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Applied and Environmental Microbiology, February 1999, p. 822-827, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Poly-3-Hydroxybutyrate in Legionella
pneumophila, an Energy Source for Survival in Low-Nutrient
Environments
Brian W.
James,*
W.
Stuart
Mauchline,
P. Julian
Dennis,
C. William
Keevil, and
Robin
Wait
Centre for Applied Microbiology and Research,
Salisbury, Wiltshire SP4 0JG, United Kingdom
Received 10 August 1998/Accepted 16 November 1998
 |
ABSTRACT |
Chloroform-soluble material was extracted from two strains of
L. pneumophila serogroup 1 following growth in continuous
culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas
chromatography-mass spectrometry. PHB yields of up to 16% of cell dry
weight were extracted from culture samples. The PHB was located in
electron-dense intracellular inclusions, which fluoresced bright yellow
when stained with the lipophilic dye Nile red. A Nile red
spectrofluorometric assay provided a more accurate and reliable
determination of the PHB content. PHB accumulation increased threefold
during iron-limited culture and was inversely related to the
concentration of iron metabolized. Chemostat-grown cells survived in a
culturable state for at least 600 days when incubated at 24°C in a
low-nutrient tap water environment. Nile red spectrofluorometry and
flow cytometry demonstrated that PHB reserves were utilized during
starvation. PHB utilization, as revealed by the decline in mean
cellular fluorescence and cell complexity, correlated with loss of
culturability. Fluorescence microscopy provided visual evidence of PHB
utilization, with a marked reduction in the number of Nile red-stained
granules during starvation. Heat shock treatment failed to resuscitate
nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of
PHB, which promote its long-term survival under conditions of starvation.
| |
INTRODUCTION |
Legionella species are
natural freshwater inhabitants and readily colonize artificial aquatic
environments, such as cooling towers and potable-water systems (9,
18, 22, 34). The environmental persistence of legionellae is
aided by their ability to adapt to a variety of different ecological
niches, either as intracellular parasites of amebae, as free-living
members of complex biofilm communities, or as planktonic cells
(19, 40, 47, 49).
Aquatic amebae play a central role in Legionella ecology by
supporting intracellular multiplication and providing protection under
suboptimal growth conditions (4, 6, 29, 30). Outside the
amebal host, legionellae encounter stressful environmental conditions,
such as limited nutrient availability (27, 28). In vitro
studies have demonstrated that Legionella pneumophila can
adapt to starvation conditions and survive in a culturable state for
prolonged periods without growth (42, 52). Intra-amebal growth is believed to promote this extracellular survival by inducing a
stress-resistant phenotype, characterized by altered morphology and
envelope composition and increased resistance to antimicrobial agents
(5, 7, 8).
Intracellular energy reserves, such as poly-3-hydroxybutyrate (PHB),
may also promote environmental persistence. PHB is a homopolymer of
3-hydroxybutyric acid, which some bacteria accumulate during unbalanced
growth to provide an endogenous source of carbon and energy (13,
45). Indirect evidence for the occurrence of PHB in L. pneumophila has been provided by Fourier transform infrared
spectroscopy of whole cells (25) and by pyrolysis mass spectrometry (MS) (51). We have demonstrated the ability of Legionella species to metabolize 3-hydroxybutyric acid and
provided preliminary chemical evidence for the presence of PHB in
chemostat cultures of L. pneumophila (31, 32).
However, the material was not rigorously characterized and its
physiological significance has not been investigated.
In this paper we describe the isolation and purification of PHB-like
material from two strains of L. pneumophila and its
characterization by nuclear magnetic resonance (NMR) spectroscopy and
gas chromatography (GC)-MS. We have investigated the physiology of PHB
formation under iron-limited and -replete conditions and have
demonstrated a relationship between PHB content and the survival of
L. pneumophila in low-nutrient environments.
| |
MATERIALS AND METHODS |
Strains and culture.
