37 °C. Aliquots of the culture were removed for measuring A600 and dioxygen (O2) consumption
at various times. O2 consumption was determined by an
oxygen electrode method using MD-1000 (Iijima Electronics Mfg. Co,
Ltd., Tokyo) with a water jacket at 37 °C. In each experiment, the
recorder was adjusted using a sodium sulfite solution and air-saturated
water (Milli-Q, Millipore) at 37 °C. The decreasing rate of oxygen
in the medium was normalized against bacterial number at each measuring
point. A normalized value was represented as relative O2
consumption.
Measurement of pH Inside Cells by DCF-DA--
A
membrane-permeable fluorescent dye, DCF-DA (LAMBDA, Graz, Austria) was
applied to cells as follows. DH5MCR cells harboring the vector or
pHis-bax were cultured in a M9CA medium containing Ap with shaking at
37 °C. At A600 = 0.1, IPTG (30 µM) was added to induce Bax expression, and immediately
an aliquot of the culture (1 ml) was removed (incubation time = 0). The remaining culture was further shaken with IPTG for 15, 30, 45, and 60 min, and at each incubation time, 1 ml of culture was removed.
The 1-ml aliquot was added to DCF-DA (5 µM) and stood at
37 °C for 15 min. After being washed with phosphate-buffered saline
twice, the cells were suspended in phosphate-buffered saline at
A600 of 0.1 and analyzed with a fluorescence
spectrophotometer F-3000 (Hitachi, Tokyo).
Detection of Superoxide by a sodA-lacZ Fusion Gene--
SOD
mutant QC774sodA-lacZ sodB-kan cells containing pHis-bax
were anaerobically cultured at 37 °C in the container used for O2 consumption experiments, while monitoring O2
levels. The cells were inoculated into L-broth supplemented with 50 mM glucose, 20 mM KNO3, and
ampicillin at 0.004 of A600. The cell suspension was then blown with nitrogen gas to remove O2. Following a
lag time of about 1 h, the cells grew with a doubling time of
around 20 min until 0.08 of A600, during which
O2 levels were undetectable. IPTG (10 µM) was
added, and after 30 min, the cells (1 ml) were cooled, harvested, and
washed. The cells were resuspended in 0.1 M potassium
phosphate buffer, pH 7.8, containing 1 mM dithiothreitol. After addition of lysozyme, the cells were disrupted by freezing and
thawing. Following DNase I treatment, the cell extracts (50 µl) were
prepared by centrifugation to precipitate debris. Two microliters of
10- and 100-fold diluted cell extracts were subjected to Galacto-Light
chemiluminescent assay for -galactosidase (Tropix, Bedford, MA)
using a luminometer, Lumat LB9507 (Berthold). Under aerobic conditions,
the cells with pHis-bax were grown in L-broth containing 10 mM glucose and ampicillin with vigorous shaking. IPTG (1 µM) was added at 0.2 of A600,
followed by the same procedure mentioned above.
Detection of Nicked DNA--
DH5MCR cells carrying pHis-bax
or the vector were cultured in L-broth at 37 °C. The cells were
incubated for indicated periods at the stationary phase. Harvested
cells were lysed with 1 N NaOH. During incubation at room
temperature, DNA was denatured, and DNA strands with nicks were
separated from intact DNA by ultracentrifugation (100,000 × g, 1 h). Denatured single strands of DNA were
precipitated by ethanol and dissolved in TE buffer. After RNase A
treatment, DNA samples were precipitated with isopropyl alcohol and
dissolved in TE buffer (10 mM Tris · Cl, pH 7.4, 1 mM EDTA, pH 8.0) (1 µl per 0.1 A600 unit). One microliter was subjected to
alkaline-agarose gel electrophoresis followed by blotting to a nylon
membrane Hybond-N+ (Amersham Pharmacia Biotech). A
32P-labeled probe was prepared by an oligo-random labeling
kit (Amersham Pharmacia Biotech) using genomic DNA of DH5
MCR as a
template. Hybridization was performed in a Rapid-hyb buffer (Amersham
Pharmacia Biotech) at 65 °C. The membrane was washed twice with
0.1 × SSC containing 0.1% SDS at 65 °C and exposed to an
imaging plate for analysis by a bioimaging analyzer BAS1500 (Fuji Film,
Ltd., Tokyo). The image was visualized by Pictrography (Fuji Film,
Ltd.).
Frequency of Rifampicin-resistant Mutants--
DH5MCR cells
were transformed with the vector or pHis-bax and plated on L-broth
plates with Ap. After incubation for 16 h at 37 °C, the
transformants from the Ap-resistant colonies were mixed. The cells
(1 × 109 or 100) were spread on L-plates containing
Ap with or without rifampicin (Rif; 50 µM), respectively.
