Previous Article | Next Article 
Journal of Bacteriology, January 2000, p. 264-271, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutational Analysis of a Role for Salicylic Acid in
Iron Metabolism of Mycobacterium smegmatis
Tadepalli
Adilakshmi,
Peter
D.
Ayling, and
Colin
Ratledge*
Department of Biological Sciences, University
of Hull, Hull HU6 7RX, United Kingdom
Received 29 June 1999/Accepted 26 October 1999
 |
ABSTRACT |
The role of salicylic acid in iron metabolism was examined in two
wild-type strains (mc2155 and NCIMB 8548) and three mutant
strains (mc21292 [lacking exochelin], SM3 [lacking
iron-dependent repressor protein IdeR] and S99 [a
salicylate-requiring auxotroph derived in this study]) of
Mycobacterium smegmatis. Synthesis of salicylate in SM3 was
derepressed even in the presence of iron, as was synthesis of the
siderophores exochelin, mycobactin, and carboxymycobactin. S99 was
dependent on salicylate for growth and failed to grow with the three
ferrisiderophores, suggesting that salicylate fulfills an additional
function(s) other than being a precursor of mycobactin and
carboxymycobactin. Salicylic acid at 100 µg/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only
under iron-limited conditions in the parent strain, mc2155,
and remained unaltered in SM3, suggesting an interaction between the
IdeR protein and salicylate. Thus, salicylate may also function as a
signal molecule for recognition of cellular iron status. Growth of all
strains and mutants with p-aminosalicylate (PAS) at 100 µg/ml increased salicylate accumulation between three- and eightfold
under both iron-limited and iron-sufficient growth conditions and
decreased mycobactin accumulation by 40 to 80% but increased
carboxymycobactin accumulation by 50 to 55%. Thus, although PAS
inhibited salicylate conversion to mycobactin, presumptively by
blocking salicylate AMP kinase, PAS also interferes with the additional
functions of salicylate, as its effect was heightened in S99 when the
salicylate concentration was minimal.
| |
INTRODUCTION |
Mycobacterium smegmatis,
a saprophytic mycobacterium, secretes two extracellular siderophores
(exochelin and carboxymycobactin) and has an intracellular siderophore
termed mycobactin (28, 30). De Voss et al. (8)
demonstrated the importance of the siderophores mycobactin and
carboxymycobactin in M. tuberculosis by showing that a
mutant incapable of producing these two siderophores failed to grow
within macrophages. Carboxymycobactin is structurally related to
mycobactin but is probably not derived from it (2). While
carboxymycobactin is the sole extracellular siderophore of pathogenic
mycobacteria (30), it is only a minor one in saprophytic mycobacteria, which use exochelin as the major siderophore for iron
acquisition (28). Salicylic acid is produced extracellularly by most mycobacteria in greatly increased quantities (up to 17 µg/ml)
when grown under iron-deficient conditions (28, 33) as
opposed to iron-sufficient conditions. Although salicylate is a
precursor of mycobactin (31), mycobactin could not spare the
requirement for salicylate in a salicylate-requiring auxotrophic mutant
of M. smegmatis (32), suggesting that salicylate
fulfills a second but unknown role in this bacterium.
Salicylate and its derivatives are important as analgesics,
antipyretics, anticoagulants, and anti-inflammatory drugs (15, 41), and in addition, salicylate is also important in plants, where it induces flowering and is involved in resistance to systemic diseases through multiple signal transduction pathways (14). The role of salicylate in iron metabolism in bacteria is much less
clearly defined. Salicylate appears to be synthesized by the members of
only four genera: Pseudomonas, Azospirillum,
Yersinia, and Mycobacterium. For the former two
genera, it has been suggested that salicylate acts as a siderophore in
its own right (21, 35, 37, 40), although this can only
happen in the absence of competing ions such as phosphate, which
quickly precipitate iron from ferrisalicylate and render the iron
inaccessible for uptake into cells (34). Thus, a role for
salicylate as a siderophore must be very limited and could not, in any
case, apply to pathogenic bacteria, which would encounter high
concentrations of phosphate in host tissues (1).
Salicylate also induces a multiple antibiotic resistance
(mar) operon in Escherichia coli (5):
it binds to the MarR (repressor) protein and thereby decreases its
affinity for operator sites containing the MarR recognition sequence in
vitro (18). Salicylate also inactivates EmrR, a MarR-like
repressor of an unrelated promoter involved in efflux of several
hydrophobic antibacterials in E. coli (38). The
elucidation of the sequence of the M. tuberculosis genome
has identified two proteins similar to MarA of E. coli (6), suggesting the operation of a mar system in
mycobacteria. Salicylate therefore acts as a regulator of metabolism in
a number of diverse systems, although its function in mycobacteria
remains unclear.
In the present study, the function of salicylic acid has been examined
in wild-type M. smegmatis mc2155 and mutant
strains SM3, mc21292, and S99. Some studies were also
performed with a second wild-type strain, NCIMB 8548. The results
confirm the central role of salicylate as the precursor of mycobactin
and carboxymycobactin and, in addition, point to an as yet unidentified
role(s) for it in the assimilation of iron.
| |
MATERIALS AND METHODS |
Strains and growth conditions.