Two strains of L. pneumophila serogroup 1 subgroup Pontiac, an environmental strain
(74/81) and a clinical isolate (Corby), were grown in tyrosine-limited
chemostat culture in ACES
[N-(2-acetamido)-2-aminoethanesulfonic acid]-buffered
chemically defined (ABCD) medium as described previously (31). Strain Corby was also grown under iron limitation, as detailed by James et al. (28). Steady-state culture samples were harvested by centrifugation and washed with deionized water before analysis.
PHB extraction and quantitation.
PHB was extracted and
purified by a modification of the method of Findlay and White
(21). In brief, 0.5 g of lyophilized washed biomass was
extracted with boiling chloroform (60 ml) in a Soxtec apparatus
(Tekator Ltd.) for up to 8 h. After being cooled, the extract was
rotary evaporated and purified to a white membrane-like solid by
sequential washing with ice-cold ethanol and cold diethyl ether. The
purified material was dehydrated to crotonic acid and quantitated
spectrophotometrically, as described by Slepecky and Law
(43). A standard curve was prepared with authentic PHB from an Alcaligenes sp. (Sigma-Aldrich Co. Ltd.).
NMR spectroscopy.
For 13C NMR, 30 mg of purified
material was dissolved in 4 ml of deuteriochloroform in a
10-mm-diameter NMR tube. Proton-decoupled NMR spectra were obtained
with a Varian FT80 Fourier transform spectrometer at ambient
temperature and a field strength of 20 MHz. Fully coupled spectra were
recorded by switching the decoupler off during acquisition and back on
during the delay period. Chemical shifts were expressed relative to
tetramethylsilane at 0 ppm, using deuteriochloroform as the secondary
reference. 1H NMR spectra were recorded at 30°C with a
Varian Unity 500-MHz spectrometer. Approximately 3 mg of sample was
dissolved in 0.8 ml of deuteriochloroform in a 5-mm-diameter NMR tube.
The spectral width was 5,000, and 19,776 data points were collected.
Spectra were processed with the manufacturer's software with a
gaussian apodization function. The spectra were referenced to
tetramethylsilane at 0 ppm via residual chloroform at 7.25 ppm.
Methanolysis and GC-MS.
A portion of the purified material
(0.5 mg) was methanolyzed (1.7 ml of methanol plus 0.2 ml of
concentrated HCl; 4 h at 100°C), and the resulting
methyl-3-hydroxybutyric acid was recovered by chloroform extraction.
GC-MS was performed with a Kratos MS80 spectrometer interfaced to a
Carlo-Erba 5160 chromatograph fitted with a 25 m-by-0.2-mm BP-1
fused-silica column (SGE Ltd., Milton Keynes, United Kingdom). Helium
was used as the carrier gas, and samples were introduced by split
injection (split ratio, 1:30) at an injector temperature of 250°C.
The column was temperature programmed from 50 to 100°C at 5°C
min
1 and then to 200°C at 20°C min1.
Electron ionization (EI) spectra were recorded at an ionization energy
of 70 eV. Chemical ionization (CI) mass spectra were obtained with
isobutane as the reagent gas.
PHB measurement by Nile red spectrofluorometry.
The PHB
contents of intact cells were measured by the Nile red
(9-diethylamino-5H-benzo [
]phenoxazine-5-one;
Sigma-Aldrich Co. Ltd.) spectrofluorometric assay of Degelau et al.
(15) with modifications. Fluorescence was measured with a
Perkin-Elmer model LS-5B luminescence spectrometer. The assay
conditions were optimized by performing a number of validation
experiments (data not shown). For sample analysis, formalin-treated
cells (1% [vol/vol] formaldehyde for 30 min) were washed,
resuspended in deionized water to a standard optical density at 540 nm
(OD540) of 0.5, and stained with 1 µl of Nile red (25 mM
in DMSO) ml1 for 60 min at 25°C. The instrument was
autozeroed against a sample of unstained bacteria, and fluorescence was
recorded at excitation and emission wavelengths of 545 and 600 nm,
respectively, with integration over a 4-s response time. The
temperature was maintained at 25°C by a thermostatically controlled
cuvette holder.
