BACKGROUND
legionella pneumophila  is a gram-negative bacterium that inhabits natural freshwater environments and man-made water systems.
lp has a broad host range and is able to replicate in different species of amoeba, ciliated protozoa and slime mold  <cit> .
importantly, it is also able to infect humans, resulting in a potentially fatal illness called legionnaires’ disease   <cit> .
human infection occurs through inhalation of aerosols originating from legionella-contaminated sources, such as cooling towers, air conditioning and heating systems, fountains and even showers  <cit> .
upon entry into alveolar macrophages, lp prevents fusion with lysosomes, modulates host cell trafficking, forms a legionella-containing vacuole and then starts intracellular multiplication  <cit> .
in recent years, the occurrence rate of ld has been reported to be on the increase in many countries  <cit> .
in the us, the incidence rate of ld increased by 192 % over the last decade  <cit> .
in europe, france, italy and spain consistently have the highest number of reported cases, with  <dig>  to  <dig>  cases per thousand in  <dig>  <cit> .
most of these are sporadic cases, either community-acquired, nosocomial or travel-associated  <cit> .
nevertheless, outbreak of ld in which a large population gets exposed to contaminated aerosols from a point-source is of great concern.
investigations of previous outbreaks show that the dispersal distance of lp from cooling towers and air scrubbers can be greater than 10 km  <cit> .
many bacteria, such as bacillus megaterium, salmonella enteritidis, staphylococcus aureus and vibrio cholera, die steadily upon exposure to freshwater  <cit> .
chandran et al.
<cit>  showed that the colony-forming units  of escherichia coli and vibrio parahaemolyticus decreased by  <dig> logs after 27 days of exposure to lake water.
in contrast, lp is able to survive for a prolonged period of time in freshwater despite the lack of nutrients .
in an earlier study, lp exposed to drinking water and creek water had a mere  <dig> log reduction in cfu counts and still maintained 3 × 106 cfu/ml after an incubation time of  <dig>  years  <cit> .
the ability of lp to survive in water for long periods is essential for the colonization of water systems, allowing it to persist until optimal conditions and permissive hosts for its growth are encountered.
survival in water is, therefore crucial for the transmission of lp to the human host.
in turn, it is important to understand the genetic factors of lp that allow its prolonged survival in water.
this knowledge may allow future development of strategies that prohibit the survival of lp in water systems and eventually help control legionella outbreaks.
lp possesses approximately  <dig> genes, in which  <dig> genes are commonly found in all six strains  <cit> .
as a human pathogen, the genes involved in virulence have been extensively studied .
two major secretion systems, the lsp type ii secretion system  and the icm/dot type ivb secretion system , translocate more than  <dig> effector proteins into the host cell and are critical for the virulence of lp  <cit> .
moreover, many other genes act as virulence factors that directly contribute to host cell infection  or as virulence regulators  .
apart from the virulence genes, a recent study has showed that at least  <dig> genes are essential for optimal growth of lp in rich medium  <cit> .
however, the functions of many lp genes still remains unknown.
to date, only a few genes are known to be important for the survival of lp in water.
söderberg et al.
<cit>  studied the survival of lspd, lspe, lspf and pild mutants in tap water and concluded that the t2ss is important for lp to maintain survival at temperatures between  <dig> and 17 °c.
recently, our group has shown that the sigma factor rpos and the stringent response  are required for the survival of lp in water  <cit> .
given the lack of knowledge and the associated risk to public health, it is necessary to identify more lp genes that are required for survival in water.
bacteria typically respond to environmental changes through transcriptomic reorganization, where they express genes that are essential for coping with the new condition and repress genes that are no longer required, or that are detrimental  <cit> .
comparison of transcriptional changes using microarrays can be used to identify candidate genes needed in a particular condition .
recently, this technique has been used to identify a new gene, irot, involved in ferrous ion transport based on the transcriptomic profile of lp in an iron restricted condition  <cit> .
in this study, we use a transcriptomic approach to identify genes that are potentially involved in the survival of lp in water.
since bacteria tend to have immediate transcriptomic responses within the first few hours upon exposure to stressful conditions  <cit> , the transcriptomic response of lp to water was studied at an early time point , an intermediate time point  and a late time point .
genes involved in adaptation and regulatory functions are induced in water, while those involved in energy metabolism and translation are repressed.
in particular, our analysis shows that bdha is strongly expressed upon exposure to water, and the deletion of bdha reduces the survival of lp in water at 37 °c.