The phenotypic
characteristics and sources of all of the strains used in the present
study are given in Table 1.
mc2155 was used as the principal wild-type strain. An
additional wild-type strain, NCIMB 8548, was used in some experiments.
The mutant SM3 was kindly supplied by Issar Smith, Public Health
Research Institute, New York, N.Y., and strain mc21292 was
kindly provided by William R. Jacobs, Jr., Howard Hughes Medical
Institute, Albert Einstein College of Medicine, Bronx, N.Y. All strains
were routinely grown in minimal medium treated for iron removal
(33) and containing the following (grams per liter):
KH2PO4, 5; glycerol, 10; asparagine, 5 (pH
7.6). Prior to inoculation, the medium was supplemented with the
following (micrograms per milliliter): Zn2+, 0.45;
Mn2+, 0.1; Mg2+, 40; Fe2+, 0.05 (for iron-deficient growth); Fe2+, 2.0 (for iron-sufficient
growth). Cultures (100 ml) were grown for 5 days at 37°C with
shaking; cell dry weights and exochelin, mycobactin, and
carboxymycobactin were estimated as previously described
(30). Salicylic acid and p-aminosalicylic acid
(PAS) were obtained from Sigma. The salicylic acid-requiring mutant S99
was isolated from wild-type strain mc2155 by
N-methyl-N'-nitro-N-nitrosoguanidine
mutagenesis (13). Transposon mutagenesis was performed by
using a temperature-sensitive system (T. Parish and N. G. Stoker,
personal communication).
Assay of salicylic acid.
Cells were harvested by
centrifugation. Culture supernatants were acidified with 10 M
H2SO4 to pH 1.5 to 2.0. The medium was filtered
and extracted three times with 0.5 volume of chloroform; the extract
was then washed with double-distilled water, and the solvent was
allowed to evaporate under reduced pressure. The residue was dissolved
in methanol-water (70:30, vol/vol)-3% (vol/vol) acetic acid and
analyzed by high-pressure liquid chromatography (HPLC) using a
Lichrosorb RP-18 reverse-phase column (250 by 3.2 mm) at room
temperature. Salicylic acid was eluted with a linear gradient of
methanol-acetic acid-water (50:0.1:50, by volume) to methanol-water
(70:30, vol/vol) running at 0.5 ml/min over 20 min and then holding at
the final concentration for a further 20 min. All solvents were
continuously degassed with helium. The eluate was continuously
monitored at 296 nm. The efficiency of extraction for all
concentrations of salicylic acid was 50 to 55%. Salicylic acid
concentration was determined from a standard curve generated by using
2.5 to 100 µg of salicylic acid; the detector response is linear
within these limits, and correction was applied for the extraction efficiency.
Separation of salicylic acid and PAS.
Salicylic acid and PAS
were extracted as described above from the supernatants of the cultures
grown in the presence of PAS and separated by HPLC at room temperature
using KH2PO4-H3PO4
(0.01 mol/liter, pH 2.3)-acetonitrile-methanol (70:25:5, by volume). The elution rate was 0.5 ml/min, and eluates were monitored by measuring A237 (see Fig. 2). Salicylic acid and
PAS concentrations were determined from the standard graphs generated
by measuring A237 and using 2.5 to 100 µg of
salicylic acid and PAS; the detector response is linear within these
limits for both compounds.
SDS-PAGE of membrane proteins of M. smegmatis.
Cell
envelope proteins were extracted from cultures grown under conditions
of iron deficiency or iron sufficiency after ultrasonication for 10 30-s periods with 15-s cooling intervals using a Dawe Soniprobe, type
7533A, as previously described (9). The proteins were analyzed by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE) (16).
| |
RESULTS AND DISCUSSION |
Isolation and characterization of a salicylic acid-requiring
mutant.
An attempt was made to isolate a salicylic acid-requiring
mutant by transposon mutagenesis using a temperature-sensitive system (T. Parish and N. G. Stoker, personal communication), but for unknown reasons, no such mutant was isolated, although around 25,000 colonies were screened and the system did produce other auxotrophs at a
frequency of 2.3 × 10
4. Thus,
N-methyl-N'-nitro-N-nitrosoguanidine
was used to mutagenize M. smegmatis mc2155 as
previously described (13). Over 20,000 colonies were screened by replica plating on solid medium containing 25 µg of salicylic acid per ml and a single salicylic acid-requiring mutant, designated S99, was found among 16 putative mutants.
Growth of S99 was directly proportional to the concentration of
salicylic acid added to the growth medium, and optimal growth
was
achieved with 100 µg of salicylic acid/ml (Fig.
1). A decline
in growth was noted with
higher concentrations of salicylic acid
under both low- and high-iron
conditions. However, the growth
of this mutant was only 60% of that
observed in the parent, even
in the presence of salicylic acid. The
growth of parent strain
mc
2155 was not affected by up to
100 µg of salicylic acid/ml under
iron-deficient or iron-sufficient
growth conditions. Salicylic
acid at 1,000 µg/ml, however, was lethal
to both of the strains,
irrespective of the iron status (Fig.
1). This
mutant produced
all of the siderophores (exochelin, mycobactin, and
carboxymycobactin)
when grown with salicylic acid (see Table
3),
suggesting that
the mutation had not affected the conversion of
salicylic acid
to mycobactin and carboxymycobactin.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 1.