Survival of L. pneumophila in tap water.
Bacteria from a tyrosine-limited chemostat culture were washed three
times in deionized water and resuspended in filter-sterilized tap water
at a density of approximately 109 CFU ml1.
Duplicate suspensions were incubated in the dark at 24°C with gentle
agitation (150 rpm). Culturability on buffered charcoal-yeast extract
(BCYE) agar and the OD540 were determined at intervals as
described previously (28). The total cell count was
determined by phase-contrast microscopy with a Thoma counting chamber
and a Leitz Dialux 20 microscope. The PHB contents of samples were monitored by the Nile red spectrofluorometric assay.
Flow cytometry.
Samples of the starved population were
stained with Nile red and analyzed with a Becton Dickinson
Immunocytometry Systems benchtop flow cytometer (FACScan) operated via
a Macintosh computer system. Real-time data acquisition and analysis
was controlled by CellQuest software, while FACSComp software was
responsible for daily setup and quality control. Calibration was
performed with CaliBRITE beads (Becton Dickinson Immunocytometry
Systems). The results for each sample were based on the analysis of
10,000 events.
Microscopy.
Dry smears of formalin-treated cells were
stained with Nile red (25 mM Nile red in DMSO, diluted 1/500 in sterile
deionized water) for 30 min, rinsed, and examined by epifluorescence
microscopy (Nikon). Cell morphology was monitored by differential
interference contrast microscopy, as described previously
(28).
Heat shock treatment of starved cells.
Samples of starved
cells were incubated at 45°C for 60 min. At 10-min intervals during
heating, 1-ml aliquots were removed and plated on BCYE agar to
determine culturability. After 60 min of heat treatment, the suspension
was incubated at 24°C for 24 h before being resampled and plated
on BCYE agar.
| |
RESULTS |
PHB accumulation by strain 74/81.
A water-insoluble white
membrane-like material was purified from hot-chloroform extracts of
strain 74/81. Its 1H decoupled 13C NMR spectrum
comprised four resonances at 169.03, 67.50, 40.69, and 19.65 ppm and
was identical to the spectrum of authentic PHB from an
Alcaligenes sp. In the fully coupled spectrum, the last three signals were split into a doublet, a triplet, and a quartet, respectively, confirming that they originated from methine, methylene, and methyl carbon atoms; the singlet at 169.03 ppm was attributed to a
carbonyl resonance. The chemical shifts and values of the coupling
constants (1JCH) were consistent
with these assignments (Table 1). In the 13C NMR spectrum of free 3-hydroxybutyric acid, the
carboxyl carbon was, as expected, more deshielded than the ester-linked
carbonyl of the polymer and resonated 7 ppm downfield. The hydroxylated carbon, being less deshielded in the free acid, was shifted 3 ppm
upfield.
A portion of the purified material was depolymerized with methanolic
HCl and analyzed by GC-MS. In the EI mass spectrum, the
molecular ion
at
m/
z 118 was extremely weak and was accompanied
by M + 1 and M
1 ions. Facile elimination of water produced
an ion
at
m/
z 100, and loss of a methoxy group yielded
m/
z 87.
The ion at
m/
z 74 originated via a
McLafferty rearrangement, due
to cleavage between C-2 and C-3, with
hydrogen transfer from carbon
4 to the charged fragment
(
33). The presence of a hydroxyl group
on carbon 3 was
suggested by a characteristic fragment at
m/
z 103 (cleavage
alpha to the hydroxylated carbon). The molecular
weight was confirmed
by the observation of a protonated molecule
at
m/
z 119 in
the CI spectrum, which eliminated water and methanol,
giving fragments
at
m/
z 101 and 87. Identical spectra and chromatographic
data were obtained from a methanolysate of authentic PHB. Several
minor
components (<2% of the intensity of the methyl-3-hydroxybutyrate
signal) were present in the methanolysate of
L. pneumophila
PHB.