The frequency of Rif-resistant mutants was represented as the ratio of
the number of colonies in the presence of Rif to that in the absence of
Rif.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A Trace Amount of Bax Causes Cell Death in E. coli-- We constructed inducible expression plasmids to overexpress the mammalian apoptosis regulatory factors, Bax, Bak, and Bcl-xL in E. coli. During the studies, the cDNAs of bax and bak were found to make smaller colonies than the vectors alone, even in the absence of an inducer, IPTG in E. coli (Fig. 1A). On the other hand, the introduction of bcl-xL cDNA, the anti-apoptotic factor, resulted in the same sized colonies as transformants of vectors alone. Regardless of the expression vector, pHis (pProEX-1; see "Materials and Methods") or pGST (pGEX-3X; see "Materials and Methods"), the same results were obtained. These results indicate that small colonies are formed by the expression of the mammalian pro-apoptotic factors. Since the vector harbors the repressor lacIq gene, the expression should be limited in the absence of the inducer. It was difficult to maintain the transformants forming the small colony, because suppressor mutant cells easily appeared. Thus, in each experiment, the transformants were freshly obtained by introduction of each plasmid into competent cells.
|
|
|
|
Fatty Acid Composition-- To explore the mechanism of cell death, fatty acid composition was examined. After a 2.5-h incubation at the stationary phase, the cells with pHis-bax or the vector were harvested. After hexane extracts of the cells following methanolysis, fatty acid methyl esters were analyzed by gas-mass spectrometry (Fig. 5). Fragmentation patterns of each compound separated by gas chromatography were searched in the National Institute of Standard and Technology reference data bases. The cells with pHis-bax increased C16 = 1 and C18 = 1 fatty acids by 8- and 4-fold, respectively, compared with those of the cells with the empty vector. Searches in the libraries identified C16 = 1 to be palmitoleic methyl ester, but C18 = 1 methyl ester is unknown with regard to the position of the double bond. On the whole, the Bax expression increased monounsaturated fatty acid composition by 6-fold from 6 to 36%. These findings suggest a specific change of the bacterial physiology was induced by the trace amount of Bax, probably due to the unusual reduction-oxidation system.
|
Increased Dioxygene Consumption by Bax Expression--
As the next
step, oxygen consumption was examined during cell growth. The cells
with pHis-bax showed that a relative increase in O2
consumption by 60% at the late log phase and then a rapid decrease
just before entering the stationary phase (Fig.
6A). The cells with the empty
vector consumed oxygen at a constant rate in the log phase, and this
rate gradually decreased following entrance into the late log and
stationary phases (Fig. 6B). O2 consumption was
inhibited completely by addition of 1 mM KCN (data not
shown), indicating that Bax activated an electron transport system
directly or undirectly. To examine the effect by the activation of the
electron transport system, a membrane-permeable fluorescent dye, DCF-DA
(29, 30), was applied to the cells expressing Bax by IPTG induction.
DCF-DA in cells is deacetylated and converted to DCF by esterase(s).
DCF gives a strong fluorescence at 525 nm in a range of alkalic but not
acidic pH. DH5MCR with pHis-bax, which was incubated with IPTG for
15 min, expressed Bax at a detectable level, and the expression level
of Bax increased to be maximized after a 45-min incubation with IPTG
(Fig. 7A, inset). After IPTG treatment for indicated time, the cells were incubated with DCF-DA for
15 min. The relative fluorescence intensity of the cells at 525 nm
became stronger depending on the expression levels of Bax (Fig.
7A). Under this condition, O2 consumption
increased by 40-50% for an hour (data not shown). The cells carrying
the empty vector did not give any increase of the fluorescent signal at
525 nm after the incubation with IPTG (Fig. 7B). These
results indicated that the inside of the cells became more alkalic by
the expression of Bax. A simple explanation for this is that Bax
accelerated the electron transport system to pump protons out and that
the membrane remained intact even on accumulation of unusual fatty acids.
|
|
Increase of Superoxide Radicals by Bax Expression--
The
increment of O2 consumption by Bax expression led us to
speculate enhanced production of superoxide. Superoxide is converted to
hydrogen peroxide by SOD (superoxide dismutase) in cells as the first
step in protection cells from oxygen radicals. E. coli has
two different genes responsible for SOD, sodA and
sodB, which encode Mn-SOD and Fe-SOD, respectively. The
sodA gene is inducible by superoxide (31, 32). A sodA
sodB double mutant QC774 is completely devoid of SOD activity
(21). Paraquat, a potential generator of superoxide, increases the
sodA expression anaerobically when E. coli is
cultured with nitrate as a terminal electron acceptor (33-35). QC774
has a mutation in the sodA gene where the endogenous promoter of the gene is fused with lacZ (21). QC774 cells
harboring pHis-bax were anaerobically grown in L-broth supplied with
glucose and potassium nitrate. -Galactosidase activity was enhanced
2-fold in cells treated with IPTG for 30 min compared with cells
untreated with IPTG (Fig. 8A).