Responses of wild-type M. smegmatis
mc2155 ( ) and salicylate-requiring mutant S99 ( ) to
salicylate under iron-insufficient ( ) and iron-sufficient
(---) conditions.
|
|
To characterize the auxotrophic nature of salicylate-requiring mutant
strain S99, the growth medium was supplemented at 5
µg/ml with
various siderophores that normally occur in wild-type
M. smegmatis. While exochelin and mycobactin failed to restore
the
growth of S99 under both low- and high-iron conditions, there
was
partial (50%) restoration of growth with carboxymycobactin
or citrate,
but only under iron-sufficient conditions and not
under iron-deficient
growth conditions (data not shown). A previous
study (
32)
prompted us to assess the influence of 2,3- and 2,4-dihydroxybenzoic
acids, which at 100 µg/ml restored the growth of this present
mutant,
but only under high-iron conditions (data not
shown).
Mutant strain S99 failed to grow with any of the siderophores, added as
ferric complexes, in the absence of salicylic acid
and grew only very
slightly when combinations of the siderophores
were used (Table
2). The growth of the mutant with
ferrisiderophores
as the sole source of iron was significantly enhanced
only when
salicylic acid was added. A combination of 2,3- or
2,4-dihydroxybenzoic
acid or citrate and ferrimycobactin (sole source
of iron) failed
to support the growth of S99 (data not shown). Parent
strain mc
2155 grew better with the individual
ferrisiderophores than it
did in iron-deficient medium, indicating that
the additional iron
in the form of ferrisiderophores contributed to the
extra growth
of the cells (Table
2).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Effects of mycobacterial ferrisiderophores as the sole
sources of iron on the growth of mutant strain S99 and parent strain
mc2155 in the presence and absence of
salicylic acida
|
|
The results in Table
2 confirm the earlier finding that mycobactin
could not replace or spare the salicylate requirement
of a
salicylate-requiring mutant (
32). In addition, the present
work shows that neither mycobactin nor carboxymycobactin, nor
exochelin, can substitute for salicylate, either individually
or in
combination. If salicylate acted simply as an extracellular
siderophore, as had been suggested with
Pseudomonas species
and
Azospirillum lipoferum (
21,
35,
37,
40), then
one of the
other mycobacterial siderophores should have been able to
substitute
for it, and if salicylate was needed only for mycobactin
and/or
carboxymycobactin synthesis, then either or both of these should
have been adequate substitutes. As this did not occur, we conclude
that
salicylate must fulfill a second function that does not involve
its
further metabolism, as no compound containing a salicylate
moiety,
other than mycobactin or carboxymycobactin, has been recognized
in
mycobacteria (
28,
31). The evidence that salicylate acts
as
a siderophore for the solubilization and uptake of Fe(III)
is, however,
extremely tenuous: in the presence of phosphate ions,
any ferric
salicylate is rapidly converted to the insoluble ferric
phosphate,
which is not transportable (
34). However, synthesis
of
salicylate is considerably increased during iron-deficient
growth, as
opposed to iron-sufficient growth. Moreover, this increased
synthesis
occurs as an early response to the onset of iron deprivation
(
26), thus firmly implying that its function, besides that
of
a mycobactin precursor, is connected with iron
metabolism.
Iron regulation in mycobacteria.
The production of salicylic
acid and the siderophores is negatively regulated by iron in the two
wild-type strains (Table 3). Dusserget et
al. (10) have shown that the synthesis of mycobactin and
exochelin is regulated by the IdeR protein when activated by iron and
that the synthesis of these siderophores is derepressed in mutant
strain SM3. We have extended these observations by showing that the
synthesis of salicylic acid and carboxymycobactin is also derepressed
in the IdeR-deficient mutant, suggesting that IdeR also mediates the
regulation of their respective biosyntheses by iron. However, the
pattern of regulation of salicylate synthesis differed from that of the
siderophores since the production of salicylate in this mutant under
high-iron conditions, unlike that of its parent strain
(mc2155), was twice as high as under low-iron conditions
(Table 3). In fact, the level of salicylic acid in the IdeR mutant was
10 times the concentration seen in the wild-type cultures grown under iron-deficient conditions. These results suggest that repression by
IdeR is probably not the sole control mechanism for the synthesis of
salicylic acid. In contrast, the siderophore levels under high-iron conditions were significantly lower than those found in iron-deficient cultures, suggesting that the derepression was only partial. In addition, the concentrations of exochelin and mycobactin were lower in
SM3 than in the wild-type strain grown under low-iron conditions, in
agreement with the findings of Dusserget et al. (10). The
concentration of carboxymycobactin was similar to that found in the
parent strain.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Production of siderophores and salicylic acid by
different strains of M. smegmatis in the presence and
absence of PAS at 100 µg/mla
|
|
These results suggest that IdeR does not account for all adaptive
responses to iron starvation and that additional mechanisms
control the
expression of the various components of the mycobacterial
iron uptake
machinery. In
E. coli, expression of
fepA,
encoding
the receptor for colicin B, which is necessary for ferric
enterochelin
uptake, has been shown to be partially derepressed in
fur mutants
while other ferric uptake genes are
constitutively expressed (
7),
suggesting that
fepA is under the control of an additional regulator.