CI MS of the two most significant of these produced spectra
characterized
by protonated molecules at
m/
z 133 and
abundant dehydration fragments
(
m/
z 115), enabling their
tentative identification as the methyl
esters of isomeric hydroxy
valerates.
PHB accumulation by strain Corby.
During studies with strain
Corby, electron-dense inclusions were observed in electron micrographs
of cells grown in iron-limited and -replete chemostat cultures.
Fluorescence microscopy after staining with Nile red revealed
yellow-fluorescing intracellular inclusions. Cells normally contained
between one and three granules (Fig. 1a).
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FIG. 1.
Fluorescence microscopy of L. pneumophila
stained with Nile red. (a) Tyrosine-limited cells containing multiple
yellow-fluorescent intracellular inclusions; (b) cells after 500 days
of starvation, illustrating a marked decrease in granule content.
|
|
Proton NMR spectra of purified chloroform extracts from iron-replete
cultures displayed three clusters of resonances. The
chemical-shift
values, multiplicities, and coupling constants
(
3JH,H) of these signals were
consistent with their identification
as methyl, methylene, and methine
protons (Table
2). The spectra
of
extracts from iron-limited cells were comparable (data not
shown).
Low PHB yields, 2 to 5% of cell dry weight, were recovered from
iron-limited and -replete cells by hot-chloroform extraction.
Additional experiments demonstrated that the efficiency of the
chloroform extraction was relatively poor, recovering less than
50% of
cellular PHB during a single extraction cycle. Improved
recovery of
cellular PHB was achieved by performing multiple extraction
cycles.
Yields of up to 16% of cell dry weight were achieved after
three
extraction
cycles.
A spectrofluorometric assay based on the lipophilic dye Nile red was
used as a more accurate and less hazardous procedure
for PHB
determination. PHB content, as determined by exhaustive
chloroform
extraction and spectrophotometric measurement, was
linearly correlated
to Nile red fluorescence (
R2 = 0.96). This
relationship (described by the equation
y = 1.0114
x + 9.1039, where
y and
x represent sample
fluorescence and PHB
content, respectively) provided the basis for a
quantitative assay
to determine the PHB contents of cell
samples.
Spectrofluorometric analysis of nine separate iron-limited and -replete
samples demonstrated a threefold increase in PHB accumulation
under
iron-limited conditions, with mean PHB yields of 12 and
4% of cell dry
weight for iron-limited and -replete cultures,
respectively. The PHB
contents of iron-limited cultures ranged
between 6 and 18% of cell dry
weight. These iron-limited cultures
were grown with different
concentrations of iron. Additional analysis
revealed that the PHB yield
was enhanced in cultures grown at
lower concentrations of iron, with an
inverse linear relationship
between the concentration of iron
metabolized by iron-limited
cultures and PHB content
(
R2 = 0.95).
Survival of L. pneumophila.
L. pneumophila Corby
survived in a culturable state in filter-sterilized tap water for at
least 600 days (Fig. 2). Culturability declined rapidly during the first week, with the number of CFU decreasing by 30%. This initial phase was followed by a more gradual decline in culturability, with approximately 25% of the population still culturable after 280 days and 10% of the population culture positive after 380 days. Spectrofluorometric analysis of Nile red-stained samples demonstrated a decrease in fluorescence (Fig. 2).
As with culturability, the most rapid decline in fluorescence occurred
during the first week of starvation (33% decrease), after which there
was a gradual decline in the rate of PHB utilization until the PHB was
depleted. A progressive decline in the OD of the suspension, which
mirrored the decrease in fluorescence, was also observed (data not
shown). The changes in culturability and turbidity were not accompanied
by a comparable decrease in the total cell count, which remained
relatively constant after an initial decline of 20% during the first
week (Fig. 2).
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FIG. 2.
