Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones.

Abstract

It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents.
We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs.
The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

Full text

Page 1

BioMed Central
Page 1 of 8
(page number not for citation purposes)
BMC Microbiology
Open Access
Research article
Effects of disruption of heat shock genes on susceptibility of
Escherichia coli to fluoroquinolones
Yuko Yamaguchi1, Toshifumi Tomoyasu1, Akiko Takaya1, Mizue Morioka2
and Tomoko Yamamoto*1
Address: 1Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263-8522
Japan and 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
Email: Yuko Yamaguchi - Yuko@p.chiba-u.ac.jp; Toshifumi Tomoyasu - tomoyasu@p.chiba-u.ac.jp; Akiko Takaya - akiko@p.chiba-u.ac.jp;
Mizue Morioka - smizue@m.ecc.u-tokyo.ac.jp; Tomoko Yamamoto* - tomoko-y@p.chiba-u.ac.jp
* Corresponding author
Abstract
Background: It is well known that expression of certain bacterial genes responds rapidly to such
stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins
encoded in these genes are important for successful survival of the organism under the hostile
conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed
to affect the organisms' susceptibility to these agents.
Results: We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a
fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether
the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with
mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK,
groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The
increased susceptibility of the lon mutant was corroborated by experiments in which the gene
encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS
response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the
lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the
depletion of Lon and the continuous induction of the SOS response in the presence of FQs.
Conclusion: The present results show that the bactericidal action of FQs is moderately affected
by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have
contributed successfully to the treatment of various bacterial infections, but their widespread use
and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our
results suggest that agents capable of inhibiting the Lon protease have potential for combination
therapy with FQs.
Background
FQs are broad-spectrum agents applicable to a range of
Gram-positive and Gram-negative infections, and they
have good oral absorbability [6]. Because of these advan-
tages, FQs have been widely used against a variety of bac-
terial infections for about two decades. They target the
Published: 12 August 2003
BMC Microbiology 2003, 3:16
Received: 19 March 2003
Accepted: 12 August 2003
This article is available from: http://www.biomedcentral.com/1471-2180/3/16
© 2003 Yamaguchi et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.

Page 2

BMC Microbiology 2003, 3http://www.biomedcentral.com/1471-2180/3/16
Page 2 of 8
(page number not for citation purposes)
type II topoisomerases, DNA gyrase and topoisomerase
IV, which are essential for controlling the topological state
of DNA during replication and transcription [12].
Bacteria are known to respond to unfavorable conditions,
e.g., exposure to toxic chemicals and physical agents,
nutrient limitation, or sudden increase in growth temper-
ature, by rapid expression of regulons related to the heat
shock, SOS, and oxidative stress responses. DNA damage
by UV irradiation, or treatment with naldixic acid, induce
both the SOS and heat shock responses [9,19]. Puromycin
has been reported to induce first the SOS and secondly the
heat shock responses; hydrogen peroxide induces the oxi-
dative stress and SOS responses; and CdCl2 strongly
induces all three stress responses. Protein induction by
these responses is widely believed to be important for the
organism's survival under hostile conditions. Analo-
gously, the proteins induced when bacterial cells are
exposed to antibiotics may affect the susceptibility of the
organisms to these agents.
The aim of present study was to identify the bacterial
responses affecting the bactericidal action of FQs. We ana-
lyzed the proteins induced in Escherichia coli by exposure
to FQs, then examined the susceptibilities to these agents
of E. coli strains with mutations in the genes encoding
these proteins.
Results and Discussion
Analysis of protein synthesis in E. coli exposed to
levofloxacin
Figure 1(A) shows a profile of proteins in E. coli cells that
were pulse-labeled after incubation for 10 min with differ-
ent concentrations of LVFX. The results show a large
increase in synthesis of 70 kDa (a), 60 kDa (b), and 40
kDa proteins (c) at 10 µg/ml LVFX. These proteins were
also induced by lower concentrations of LVFX, but
increasing concentrations increased the rate of synthesis.
Comparison of the protein profiles of LVFX-treated, tem-
perature-shifted (42°C) and unshifted (30°C) bacteria
indicates that the 70 kDa (a) and 60 kDa (b) bands may
Proteins synthesized in E. coli exposed to LVFX
Figure 1
Proteins synthesized in E. coli exposed to LVFX. (A) Exponentially-growing cells were incubated with 1, 2, or 10 µg/ml
of LVFX for 10 min and then labeled with [35S]-methionine and cysteine for 1 min at 30°C. Heat shock culture was prepared as
described in Methods. (B) After incubation with 10 µg/ml of LVFX, aliquots (100 µl) of the cultures were taken at indicated
times and labeled for 1 min at 30°C. Equal amounts of acid-precipitable counts were applied to each lane and the proteins were
separated by SDS-PAGE. The gels were dried and then exposed to films (A and B). The protein bands alphabetized in (B) were
quantified using a BAS2000A image analyzer and the relative rates of synthesis are shown in (C).