It is
now clear that there are complex regulatory mechanisms for
iron
acquisition in some bacteria; for instance, multiple regulatory
elements, in addition to Fur, have been described in
Campylobacter jejuni (
39),
Vibrio
anguillarum,
P. putida, and
P. aeruginosa (
7). This view is further supported by the identification of
multiple iron-dependent regulators in the
M. tuberculosis
genome
(
6,
44).
Interestingly, salicylate synthesis was also considerably increased in
the exochelin-negative mutant mc
21292 under both
iron-sufficient and iron-deficient conditions
(Table
3), although
salicylate was at its highest concentration
in cells grown under
iron-deficient conditions. This mutant also
produced 55% more
carboxymycobactin than its parent, allowing
it to compensate for the
lack of exochelin, and this, in turn,
led to a decrease in the
mycobactin levels in the cells. The increased
production of
carboxymycobactin by mc
21292 also explains how it is able
to grow in the absence of exochelin
(see also reference
11). Mycobactin and carboxymycobactin are
derived
from the same, as yet unidentified, precursor. The high
concentration
of salicylate present in the mc
21292 mutant suggests that
the cells now have to rely entirely
on the secondary routes of iron
uptake in the absence of exochelin.
Higher concentrations of salicylate
may therefore be necessary
to ensure maximum synthesis of
carboxymycobactin, as well as optimal
assimilation of iron into the
cells.
PAS toxicity and resistance mechanism.
PAS is a potent
antitubercular drug (3, 43). While M. tuberculosis is much more sensitive to inhibition by PAS than is M. smegmatis, which can tolerate high concentrations of PAS,
its site of action remains unknown. We have now investigated the effect of PAS on the synthesis of siderophores and salicylic acid in M. smegmatis. All strains were grown with 100 µg of PAS/ml and without PAS, and the cultures were then analyzed for formation of the
siderophores. Salicylic acid was separable from PAS by HPLC (Fig.
2) and could therefore be quantified in
its presence.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 2.
Separation of salicylic acid (SA) and PAS by HPLC with
ethyl 4-hydroxybenzoic acid (EHBA) as the internal standard. Eluants
were monitored by measuring A237.
|
|
PAS at 100 µg/ml prevented the growth of mutant S99 on salicylic acid
under both low- and high-iron conditions (Table
3).
In contrast, the
growth of all of the other strains was only slightly
affected (25 to
35% inhibition) and a 3- to 10-fold salicylic
acid increase occurred
in all strains that were able to grow in
the presence of PAS,
irrespective of the iron status of the medium.
The production of
carboxymycobactin increased by 50 to 55% in
the two wild-type strains,
as well as in the mutants SM3 and mc
21292 in the presence
of PAS, whereas the mycobactin levels decreased
(40 to 80%) in all of
these cells under iron-deficient growth
conditions. PAS (1 µg/ml)
completely inhibited the growth of mutant
strain S99 under
iron-deficient growth conditions (Fig.
3). An
increase in the concentration of
salicylic acid in the medium
counteracted the toxic effects of PAS
partially, but only under
high-iron conditions. The present results
support and extend the
initial proposals that PAS is an antimetabolite
of salicylic acid
(
3,
29). The overproduction of salicylate
in
M. smegmatis in the presence of PAS (Table
3) indicates
that PAS probably
acts as an analog of salicylate and blocks the action
of salicylate
kinase, which forms salicyloyl-AMP from salicylate as the
first
step in mycobactin and carboxymycobactin synthesis
(
25). However,
this inhibition was not complete, as
indicated by the continued
synthesis of mycobactin and
carboxymycobactin under iron-restricted
conditions (Table
3), and
although mycobactin formation was decreased
by this action of PAS, the
synthesis of carboxymycobactin was
increased. Overall, however, there
was a diminished flux of salicylate
along this pathway to the common
precursor of mycobactin and carboxymycobactin.
Extremely high
concentration of salicylate accumulated in both
SM3 (IdeR mutant) and
mc
21292 (exochelin-deficient mutant) after their growth in
the presence
of PAS, reaching between about 5 and 8 mg/100 ml,
respectively
(Table
3). This increase could be attributed to the dual
effect
of the lack of repression of salicylate synthesis in both SM3
and exochelin-deficient strains and the probable inhibition of
salicylate kinase by PAS.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Influence of exogenous salicylate on the growth of S99
in the absence of PAS () and in the presence of 1 () or 5 ( )
µg of PAS per ml under iron-deficient () and iron-sufficient
(---) conditions.
|
|
The increased sensitivity of the salicylate auxotroph S99 to PAS also
suggests that PAS is an antagonist of salicylic acid.
With little
salicylate being available in this mutant and with
PAS gaining access
to the cell via an uptake system separate from
that of salicylate
(
3), PAS was clearly able to exert its inhibitory
action
without competition from salicylate. Even when an equal
amount of
salicylate was added with PAS in the medium, no growth
took place
(Table
3). However, when the concentration of PAS
was decreased to 1 µg/ml, growth could be restored partially by
adding salicylate at 100 µg/ml, but only under iron-sufficient
conditions (Fig.
3). These
results are explainable by salicylate
and PAS having separate uptake
systems and PAS being able to outcompete
salicylate in whatever
function salicylate normally fulfills.