Survival of L. pneumophila in sterile tap
water.  , Culturability on BCYE agar;  , Nile red fluorescence;
 , total cell count.
|
|
Figure
3 illustrates how the relationship
between culturability and fluorescence changed with time. Both
culturability and
fluorescence declined markedly during the first week,
after which
there was a marked reduction in the rate at which both
parameters
changed. Over the next 50 days, fluorescence decreased by
40%
while culturability only declined by approximately 10% (Fig.
3).
After 400 days of starvation, no further decline in fluorescence
was
detected while culturability continued to decline independently.
A
repeat experiment performed with a different culture sample
of the same
strain produced similar results.
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FIG. 3.
Relationship between culturability and Nile red
fluorescence during starvation. Days zero to 5 (a), 6 to 56 (b), and 57 to 600 (c) following incubation in tap water are shown. Fluorescence
and culturability data from Fig. 2 were expressed as percentages of
their respective values at day zero to illustrate the relationship
between PHB utilization and loss of culturability during starvation.
|
|
Flow cytometry of Nile red-stained samples confirmed a decrease in
cellular fluorescence during starvation. At day 0, the
fluorescence
intensities of 98% of the cells were greater than
10 (within the gated
region M1 [Fig.
4a]). However, after
490
days, the fluorescence intensities of approximately 90% of the
population had declined to the background level of the unstained
sample
(Fig.
4b). There was a gradual decline in cellular fluorescence
during
starvation, in close agreement with the fluorometric data
(data not
shown). A linear relationship was detected between the
decrease in
culturability and mean cellular fluorescence (
R2 = 0.96) when both were expressed as percentages of their respective
values at day zero (data not shown).
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FIG. 4.
Changes in cellular fluorescence and population
distribution during starvation as detected by flow cytometry. The
histogram plots (a and b) illustrate the fluorescence intensities
recorded at 580 ± 25 nm for samples collected at day zero (a) and
after 490 days of starvation (b). The dot plots (c and d) illustrate
the relative cell complexities and size distributions (side scatter is
representative of relative complexity and granularity; forward scatter
is representative of relative cell length) for samples collected at day
zero (c) and day 490 (d). All plots are based on the analysis of 10,000 events.
|
|
Flow cytometry also revealed changes in cell complexity and size during
starvation. Initially, a broad distribution of cell
complexity and size
was observed (Fig.
4c). After 490 days of
starvation, the mean cell
complexity (
y-mean) and mean cell length
(
x-mean)
were reduced by 80 and 30%, respectively (Fig.
4d). This
trend was
confirmed by analysis of the gated region R2, which
represents small
cells with low internal complexity. The percentage
of cells in this
region increased from 7% at day zero to 86% after
490 days of
starvation (Fig.
4c and d). A linear relationship
was also observed
between the decline in culturability and mean
cell complexity
(
R2 = 0.97 [data not shown]).
Microscopic examination of the starved cells revealed changes in
morphology. At day zero the majority of cells contained between
one and
three brightly fluorescing intracellular granules (Fig.
1a). After 500 days of starvation, a marked decrease in granule
content was observed,
with many cells devoid of granules (Fig.
1b). Differential interference
contrast microscopy also revealed
some alterations in cellular
morphology in response to starvation.
At day zero, most cells appeared
to be intact, but after 600 days
of starvation much cell debris was
observed (data not
shown).
A heat shock experiment was performed after 600 days of starvation to
investigate whether nonculturable cells could be resuscitated.
Heating
cells to 45°C for up to 60 min, prior to plating them
on BCYE agar,
failed to increase culturability. Indeed, a 30%
decline in recovery
was observed after 60 min of heat shock. The
suspension was also
incubated at 24°C after heat treatment, and
culturability was
monitored after 24 h. Again, no increase in
culturability was
detected.
| |
DISCUSSION |
PHB formation by L. pneumophila.
We previously reported
the presence of electron-dense cytoplasmic inclusions in chemostat
cultures of strain 74/81 grown at different temperatures and provided
preliminary chemical evidence for the presence of PHB (31).