Page 3

BMC Microbiology 2003, 3 http://www.biomedcentral.com/1471-2180/3/16
Page 3 of 8
(page number not for citation purposes)
be heat shock proteins. Figures 1(B) and 1(C) show the
time-course of synthesis of these proteins after incubation
with 10 µg/ml LVFX for 60 min. Synthesis of the 70 kDa
and 60 kDa proteins appears to have accelerated for 15
min and then stopped. In contrast, induction of the 40
kDa protein continued up to 45 min after exposure to
LVFX, suggesting that the induction mechanism is differ-
ent from that for the 70 and 60 kDa proteins.
The proteins were separated on two-dimensional gels to
facilitate identification (Figure 2). Visual scanning of the
autoradiogram showed that synthesis of at least 16 pro-
teins was markedly elevated in bacterial cells exposed to
LVFX compared with untreated cells. The spots numbered
on the gel are proteins that could be identified by mass
spectrometric analysis. Among these, heat shock proteins
are identified as follows: ClpB (spot 1), DnaK (spot 2),
GroEL (spot 3), HtpG (spot 4), HslU (spot 5) and GrpE
(spot 6). Enhanced production of the small heat shock
proteins GroES, IbpA, and IbpB was also observed when
12.5 % SDS-PAGE was used as the second dimension
(results not shown). Phosphogluconate dehydrogenase
(spot 7) and phosphoglycerate kinase (spot 8) were also
identified.
The 40 kDa protein showing the most marked induction
in Figure 1 does not appear on the two-dimensional gel
(Figure 2). We therefore fractionated the proteins from
LVFX treated E. coli cells along with those from control
cells. As shown in Figure 3, the 40 kDa protein seems to
be largely associated with high salt-soluble proteins (lanes
6 and 8). A portion of the high salt-soluble fraction was
separated by two-dimensional gel electrophoresis; the
spot corresponding to the LVFX-induced 40 kDa protein
Two-dimensional gel electrophoresis patterns of proteins synthesized in E. coli exposed to LVFX
Figure 2
Two-dimensional gel electrophoresis patterns of pro-
teins synthesized in E. coli exposed to LVFX. Bacterial
cells were exposed to 10 µg/ml of LVFX for 15 min and then
pulse-labeled for 1 min. A portion of the protein samples
from untreated cells (A) or LVFX-treated cells (B) was sub-
jected to two-dimensional gel electrophoresis using 10 %
SDS-polyacrylamide gel as the second dimension. Spots
enclosed in circles represent the proteins induced during
exposure to LVFX. The proteins numbered are interpreted
in the text.
Fractionation of proteins synthesized in E. coli exposed to LVFX
Figure 3
Fractionation of proteins synthesized in E. coli
exposed to LVFX. Proteins from cultures labeled in the
presence of 10 µg/ml LVFX were fractionated as described in
Methods and separated by SDS-PAGE (A). The protein sam-
ples from untreated cells were run in lanes 1, 3, 5, 7 and 9.
The samples from LVFX-treated cells were in lanes 2, 4, 6, 8
and 10. Lanes contain protein fractions as follows: lanes 1
and 2, whole cell lysates; lanes 3 and 4, cytoplasmic and peri-
plasmic protein fractions; lanes 5 and 6, membrane-associ-
ated protein fractions. The membrane-associated proteins
were solubilized in 1 M NaCl and then dialyzed described in
Methods. After centrifugation, the pellets were solubilized in
lysis buffer and applied in lanes 7 and 8. The supernatants
were applied in lanes 9 and 10. Portions of the protein sam-
ples applied in lanes 7 and 8 were also subjected to two-
dimensional gel electrophoresis and the results are shown in
(B) and (C), respectively.