The addition of mycobactin to a
previous salicylate auxotroph
treated with PAS did not restore its
growth (
3), again indicating
that salicylate has a role
beyond mycobactin synthesis. As a mycobactin-requiring
auxotroph of
M. smegmatis also retained this high sensitivity
to PAS,
even in the presence of mycobactin (
3), PAS could also
act
at additional sites other than the conversion of salicylate
to
mycobactin.
The much higher reported PAS sensitivity of pathogenic mycobacteria,
where 1.0 µg/ml is typically sufficient to cause inhibition
(
43), might possibly be due to the lower concentrations of
salicylate
synthesized by them.
M. bovis BCG grown under
iron-deficient conditions
accumulated only about 20% of the salicylate
seen with
M. smegmatis (
33), but this could also
have been due to increased uptake
of PAS via the
p-aminobenzoate permease (
3). Either case would
lead to a higher intracellular concentration of PAS and a higher
PAS-salicylate ratio in the cells, which is clearly crucial for
effective
inhibition.
Expression of the 29-kDa envelope protein.
The uptake of
ferrisiderophore complexes is mediated by specific proteins that occur
on the outer membrane surface of microbial cells (23). In
order to study the consequence of mutation on the expression of these
proteins, the envelope protein profile was analyzed in all of the
strains. The 29-kDa iron-regulated envelope protein described by Hall
et al. (12) and considered by Dover and Ratledge
(9) to be a possible exochelin-binding protein was found to
be constitutively expressed in the NCIMB wild-type strain, irrespective
of the iron status of the medium (see lanes 1 and 4 in Fig. 5C), in
accordance with Dover and Ratledge (9). The N-terminal
sequence of this protein shows 75% homology to that of a 29-kDa
protein of M. tuberculosis which has been reported to show
increased expression under iron-sufficient cells (4).
Surprisingly, all of the strains derived from mc2155 showed
increased expression of this 29-kDa protein under high-iron conditions,
with the single exception of the S99 mutant strain, in which diminished
synthesis was evident, irrespective of the iron status of the growth
medium (lanes 7 and 8 of Fig. 4).
View larger version (119K):
[in this window]
[in a new window]
|
FIG. 4.
SDS-PAGE profile of cell envelope proteins of M. smegmatis mutant SM3 (lanes 1 and 2), parent mc2155
(lanes 3 and 4), mutant mc21292 (lanes 5 and 6), and mutant
S99 (lanes 7 and 8) under iron-deficient and -sufficient conditions,
respectively. The arrow indicates the 29-kDa iron-regulated envelope
protein. Molecular masses (kilodaltons) are shown on the right.
|
|
To investigate whether the decrease in the 29-kDa protein was the
result of a pleiotropic effect of the mutation in strain
S99, or the
consequence of the high concentration (100 µg/ml)
of exogenous
salicylic acid required for its growth, strain SM3
and parental strain
mc
2155 were each grown in the presence of 25 and 100 µg
of salicylic
acid per ml under low- and high-iron conditions (Fig.
5A and B).
There was a marked decrease of
the 29-kDa protein in mc
2155 grown with 25 µg of
salicylic acid/ml and complete repression
of the protein with 100 µg
of salicylic acid/ml. However, the
effect of salicylic acid was seen
only under iron-deficient conditions,
leaving the 29-kDa protein
unaltered under iron-sufficient conditions.
There was no significant
change in this protein in SM3, the IdeR-deficient
mutant. The results
obtained with wild-type strain NCIMB 8548
(Fig.
5C) were similar to
those obtained with mc
2155.
View larger version (112128100K):
[in this window]
[in a new window]
|
FIG. 5.
SDS-PAGE profile of cell envelope proteins of M. smegmatis. The effect of exogenous salicylic acid on the 29-kDa
protein in the SM3 mutant (lanes 1 to 4) and the parent strain
mc2155 (lanes 5 to 8) is shown. Lanes: 1 and 5, low iron; 2 and 6, low iron plus salicylic acid; 3 and 7, high iron; 4 and 8, high
iron plus salicylic acid. Salicylic acid was added at 25 (A) and 100 (B) µg/ml. (C) Wild-type M. smegmatis NCIMB 8548 grown
with different concentrations of salicylic acid under iron-deficient
(lanes 1 to 3) and iron-sufficient (lanes 4 to 6) conditions. Lanes: 1 and 4, no salicylic acid; 2 and 5, 50 µg of salicylic acid/ml; 3 and
6, 100 µg of salicylic acid/ml. Lanes M contained marker proteins
whose molecular masses (kilodaltons) are shown beside the panels.
|
|
These results suggest a possible interaction between salicylic acid and
the regulatory protein IdeR to bring about repression
of the synthesis
of the 29-kDa protein. Surprisingly, PAS, like
salicylic acid, also
repressed the formation of this protein in
the wild-type
mc
2155 strain, and again, this effect was only seen under
iron-deficient
conditions (Fig.
6). The
combination of PAS and salicylic acid
led to a decrease in the 29-kDa
protein even under iron-sufficient
conditions.
View larger version (81K):
[in this window]
[in a new window]
|
FIG. 6.
SDS-PAGE profile of cell envelope proteins of parent
strain mc2155 grown with 100 µg of PAS ml1
under low- and high-iron conditions in the absence of salicylic acid
(lanes 1 and 2) and in the presence of salicylic acid (lanes 3 and 4).