In the current study, a white solid was purified from hot-chloroform
extracts of strain 74/81. Carbon 13 NMR spectroscopy provided strong
evidence that this material is a polymer of 3-hydroxybutyric acid. The
chemical shifts and values of the coupling constants were in close
agreement with published solution values and with those obtained by
cross-polarization magic-angle spinning NMR and in vivo NMR experiments
(2, 3, 16, 17). Material purified from strain Corby also
yielded 1H NMR spectra with coupling constants and chemical
shifts consistent with PHB (17, 38). Taken together with
GC-MS analysis, these data establish the structure of the purified
chloroform-extractable material as that of PHB.
Intracellular granules were observed in electron micrographs for
iron-limited and -replete samples, in agreement with previous
reports
of granule formation by
L. pneumophila (
11,
31,
39).
These inclusions fluoresced bright yellow after being
stained
with Nile red, confirming their lipid nature. Nile red is a
lipophilic
stain which produces yellow-gold fluorescence in hydrophobic
environments,
and it has been used extensively for the detection of
neutral
and polar lipid inclusions in eukaryotic cell types (
10,
23,
24). Nile blue A, a basic oxazime dye containing traces of
the
oxidized product Nile red, has been used to visualize PHB granules
in
Bacillus megaterium and
Azobacter chroococcum
(
35).
The efficiency of hot-chloroform extraction was relatively poor, and
multiple extraction cycles were required to obtain a
more accurate
quantitation of cellular PHB. By performing multiple
extraction cycles,
we demonstrated that
L. pneumophila can accumulate
more
significant PHB reserves than was previously thought (
31).
Poor PHB recovery by chloroform extraction has been reported previously
for studies with
Alcaligenes eutrophus (
37).
A Nile red spectrofluorometric assay was found to be a more accurate
and reliable procedure for determining the PHB content
of intact cells,
in agreement with the observations of Degelau
et al. (
15).
PHB accumulation was enhanced during iron-limited
growth, consistent
with the physiology of PHB formation, which
is promoted during
unbalanced growth when an essential nutrient
other than the carbon and
energy source is limiting (
13,
14,
45). Furthermore, the PHB
content of iron-limited cells was
inversely related to the
concentration of iron metabolized. This
reflects the increased
concentration of excess carbon source which
becomes available at lower
concentrations of iron and can be channelled
into PHB
formation.
PHB synthesis by
L. pneumophila is likely to proceed via
pyruvate and acetyl-coenzyme A (CoA). Acetyl-CoA acyl transferase,
CoA-SH, and NADH are key regulatory elements in the PHB biosynthetic
pathway. During balanced growth, CoA-SH levels are high and polymer
synthesis is inhibited. However, when growth is limited by an
essential
nutrient other than the carbon and energy source, the
NADH
concentration increases, resulting in inhibition of early
enzymes of
the tricarboxylic acid cycle, namely, citrate synthase
and isocitrate
dehydrogenase. This leads to an accumulation of
acetyl-CoA, which
relieves the inhibition exerted by CoA-SH, leading
to 3-hydroxybutyric
acid formation (
13,
45). Therefore, excess
metabolic
intermediates are channelled into PHB formation in a
reductive process,
which also regenerates reduced
cofactors.
PHB and survival of L. pneumophila in a low-nutrient
environment.
The present study demonstrates that L. pneumophila can maintain a vegetative state for prolonged periods
(at least 600 days) when incubated in a low-nutrient environment. This
is consistent with previous studies reporting the survival of L. pneumophila under starvation conditions (26, 42, 50,
52).
Nile red spectrofluorometry and flow cytometry demonstrated that
intracellular PHB reserves were utilized during starvation.
This was
also demonstrated by a decline in the mean cell complexity
and culture
turbidity, which represented the decrease in cell
refractivity due to
the loss of electron-dense intracellular granules
(
44).
Fluorescence microscopy provided visual evidence of PHB
utilization,
with a marked reduction in the granule content of
the cells during
starvation.