Page 4

BMC Microbiology 2003, 3http://www.biomedcentral.com/1471-2180/3/16
Page 4 of 8
(page number not for citation purposes)
was subjected to mass spectrometric analysis and identi-
fied as RecA. RecA is usually cytosolic in E. coli and binds
to single-stranded regions of damaged DNA [14]. Detec-
tion of a large amount of RecA protein in the high salt-sol-
uble fraction suggests that when it is synthesized in
abnormally large amounts, it aggregates with various
denatured and misfolded proteins.
In E. coli, the expression of heat shock genes is positively
regulated at the transcriptional level by the heat shock
specific sigma subunit of RNA polymerase, σ32. In addi-
tion, the heat shock response is negatively autoregulated
by the DnaK chaperone machine that interferes with the
efficient binding of σ32 to the RNA polymerase core,
turning off the response [2,4,16]. Heat-induced aggrega-
tion of proteins induces the expression of the heat shock
regulon through the titration of the DnaK chaperone by
the aggregates [4,16]. Therefore, in these experiments, the
induction of the E. coli heat shock regulon could have
been triggered by protein aggregates accumulated after
incubation with LVFX.
Antibiotic susceptibility of E. coli strains with mutations in
heat shock genes
The heat shock proteins comprise chaperones such as the
DnaK/DnaJ/GrpE, GroEL/GroES and IbpA/IbpB systems,
and ATP-dependent proteases such as Lon, ClpXP and
HslVU. Whereas the chaperones are abundantly synthe-
sized, the levels of the proteases are relatively low even
under inducing conditions. In these experiments, pro-
teases were not present in sufficient amounts to identify
by mass spectrometry in LVFX-treated cells. However, all
the heat shock genes in one regulon under the control of
σ32 would be simultaneously induced in cells exposed to
LVFX.
To examine the involvement of heat shock proteins in the
susceptibility of E. coli to FQs, we constructed strains with
mutations in various heat shock genes and then tested
their susceptibility to LVFX (Figure 4A and 4B). Among
the chaperone mutants, ∆dnaK and groEL- exhibited
increased susceptibility. However, a mutation in the small
chaperone, IbpAB, did not affect susceptibility. Among
the protease mutants, ∆clpP, ∆clpX, ∆clpA, ∆hslVU and
∆htrA all showed the same susceptibility to LVFX as the
wild type. In contrast, ∆lon showed a greatly increased sus-
ceptibility. This hypersusceptibility of the ∆lon mutant
was corroborated by providing a functional lon using a
low copy-number-vector (Figure 4C and 4D). The results
suggest that the hypersensitivity of the strain to LVFX is
largely due to the disruption of lon. ∆lon showed a simi-
larly increased sensitivity to other FQs such as cipro-
floxacin, sparfloxacin and sitafloxacin (data not shown).
As expected, a mutation in rpoH, which encodes σ32, also
resulted in increased susceptibility of E. coli to LVFX.
Chaperones and proteases are essential for de novo fold-
ing and quality control of proteins, acting by preventing
protein aggregation and by refolding or degrading mis-
folded proteins. Therefore, the increased susceptibility of
the chaperone and lon mutants can partly be explained by
the disruption of the quality control system in E. coli
cytosol.
Effect of sulA-disruption on the hypersusceptibility of the
∆lon mutant to fluoroquinolones
The Lon protease degrades SulA, which inhibits separa-
tion by binding to FtsZ, a key cell division protein [1].
Susceptibility of chaperone- or protease-deficient mutants of
E. coli to LVFX
Figure 4
Susceptibility of chaperone- or protease-deficient
mutants of E. coli to LVFX. Bacterial cultures were
diluted from 10-3 to 10-7 and then spotted on to agar plates
prepared by the standard agar doubling dilution method. (A)
and (C) are the results with drug-free agar plates. (B) and (D)
are the results with agar plates containing 0.0125 µg/ml
LVFX. Abbreviation: plon+, a complementing plasmid carrying
the intact lon gene [15].