Molecular mass marker (lane M) sizes are shown on the left in
kilodaltons.
|
|
Conclusions.
Our results lead us to suggest that salicylate
fulfills three distinct roles. First, salicylate acts as the precursor
of mycobactin and carboxymycobactin, and PAS clearly inhibits the first
reaction of this pathway, causing additional accumulation of
salicylate. Second, salicylate is probably involved in the
intracellular transfer of iron from the siderophores exochelin,
carboxymycobactin, or mycobactin following the reduction of Fe(III) to
Fe(II), which allows the iron to be desequestered (19).
Ferrosalicylate could then transfer the iron into bacterioferritin,
which acts as the cytoplasmic store of iron (28), and also
into apoproteins and porphyrins. If this second role were proved to be
correct, then this could explain the dependency of the salicylate
auxotroph S99 on salicylate for utilization of the iron from
ferrisiderophores (Table 2). Interestingly, when M. smegmatis was grown under iron-sufficient conditions, which is
when the concentration of bacterioferritin would be optimal, either
2,3- or 2,4-dihydroxybenzoic acid could restore the growth of the S99
auxotroph. Thus, as these two phenolic acids would serve to complex
Fe(II) similarly to salicylate, the loading of iron into
bacterioferritin might be accomplished by alternatives to salicylate.
Citrate, which also supported limited growth of the auxotroph under
iron-sufficient conditions, may also be capable of acting as an Fe(II)
transfer agent. This role of salicylate, as a means of moving and
transferring Fe(II) from molecule to molecule, may not be entirely
stringent, but as under iron-deficient conditions, the dihydroxybenzoic
acids and citrate do not substitute for salicylate, this suggests that
salicylate then fulfills a further and separate role under such conditions.
The third role for salicylate could be as a signal molecule to
recognize the iron status within the cell. Salicylate would
then be
expected to act together with the IdeR protein that is
involved in the
regulation of a number of genes associated with
iron metabolism
(
10). Support for this hypothesis comes from
the expression
of the 29-kDa envelope protein. Antibodies against
this protein
prevented iron uptake into whole cells in the presence
of
ferriexochelin (
12), suggesting that it is involved in iron
uptake as a putative exochelin receptor (
9). The N terminus
of this protein is distinctive (
24) and shows 75 to 90%
homology
to several proteins: N termini of a DNA-binding protein (HupB)
(
6), 28- and 29-kDa proteins from
M. tuberculosis
(
4), a
histone-like DNA-binding protein from
M. smegmatis (
17), and
a 40-kDa outer membrane protein of
M. smegmatis (
22). The repression
of the 29-kDa
protein by salicylate suggests a commonality with
salicylate binding to
the repressors MarR and EmrR of
E. coli and inhibition of
their function (
18,
38). As there are reports
of
mar-like systems in mycobacteria (
20), salicylic
acid could
also modulate gene expression in mycobacteria, perhaps by
interacting
with IdeR, and thereby serve as an iron signal
molecule.
Cloning and characterization of the putative salicylate synthetase
gene(s) from
M. tuberculosis are under way, and preliminary
studies indicate that
entD (
6) could be involved
in the formation
of salicylate in mycobacteria. However, it is possible
that both
entD and
trpE2 (
mbtI)
(
6,
25) are involved in the formation
of
salicylate.
| |
ACKNOWLEDGMENTS |
We thank the University of Hull for a Sir Brynmor Jones Research
studentship to T.A.
We thank Maureen Ewing for technical assistance and Neil Stoker and
Tanya Parish for generous hospitality in the Stoker laboratory and
assistance with the transposon mutagenesis system.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom. Phone: 44-1482-465243. Fax: 44-1482-465458. E-mail:
c.ratledge{at}biosci.hull.ac.uk.
| |
REFERENCES |
| 1.
|
Barclay, R., and P. R. Wheeler.
1989.
Metabolism of mycobacteria in tissues, p. 137-196.
In
C. Ratledge, J. L. Stanford, and J. M. Grange (ed.), The biology of the mycobacteria, vol. 3. Academic Press Ltd., London, United Kingdom.
|
| 2.
|
Barclay, R., and C. Ratledge.
1983.
Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium.
J. Bacteriol.
153:1138-1146[Abstract/Free Full Text].
|
| 3.
|
Brown, K. A., and C. Ratledge.
1975.
The effect of p-aminosalicylic acid on iron transport and assimilation in mycobacteria.
Biochim. Biophys. Acta
385:207-220[Medline].
|
| 4.
|
Calder, K. M., and M. A. Horwitz.
1998.
Identification of iron-regulated proteins of Mycobacterium tuberculosis and cloning of tandem genes encoding a low iron-induced protein and a metal transporting ATPase with similarities to two-component metal transport systems.
Microb. Pathog.
24:133-143[CrossRef][Medline].
|
| 5.
|
Cohen, S. P.,
S. B. Levy,
J. Foulds, and J. L. Rosner.
1993.
Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway.
J. Bacteriol.
175:7856-7862[Abstract/Free Full Text].
|
| 6.
|
Cole, S. T., et al.
1998.
Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature
393:537-544[CrossRef][Medline].
|
| 7.
|
Crossa, J. H.
1997.
Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria.
Microbiol. Mol. Biol. Rev.