The response of the culture to starvation changed with time. The marked
decline in fluorescence during the first week probably
represents
expenditure of carbon and energy reserves associated
with adapting to a
low-nutrient environment. The associated loss
of culturability probably
represents those cells that were unable
to adapt to the new
environment. The subsequent survival stage
was characterized by a
marked reduction in the rate of PHB utilization,
indicating that the
metabolic activity of the cells had become
more closely coupled to
their energy requirement for maintenance
of a vegetative state. Little
loss of culturability occurred during
this period, indicating
adaptation to starvation conditions, with
PHB promoting survival. After
prolonged starvation, changes in
culturability became independent of
PHB utilization. It is possible
that extracellular nutrients released
by cell death and lysis
were contributing to survival at this stage.
Alternatively, the
spectrofluorometric assay may not have been
sufficiently sensitive
to detect very low rates of residual PHB
utilization associated
with a very low metabolic activity of the
surviving cells. Consistent
with the latter possibility, a linear
relationship was demonstrated
between the decreases in mean cellular
fluorescence and culturability
(both expressed as percentages),
confirming that PHB utilization
correlated with loss of culturability.
Therefore, this study demonstrates
that PHB is an important energy
reserve, which supports the long-term
survival of
L. pneumophila in a culturable state in a low-nutrient
environment.
In the natural habitat, the survival of
L. pneumophila will
be influenced by other parameters, such as temperature and
changes in
nutrient availability, which may affect the rates of
PHB accumulation
and
utilization.
Several studies have noted the formation of inclusions resembling PHB
granules by
L. pneumophila during intracellular growth
in
aquatic amebae (
1,
20,
41,
48). Restricted availability
of
essential nutrients, such as iron, is likely to promote PHB
formation
by
L. pneumophila inside the amebal host. Indeed, this
study
has demonstrated that PHB accumulation is promoted during
iron-limited
growth in vitro. Therefore, as well as providing
protection and
supporting proliferation in hostile environments,
amebae may also
contribute to the environmental persistence of
legionellae by inducing
a PHB-rich phenotype, which is physiologically
more prepared for
extracellular survival in low-nutrient
environments.
Although this study demonstrates that PHB depletion correlates with
loss of culturability, it is possible that these cells
entered a
viable-but-nonculturable (VBNC) state.
L. pneumophila has
been reported to enter a dormant state when exposed to low-nutrient
environments (
26,
36). Colbourne and Dennis (
12)
suggested
that dormant
L. pneumophila cells were
resuscitated by heat shock
treatment. However, in the present study
comparable heat shock
treatment failed to revive potentially
nonculturable cells. A
recent study by Steinert and colleagues
(
46), using both acridine
orange staining and 16S rRNA
hybridization, demonstrated that
L. pneumophila entered a
viable-but-nonculturable state following
starvation, from which it
could be resuscitated by the addition
of
Acanthamoeba
castellanii. Therefore, in the present study it
is possible that
the appropriate physicochemical stimuli to induce
resuscitation were
not
applied.
In conclusion, this study demonstrates that
L. pneumophila
accumulates considerable intracellular reserves of PHB, which support
its long-term survival in a culturable state under starvation
conditions. This endogenous energy reserve is likely to play an
important role in promoting the persistence of legionellae in
stressful
low-nutrient environments outside the amebal
host.
| |
ACKNOWLEDGMENTS |
We thank B. Mulloy, National Institute for Biological Standards
and Control, United Kingdom, for performing proton NMR analysis.
This work was supported by project grant JR 121/2790 from the
Department of Health, United Kingdom.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre for
Applied Microbiology and Research, Salisbury, Wiltshire SP4 0JG, United Kingdom. Phone: 1980 612100. Fax: 1980 612731. E-mail:
brian.james{at}CAMR.org.UK.
Present address: Thames Water Utilities, Reading, Berkshire, RG1
8DP, United Kingdom.
Present address: Matilda and Terence Kennedy Institute for
Rheumatology, London, W6 8LH, United Kingdom.
| |
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Abstract
Introduction