RESULTS
survival of lp in fraquil
in order to ensure reproducibility, an artificial freshwater medium was used to perform the transcriptomic analysis.
the freshwater medium fraquil was selected for this purpose since it mimics the composition of freshwater in north america  <cit> .
the survival of the wild-type strain jr <dig> in fraquil at 25 °c is shown in fig.
1a.
the population of lp in fraquil was stable for at least five weeks.fig.
<dig>
lp survives well in water at 25 °c.
a cfu counts of jr <dig> during five weeks of exposure to fraquil.
dl indicates the detection limit at 100 cfu/ml.
b cfu counts of jr <dig> cultured in aye broth or exposed to fraquil.
all samples were at an od <dig> of  <dig> .
data shows the mean +/− sd of three biological replicates.
different letters on the bars indicate significant differences between the samples according to tukey's test .
c percentage of live, undefined or dead jr <dig> cells cultured in aye broth or exposed to fraquil.
live/dead staining was used with flow cytometry to determine the status of  <dig> cells in each sample.
d forward scatter  of jr <dig> cultured in aye broth or exposed to fraquil.
each sample had three biological replicates and the mean fsc signal of  <dig> cells in each replicate was detected by flow cytometry
for the transcriptomic analysis, jr <dig> was first cultured in aces-buffered yeast extract  broth to exponential phase in triplicate and 1 ml samples were harvested to serve as controls.
then, the cells were washed three times with fraquil and re-suspended in fraquil at a final od <dig> of  <dig> .
the three re-suspensions were transferred to vessels of a biostat® q plus bioreactor to control temperature and dissolved oxygen.
samples were taken after  <dig>   <dig> and 24 h of exposure  and compared to controls grown in aye broth.
at each time point, we performed both cfu counts and live/dead staining.
although both aye samples and fraquil samples were at the same optical density, the cfu counts of jr <dig> exposed to fraquil were consistently lower, albeit by a very small margin, than jr <dig> from aye broth .
there were no significant differences in cfu counts between the fraquil samples at the different time points tested.
flow cytometry analysis of live/dead staining was used to evaluate the percentage of viable cells and showed that there were no differences between samples .
taking into account cfu counts and cell staining, lp showed no significant survival defects within the first 24 h of exposure to fraquil.
based on the flow cytometry data, a steady reduction in the forward scatter  was observed in jr <dig> upon exposure to fraquil .
the lowest average fsc signal was observed in fraquil samples after 24 h .
since the fsc signal is proportional to particle size  <cit> , this result indicates that lp undergoes a gradual reduction in cell size after exposure to water.
transcriptomic response of lp exposed to water
in order to understand the genetic regulation of lp during short-term exposure to water, we performed a transcriptomic analysis through dna microarray hybridization.
rna was extracted from exponential phase  and fraquil-treated  samples and the data of each treatment was compared with the control.
compared to jr <dig> growing in aye broth, the expression of  <dig> annotated genes and  <dig> predicted srna encoding sequences changed significantly , in at least one time point  after exposure to fraquil .
a progressive transcriptomic change over time is clearly seen in the heat map showing the induction and repression of genes .
the percentages of significantly up- and down-regulated genes increased from  <dig> to 6 h of water exposure, while more genes were significantly down-regulated than up-regulated after 24 h .
there were  <dig>  % up-regulated genes and  <dig>  % down-regulated genes after 2 h of water exposure, demonstrating the rapid transcriptional responses of lp upon exposure to fraquil.fig.
<dig> progressive transcriptomic changes of lp in water.
the global gene expression of jr <dig> after  <dig>   <dig> and 24 h exposure to fraquil in comparison with the control cultured in aye broth is shown in the heat map.
the hierarchical clustering shows the similarities between samples.
the genes that were up-regulated are shown in red and those down-regulated are shown in green
fig.