Under aerobic conditions, induced Bax enhanced the promoter activity to
some extent (Fig. 8B), probably because the background is
relatively high. These results suggested that expression of Bax caused
an increase of superoxide in cells.
|
Nicked DNAs and Mutants--
Generation of superoxide results in
generation of other ROS. Damage of DNA was examined by two methods: by
extent of degraded DNA and by the increase of the mutation frequency.
The cells with pHis-bax or the empty vector were grown in L-broth.
After various periods of incubation at the stationary phase, the cells
were solubilized in a alkali solution. Denatured single-stranded DNAs were subjected to alkaline-agarose gel electrophoresis and detected by
Southern blot analysis (Fig. 9). The
cells with pHis-bax had markedly increased amounts of nicked DNA after
1 h in the stationary phase, while the cells with the empty vector
did not. As the next criteria of damage of DNA, the frequency of
mutations of the cells with pHis-bax was compared with that of the
cells with the vector (Fig. 10).
DH5MCR cells carrying pHis-bax gave 63 rifampicin-resistant (Rifr) colonies per 1010 cells, while the cells
harboring the empty vector gave 2.1 Rifr colonies per
1010 cells. Thus, Bax expression increased the frequency of
DNA mutation by 30-fold. Taken together, trace amounts of Bax appear to
kill bacteria by damaging the DNA.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We reported here that a trace amount of the human Bax protein halted the growth and then caused the death of E. coli cells accompanied by some physiological changes, including increases in monounsaturated fatty acids, O2 consumption, superoxide radicals, nicked DNA, and the frequency of mutations. Bax easily mutated the host and plasmid DNA to give colonies of normal size. Therefore, so far, it might be difficult to find these phenomena.
It was shown that the Bax protein is lethal in S. cerevisiae and S. pombe (13, 16, 18). However, the amount of Bax expressed was not described. In the case of E. coli, a trace amount (0.01% of total protein) was sufficient to kill the host. Two criteria were applied to distinguish death from growth arrest: a viable bacteria-specific fluorescence dye system and a decrease of colony formation ability (Fig. 1). These results indicated that E. coli cells carrying pHis-bax are easily killed, and the process does not involve cell lysis. This death appears to be specifically caused by the mammalian pro-apoptotic factors, because Bcl-xL did not affect E. coli growth, unlike Bax and Bak. Bcl-xL had a modest inhibitory effect on Bax in E. coli. When Bcl-xL was expressed in excess of Bax, Bcl-xL effectively abolished the Bax cytotoxicity (Fig. 2). On the other hand, Bcl-xL antagonized the cytotoxicity to the less extent, when bcl-x was tandemly located downstream of bax (Figs. 1 and 3). In the latter, the expression of Bcl-xL was nearly the same as that of Bax and the trace amount (data not shown). Most of the Bax protein molecules in the cell could avoid association with Bcl-xL. Again, it was underscored that Bcl-xL inhibited the Bax cytotoxicity through an interaction with BH3 of Bax (Fig. 2) as shown in mammalian cells (25). In addition, we have recently identified the region of Bax lethal to E. coli and found that the region is responsible for inducing apoptosis (22).
During culture in L-broth, a trace of Bax protein was detected at 2 h before cessation of growth and the amount increased gradually with vigorous shaking (Fig. 3). At the point at which cell growth stopped, the amount of Bax expressed in a single cell corresponded to no more than 0.01% of total E. coli protein. It is well known that the depletion of glucose in medium elevates cAMP levels in cells to make a complex with a cAMP receptor protein. In turn, this complex can activate lac promoter. It is likely that depletion of glucose contained in yeast extract results in the expression of a trace amount of Bax protein.
Bax expression enhanced O2 consumption of E. coli cells by 60% for 45 min just before it completely stopped the growth (Fig. 6). Since KCN inhibited the O2 consumption, a respiratory chain was activated (data not shown). It is not likely that the Bax protein directly binds to oxygen to consume oxygen, because computer analysis showed that Bax does not share any motif with proteins which bind to dioxygene. It is unknown how Bax activates the respiratory chain. Enhanced generation of superoxide by Bax expression can be also explained by activation of the respiratory chain. Otherwise, a redox imbalance due to Fe2+ or NADPH depletion in cells may take place in response to Bax expression (36, 37).