61:319-336[Abstract].
|
| 8.
|
De Voss, J. J.,
K. Rutter,
B. G. Schroeder, and C. E. Barry, III.
1999.
Iron acquisition and metabolism by mycobacteria.
J. Bacteriol.
181:4443-4451[Free Full Text].
|
| 9.
|
Dover, L. G., and C. Ratledge.
1996.
Identification of 29 kDa protein in the envelope of Mycobacterium smegmatis as a putative ferri-exochelin receptor.
Microbiology
142:1521-1530[Abstract/Free Full Text].
|
| 10.
|
Dussurget, O.,
M. Rodriguez, and I. Smith.
1996.
An ideR mutant of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative stress response.
Mol. Microbiol.
22:535-544[CrossRef][Medline].
|
| 11.
|
Fiss, E. H.,
S. Yu, and W. R. Jacobs, Jr.
1994.
Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways.
Mol. Microbiol.
14:557-569[CrossRef][Medline].
|
| 12.
|
Hall, R. M.,
M. Sritharan,
A. J. M. Messenger, and C. Ratledge.
1987.
Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake.
J. Gen. Microbiol.
133:2107-2114[Abstract/Free Full Text].
|
| 13.
|
Holland, K. T., and C. Ratledge.
1971.
A procedure for selecting and isolating specific auxotrophic mutants of Mycobacterium smegmatis.
J. Gen. Microbiol.
66:115-118[Abstract/Free Full Text].
|
| 14.
|
Klessig, D. F., and J. Malamy.
1994.
The salicylic acid signal in plants.
Plant Mol. Biol.
26:1439-1458[CrossRef][Medline].
|
| 15.
|
Kopp, E., and S. Ghosh.
1994.
Inhibition of NF- B by sodium salicylate and aspirin.
Science
265:956-959[Abstract/Free Full Text].
|
| 16.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[CrossRef][Medline].
|
| 17.
|
Lee, B. H.,
B. Murugasu-Oei, and T. Dick.
1998.
Upregulation of a histone-like protein in dormant Mycobacterium smegmatis.
Mol. Gen. Genet.
260:475-479[CrossRef][Medline].
|
| 18.
|
Martin, R. G., and J. L. Rosner.
1997.
Fis, an accessorial factor for transcriptional activation of the mar (multiple antibiotic resistance) promoter of Escherichia coli in the presence of the activator MarA, SoxS, or Rob.
J. Bacteriol.
179:7410-7419[Abstract/Free Full Text].
|
| 19.
|
McCready, K. A., and C. Ratledge.
1979.
Ferrimycobactin reductase activity from Mycobacterium smegmatis.
J. Gen. Microbiol.
113:67-72.
|
| 20.
|
McDermott, P. F.,
D. G. White,
I. Podglajen,
M. N. A. Lekshun, and S. B. Levy.
1998.
Multidrug resistance following expression of the Escherichia coli marA gene in Mycobacterium smegmatis.
J. Bacteriol.
180:2995-2998[Abstract/Free Full Text].
|
| 21.
|
Meyer, J. M.,
P. Azelvandre, and C. Georges.
1992.
Iron metabolism in Pseudomonas: salicylic acid, a siderophore of Pseudomonas fluorescens CHA0.
Biofactors
4:23-27[Medline].
|
| 22.
|
Mukhopadhyay, S.,
D. Basu, and P. Chakrabarti.
1997.
Characterization of a porin from Mycobacterium smegmatis.
J. Bacteriol.
179:6205-6207[Abstract/Free Full Text].
|
| 23.
|
Neilands, J. B.
1982.
Microbial envelope proteins related to iron.
Annu. Rev. Microbiol.
36:285-309[CrossRef][Medline].
|
| 24.
|
Nixon, G.
1999.
Studies in the iron metabolism of mycobacteria. Ph.D. thesis.
University of Hull, Hull, United Kingdom.
|
| 25.
|
Quadri, L. E.,
J. Sello,
T. A. Keating,
P. H. Weinreb, and C. T. Walsh.
1998.
Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin.
Chem. Biol.
5:631-645[CrossRef][Medline].
|
| 26.
|
Ratledge, C.
1969.
The biosynthesis of salicylic acid in Mycobacterium smegmatis via the shikimic acid pathway.
Biochim. Biophys. Acta
192:148-150[Medline].
|
| 27.
|
Ratledge, C.
1982.
Nutrition, growth and metabolism, p. 186-212.
In
C. Ratledge, and J. L. Stanford (ed.), Biology of the mycobacteria, vol. 1. Academic Press Ltd., London, United Kingdom.
|
| 28.
|
Ratledge, C.
1999.
Iron metabolism, p. 260-286.
In
C. Ratledge, and J. Dale (ed.), Mycobacteria: molecular biology and virulence. Blackwell Science Publishers Ltd., Oxford, United Kingdom.
|
| 29.
|
Ratledge, C., and K. A. Brown.
1972.
Inhibition of mycobactin formation in Mycobacterium smegmatis by p-aminosalicylate. A new proposal for the mode of action of p-aminosalicylate.
Am. Rev. Respir. Dis.
106:774-776[Medline].
|
| 30.
|
Ratledge, C., and M. Ewing.
1996.
The occurrence of carboxymycobactin, the siderophore of pathogenic mycobacteria, as a second extracellular siderophore in Mycobacterium smegmatis.