<dig> many lp genes are shut down after 24 h of exposure to water.
a percentage of genes significantly up-regulated or down-regulated after  <dig>   <dig> and 24 h of exposure to fraquil.
the numbers on the top of each bar represent the number of genes differently expressed  over a total of  <dig> annotated genes from the original genome annotation.
cluster of orthologous groups analysis of jr <dig> genes after  2 h,  6 h and  24 h of exposure to fraquil is shown.
up-regulated genes are shown in red and down-regulated genes are shown in green
genes that were differentially expressed after water exposure were categorized into orthologous groups.
after  <dig> and 6 h of water exposure, seven of the  <dig> orthologous groups had a higher percentage of up-regulated genes .
these orthologous groups are “chemotaxis/motility/cell division”, “detoxification/adaptation”, “icm/dot effector”, “signal transduction/other regulatory functions”, “transcription”, “unclassified/unknown/hypothetical” and “viral functions/phage/transposases”.
in contrast, all  <dig> orthologous groups showed major down-regulation of genes after 24 h of water exposure .
at each time point, “energy metabolism”, “translation” and “transport and binding” remained the top three orthologous groups with the highest percentage of down-regulated genes.
examples of genes in these orthologous groups are listed in table  <dig> and  <dig> and will be discussed later.table  <dig> selected genes significantly down-regulated in water
lpg2610
ftsa
lpg2611
ftsq
lpg2615
ftsw
lpg2988
atpb
lpg2987
atpe
lpg2989
atpi
lpg2984
lpg2982
atpd
lpg2981
atpc
lpg2983
atpg
lpg2985
atph
lpg2896
lpg2897
lpg2898
lpg2789
nuoa
lpg2788
nuob2
lpg2787
nuoc
lpg2782
nuoh
lpg2781
nuoi
lpg0528
sdhc
lpg0530
sdha
lpg0531
sdhb
lpg2704
petb
lpg2705
peta
lpg1912
lets
lpg2646
leta
lpg0791
mip
lpg0354
rpoa
lpg0322
rpob
lpg0323
rpoc
lpg2667
rpoh
lpg0351
rpsm
lpg2636
rpst
lpg1592
rpsf
lpg0325
rps7
lpg0348
rplo
lpg0336
rplp
lpg0479
rpmb
lpg0344
rplf
lpg0326
fusa
lpg1713
tsf
lpg1770
infa
lpg2713
infc
lpg2292
lpg0797
lpg1929
lpg1866
lpg1658
lpg1691
lpg1917
lpg0970
lpg0228
lpg0049
lpg2658
feoa
lpg2657
feob
lpg2686
dota
lpg2675
dotc
lpg2674
dotd
lpg0447
lpha
lpg0456
icmb
lpg0453
icmc
lpg0458
icmf
lpg0459
icmh
lpg0455
icmj
lpg0450
icmk
lpg0449
icml
lpg0708
lpg0383
lpg0448
icmm
lpg0446
icmo
lpg0444
icmq
lpg0443
icmr
lpg0442
icms
lpg0441
icmt
lpg2687
icmv
lpg2688
icmw
lpg2689
icmx
lpg0927
pilm
lpg0928
piln
lpg0930
pilp
aonly values that were significantly different than the control are shown
lpg1790
flio
lpg1221
flgg
lpg1782
flia
lpg1788
fliq
lpg1784
flhf
lpg1219
flge
lpg1222
flgh
lpg2318
mota
lpg1780
motb
lpg1781
motc
lpg1792
flim
lpg1223
flgi
lpg1340
flic
lpg2349
ahpd
lpg2350
ahpc
lpg2965
ahpc
lpg2024
dnaj
lpg2817
yrfi
lpg2151
lpg1492
lpg2011
lpg2348
sodc
lpg0935
lpg1488
legc5
lpg2144
legau13
lpg0096
ceg4
lpg2591
ceg33
lpg1290
lem8
lpg1496
lem10
lpg0376
sdha
lpg0621
sida
lpg2157
sdea
lpg2830
legu2
lpg1976
legg1
lpg2316
bdha
ssrs
rsmy
rsmz
lpg2146
lpg0230
pled
lpg2734
lpg0029
rre41
lpg1025
yege
lpg0208
pkn5
lpg1206
lpg0156
lpg1336
enha
lpg1386
lpg2641
enha
lpg0910
enha
lpg2640
enhb
lpg1172
lpg1356
lpg0645
lpg2441
lpg0477
lpg0954
mfd
lpg0586
lpg2723
lpg1486
lpg2167
lpg2524
lpg2138
lpg2140
lpg0453
icmc
lpg0472
lpg0454
icmd
lpg0446
icmo
lpg0445
icmp
lpg0441
icmt
lpg2687
icmv
lpg1616
uup
lpg0257
lpg0429
oprm
lpg2189
ygjt
lpg2563
lpg2120
lpg2363
aonly values that were significantly different than the control are shown
validation of microarray results by rt-qpcr
to validate the results of the microarray analysis, the expression profiles of ten different genes were confirmed by reverse transcription quantitative pcr  using 16 s rrna as an internal control  <cit> .