Page 5

BMC Microbiology 2003, 3http://www.biomedcentral.com/1471-2180/3/16
Page 5 of 8
(page number not for citation purposes)
SulA is a SOS response protein induced by DNA damage
after UV-irradiation and quinolone-treatment [23]. We
examined the possibility that the hypersensitivity of the
∆lon mutant is due to the accumulation of SulA protein.
The sulA gene was disrupted in both wild-type and ∆lon-
mutant cells and then the resultant double mutants were
compared for susceptibility to LVFX. As shown in Figure 5,
the hypersusceptibility of ∆lon was suppressed by the sub-
sequent disruption of sulA, suggesting that the increased
sensitivity is due to the inhibition of cell division by
abnormal accumulation of SulA. It therefore appears that
in ∆lon, depletion of the Lon protease and continuous
induction of the SOS response in the presence of LVFX
result in abnormally high levels of the cell division
inhibitor SulA. Complementation analysis also indicates
that continuous induction of SulA by FQs contributes to
quinolone killing. This hypothesis is supported by the
finding that the ∆sulA mutant is less sensitive to LVFX than
the wild type (Figure 5). Moreover, disruption of lon did
not affect the susceptibility of bacteria to antibiotics other
than quinolones, such as ampicillin, cephalothin, strepto-
mycin, polymixin-B or rifampicin (our unpublished
data).
Effect of the lon mutation in fluoroquinolone-resistant
mutants
Bacterial quinolone resistance is usually due to mutations
in the genes for targets of these agents: DNA gyrase
encoded by gyrA and gyrB, and topoisomerase IV encoded
by parC and parE. In several species of Enterobacteriaceae,
decreased susceptibility or resistance to FQs is associated
with specific point mutations in gyrA [21,22]. An addi-
tional mutation in parC results in greater resistance
[10,20]. We examined whether the ∆lon mutation affects
the resistance to LVFX in E. coli gyrA and parC mutants.
In Figure 6, strain CS5086 has a gyrA mutation (Ser83 to
Leu). To construct a gyrA and parC double mutant,
CS5086 was initially treated with mini F-parC1 (Gly78 to
Asp, Val253 to Ile). Then the chromosomal parC gene of
the resultant strain was replaced with parC::Cm by P1
transduction; lon was subsequently disrupted in both the
gyrA and the gyrA, parC1 mutants. As shown in Figure 6,
∆lon decreased the resistance of both gyrA and gyrA, parC1
to LVFX.
Effect of recA mutation on susceptibility of E. coli to
fluoroquinolone
Since one of the proteins strongly induced by LVFX is
RecA (Figure 1 and Figure 3), the role of RecA in the bac-
tericidal action of the agent was examined by susceptibil-
ity testing of a E. coli recA mutant along with the isogenic
wild type. Figure 7 shows that the recA mutation increases
Susceptibility of E. coli ∆lon and ∆sulA double mutant to LVFX
Figure 5
Susceptibility of E. coli ∆lon and ∆sulA double mutant
to LVFX. (A), (B), (C) and (D) show the results of spot
testing with agar plates containing 0 µg/ml, 0.0125 µg/ml,
0.025 µg/ml and 0.05 µg/ml LVFX, respectively.
Levels of LVFX-resistance of E. coli gyrA, ∆lon and gyrA, parC,
∆lon mutants
Figure 6
Levels of LVFX-resistance of E. coli gyrA, ∆lon and
gyrA, parC, ∆lon mutants. (A), (B), (C) and (D) show the
results of spot testing with the agar plates containing 0 µg/ml,
0.0125 µg/ml, 0.025 µg/ml, and 0.10 µg/ml LVFX, respec-
tively. Bacterial strains used are as follows: a, W3110 (lon+)
and CS5140 (∆lon); b, CS5086 (lon+) and CS5216 (∆lon); c,
CS5217 (lon+) and CS5218 (∆lon).