Microbiology
142:2207-2212[Abstract/Free Full Text].
|
| 31.
|
Ratledge, C., and M. J. Hall.
1970.
Uptake of salicylic acid into mycobactin S by growing cells of Mycobacterium smegmatis.
FEBS Lett.
10:309-312[CrossRef][Medline].
|
| 32.
|
Ratledge, C., and M. J. Hall.
1972.
Isolation and properties of auxotrophic mutants of Mycobacterium smegmatis requiring either salicylic acid or mycobactin.
J. Gen. Microbiol.
72:143-150[Abstract/Free Full Text].
|
| 33.
|
Ratledge, C., and F. G. Winder.
1962.
The accumulation of salicylic acid by mycobacteria during growth on iron-deficient medium.
Biochem. J.
84:501-506[Medline].
|
| 34.
|
Ratledge, C.,
L. P. Macham,
K. A. Brown, and B. J. Marshall.
1974.
Iron transport in Mycobacterium smegmatis: a restricted role for salicylic acid in the extracellular environment.
Biochim. Biophys. Acta
372:39-51[Medline].
|
| 35.
|
Saxena, B.,
M. Modi, and V. V. Modi.
1986.
Isolation and characterization of siderophores from Azospirillum lipoferum D-2.
J. Gen. Microbiol.
132:2219-2224.
|
| 36.
|
Snapper, S. B.,
R. E. Melton,
S. Mustafa,
T. Kieser, and W. R. Jacobs, Jr.
1990.
Isolation and characterisation of efficient plasmid transformation mutants of Mycobacterium smegmatis.
Mol. Microbiol.
4:1911-1919[Medline].
|
| 37.
|
Sokol, P. A.,
C. J. Lewis, and J. J. Dennis.
1992.
Isolation of a novel siderophore from Pseudomonas cepacia.
J. Med. Microbiol.
36:184-189[Abstract/Free Full Text].
|
| 38.
|
Sulavik, M. C.,
L. F. Gambino, and P. F. Miller.
1995.
The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: proto-typic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds.
Mol. Med.
1:436-446[Medline].
|
| 39.
|
Van Vliet, A. H. M.,
K. G. Wooldridge, and J. M. Ketley.
1998.
Iron-responsive gene regulation in a Campylobacter jejuni fur mutant.
J. Bacteriol.
180:5291-5298[Abstract/Free Full Text].
|
| 40.
|
Visca, P.,
A. Ciervo,
V. Sanfilippo, and N. Orsi.
1993.
Iron-regulated salicylate synthesis by Pseudomonas spp.
J. Gen. Microbiol.
139:1995-2001[Abstract/Free Full Text].
|
| 41.
|
Weismann, G.
1991.
Aspirin.
Sci. Am.
264:58-64[Medline].
|
| 42.
|
Wheeler, P. R., and C. Ratledge.
1994.
Metabolism of Mycobacterium tuberculosis, p. 353-385.
In
B. R. Bloom (ed.), Tuberculosis: pathogenesis, protection, and control. American Society for Microbiology, Washington, D.C.
|
| 43.
|
Winder, F. G.
1964.
The antibacterial action of streptomycin, isoniazid and PAS, p. 111-149.
In
V. C. Barry (ed.), Chemotherapy of tuberculosis. Butterworth & Co. Ltd., London, United Kingdom.
|
| 44.
|
Wong, D. K.,
B.-Y. Lee,
M. A. Horwitz, and B. W. Gibson.
1999.
Identification of Fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry.
Infect. Immun.
67:327-336[Abstract/Free Full Text].
|
Journal of Bacteriology, January 2000, p. 264-271, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Dellagi, A., Segond, D., Rigault, M., Fagard, M., Simon, C., Saindrenan, P., Expert, D.
(2009). Microbial Siderophores Exert a Subtle Role in Arabidopsis during Infection by Manipulating the Immune Response and the Iron Status. Plant Physiol.
150: 1687-1696
[Abstract]
[Full Text]
-
Miethke, M., Marahiel, M. A.
(2007). Siderophore-Based Iron Acquisition and Pathogen Control. Microbiol. Mol. Biol. Rev.
71: 413-451
[Abstract]
[Full Text]
-
Miranda-CasoLuengo, R., Duffy, P. S., O'Connell, E. P., Graham, B. J., Mangan, M. W., Prescott, J. F., Meijer, W. G.
(2005). The Iron-Regulated iupABC Operon Is Required for Saprophytic Growth of the Intracellular Pathogen Rhodococcus equi at Low Iron Concentrations. J. Bacteriol.
187: 3438-3444
[Abstract]
[Full Text]
-
Gaille, C., Kast, P., Haas, D.
(2002). Salicylate Biosynthesis in Pseudomonas aeruginosa. PURIFICATION AND CHARACTERIZATION OF PchB, A NOVEL BIFUNCTIONAL ENZYME DISPLAYING ISOCHORISMATE PYRUVATE-LYASE AND CHORISMATE MUTASE ACTIVITIES. J. Biol. Chem.
277: 21768-21775
[Abstract]
[Full Text]
-
Adilakshmi, T., Ayling, P. D., Ratledge, C.
(2000). Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis. Microbiology
146: 633-638
[Abstract]
[Full Text]
Top
Abstract
Introduction