these ten genes were selected from eight different orthologous groups.
according to the microarray data, six of the selected genes  and lpg2524) were significantly up-regulated in water at two or all three time points, while the remaining four genes  were significantly down-regulated .
the rt-qpcr results for these  <dig> genes are shown in fig.
4b.
despite some differences, the general expression profiles were conserved between the microarray and rt-qpcr data, which validates the former.fig.
<dig> microarray data is validated by rt-qpcr.
transcriptomic changes of ten selected jr <dig> genes upon water exposure shown by  microarray analysis and  rt-qpcr.
results are shown as the log <dig> ratio between the samples exposed to fraquil for  <dig>   <dig> or 24 h and the control cultured in aye broth
antibiotic resistance of lp subsequent to water exposure
many genes involved in detoxification and adaptation were induced in water, including some associated with antibiotic resistance, such as an aminoglycoside 6-adenylyltransferase, a spectinomycin phosphotransferase, an erythromycin transporter and several efflux pumps .
the induction of these genes may increase the resistance of water-exposed lp to antibiotics.
to investigate this hypothesis, we compared the antibiotic resistance of lp cultured in aye broth to exponential phase and those exposed to fraquil for 24 h. in both cases, we compared the cfu counts with and without the addition of a β-lactam , two aminoglycosides  and a macrolide .
in order to determine the impact of temperature on antibiotic resistance, lp was grown in aye and exposed to fraquil at both 25 °c and 37 °c prior to testing resistance.
there were no significant differences in the cfu counts between aye-grown and fraquil-exposed lp treated with ampicillin .
lp grown in aye at 37 °c was more susceptible to erythromycin than lp grown in aye at 25 °c or lp exposed to fraquil at either temperature.
lp exposed to fraquil was significantly more resistant to gentamycin than lp grown in aye broth, regardless of the temperature.
moreover, exposure to 37 °c seems to slightly increase susceptibility to gentamycin.
lp was more resistant to kanamycin after exposure to fraquil than when grown in aye, and this difference was more pronounced at 37 °c.
overall, lp is more resistant to aminoglycosides after exposure to fraquil, which is consistent with our prediction based on the transcriptomic data.
incubation temperature also seems to affect antibiotic resistance, especially against erythromycin.fig.
<dig>
lp exposed to water is more resistant to antibiotics.
cfu changes of lp cultured in aye broth or exposed to fraquil at  <dig> or 37 °c after antibiotic treatments.
the antibiotics used include ampicillin, erythromycin, gentamycin and kanamycin, all at 100 μg/ml.