Generation of superoxide results in high levels of ROS, including hydrogen peroxide and hydroxy radical, which are known to cause damage to DNA, proteins, and membranes. Enhanced conversion of superoxide to other ROS probably accounts for the increase of nicked DNA caused by Bax expression, although we could not detect any substantially damaged products such as 8-hydroxydeoxyguanosine and peroxidized lipid and any radicals by electron spin resonance spectrometry (data not shown).
It is very interesting that in cells expressing Bax, the composition of monounsaturated fatty acids increased 6-fold (Fig. 5). Some bacteria such as E. coli and Pseudomonas synthesize monounsaturated fatty acids via an anaerobic pathway, whereas in others, such as Bacillus and Corynebacterium, and in animals, synthesis is via an aerobic pathway (38). In the aerobic pathway, desaturase(s) uses oxygen molecules in a monooxygenase-type reaction. Bax expression may activate the enzymes involved in the synthesis of monounsaturated fatty acids in the anaerobic pathway. It is well known that the higher the content of unsaturated fatty acids, the more flexible the cell membrane. The cells expressing Bax were expected to be more sensitive to osmotic pressure. Scanning electron microscopy revealed that the cells expressing Bax enlarged along both axes (Fig. 4).
Recently the x-ray structures of renatured (39) and native (27) truncated Bcl-xL protein were resolved. The three-dimensional fold of Bcl-x is similar to that of the bacterial toxins diphtheria toxin and colicin A (27, 39). Like these bacterial toxins, Bcl-xL was also shown to form an ion channel in synthetic lipid membrane with selectivity for K+ and Na+ (40). These proteins appear to insert and be internalized into a membrane by a very similar multistep mechanism. Bax may insert into a membrane more easily than Bcl-xL, because Bax has a high sequence homology with Bcl-xL, and its expected hydrophobic cleft, formed by BH1, BH2 and BH3, is bigger than that of Bcl-xL (27). In addition, Bax structure lacks two hydrogen bonds stabilizing the central helices, suggesting that Bax possesses a greater potential for membrane insertion than either Bcl-2 or Bcl-xL (27). As expected, Bax has recently been shown to form an ion channel at neutral pH in synthetic lipid membrane, whose activity was inhibited by Bcl-2 (41). The results of DCF-DA should be interpreted with caution. One plausible explanation is as follows: Bax formed an ion channel and enhanced the transport of specific cations (not proton) or anions, which diminished the membrane potential as a result. To compensate the membrane potential, the respiratory chain may be activated to enhance the proton pumping activity, leading the inside of the cell to become alkaline. The activated respiration may lead to oxygen radical formation.
Bcl-2 inhibits the release of cytochrome c from mitochondria (42, 43) and the loss of mitochondrial membrane potential (44-46). It can be explained as that Bax associates with Bcl-2 and Bcl-xL to abolish their activities, resulting in cell death. It has been proposed that Bcl-2 functions as an inhibitor of the anti-oxidant pathway. This is also explained as that Bax generates superoxide and that Bcl-2 antagonizes Bax. In fact, Xiang et al. (20) reported that inducible expression of Bax causes apoptosis without the need for stimulus and increases ROS. Recently, we have isolated an E. coli mutant that suppresses cell death even on expression of Bax, in which the RNase E gene is split into 5'- and 3'-regions. The truncated RNase E also made cells resistant to paraquat, a generator of superoxide.2 Therefore, it is likely that Bax induces the oxygen radical in E. coli. We show the region lethal to bacteria is common to a region inducing apoptosis in mammalian cells (22). The findings described in this study offer a useful approach using E. coli for investigating the molecular mechanism of apoptosis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Naomitsu Eguchi (Meiji College of Pharmacy, Tokyo) for his technical help with gas-mass spectrometry and Seiko Egawa (Institute of Gerontology, Nippon Medical School) for her technical assistance with scanning electron microscopy. We express thanks to Dr. S. Yonei (Kyoto University, Faculty of Science) for giving a SOD mutant QC774sodA-lacZ sodB-kan strain.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Helix Research Institute Inc., Yana,
Kisarazu-city, Chiba-ken 292-0812, Japan.
§ To whom correspondence should be addressed. Tel.: 81-44-733-9267; Fax: 81-44-733-1877; E-mail: ohta{at}nms.ac.jp.
1
The abbreviations used here are: ROS, reactive
oxygen species; Ap, ampicillin; PCR, polymerase chain reaction; IPTG,
isopropyl--D-thiogalactopyranoside; Rif, rifampicin;
DCF-DA, 2',7'-dichlorofluorescein diacetate; DCF,
2',7'-dichlorofluorescein; SOD, superoxide dismutase.
2 R. Nanbu-Wakao, S. Asoh, K. Nishimaki, Y. Ishibashi, R. Tanaka, and S. Ohta, submitted for publication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|