5x indicates a five times concentration of erythromycin and gentamycin  were used as well.
the data are expressed as the log transformation of cfus in treated wells  divided by the cfus in the control wells without antibiotics .
a negative value indicates a cfu reduction upon treatment.
data shows the mean +/− sd of three biological replicates.
different letters on the bars indicate significant differences between different samples in a particular treatment according to tukey's test .
cfu changes between different treatments were not compared
importance of bdha for the survival of lp in water
genes that are significantly induced upon exposure to fraquil may be important for lp to survive in water.
upon this premise, the highly up-regulated bdha gene was selected for further characterization.
a deletion mutant of bdha  and its complement  were constructed.
for the complementation, bdha was cloned downstream of the ptac promoter on pmmb207c.
the wild-type strain ks <dig>  Δbdha, spf <dig> without isopropyl-β-d-thiogalactopyranoside   and spf <dig> with 1 mm iptg  were exposed to fraquil at  <dig> and 37 °c.
cfu counts were monitored weekly and their survival was also assessed by live/dead staining after 19 weeks of exposure, when the cfus of all strains at 37 °c dropped below the detection limit.
no survival defects were observed in the mutant strain compared to the wild-type or the complements in fraquil at 25 °c .
however, a minor reduction  in the cfu counts was observed in all strains after 20 weeks of water exposure.
an overall faster drop in cfus was observed in all strains at 37 °c, compared to 25 °c .
the bdha mutant strain showed a more rapid reduction in cfus than the wild-type strain.
this phenotype was complemented by expression of bdha from the ptac promoter in the presence of iptg .
the cfu count of spf <dig> without iptg was similar to the bdha mutant strain.
therefore, bdha seems to be required for the survival of lp in water at 37 °c.fig.
<dig>
bdha is important for long-term survival of lp in water.
weekly cfu counts of the wild-type ks <dig>  Δbdha and its complement spf <dig> with and without iptg exposed to fraquil at  25 °c and  37 °c.
1 mm iptg was used to induce bdha on the plasmid of the complemented strain.
data show the mean +/− sd of three biological replicates.
dl: detection limit.
c percentage of live, undefined or dead cells of ks <dig>  Δbdha and its complement spf <dig> with and without iptg.
live/dead staining analysed by flow cytometry was performed after the strains were exposed to fraquil for 19 weeks at  <dig> and 37 °c
since the cfu counts of all strains exposed to 37 °c decreased below the detection limit after 19 weeks of water exposure, we sought to determine if the cells entered a viable but non-culturable  state at this time point  <cit> .
to this end, we used a live/dead staining procedure together with flow cytometry.
at 25 °c, no significant differences in the percentage of viable cells were observed between strains , which is consistent with the cfu counts.
on the other hand, the wild-type strain showed a small fraction of dead cells at 37 °c, which is not significant compared to 25 °c.
in comparison, only 61 % cells of the bdha mutant strain were stained as viable.
the complemented strain exposed to water in the presence of iptg had a higher percentage of viable cells but was still lower than that of the wild-type strain.
taken together with the cfu counts, our results show that lp enters a vbnc state after 19 weeks of exposure to fraquil at 37 °c.
regulation of bdha by rpos
hovel-miner et al.
<cit>  showed that the expression of bdha in a rich medium is regulated by rpos when lp reaches the post-exponential phase.
moreover, transcriptomic analysis of the rpos mutant in water suggests that the expression of bdha is rpos-dependant  <cit> .
therefore, we investigated this possibility of rpos regulation of bdha by using a green fluorescent protein  reporter assay  <cit> .
briefly, a plasmid carrying the pbdha-gfp transcriptional fusion was constructed  and transformed into jr <dig> and the rpos mutant .
jr <dig> harbouring the pxdc <dig> plasmid, which expresses gfp from the ptac promoter in the presence of iptg, was used as positive control.
jr <dig> and rpos mutant strains with a plasmid containing no promoter upstream of the gfp encoding sequence  served as negative controls.
each strain was exposed to aye broth or fraquil for 24 h and the level of gfp in terms of green fluorescence signal was measured by flow cytometry.
a significantly higher level of gfp was found to be expressed in ks <dig> psf <dig> exposed to fraquil than that exposed to aye broth, suggesting that the promoter of bdha was more highly induced after 24 h in water .
this induction is consistent with the up-regulation of bdha  observed in jr <dig> exposed to water for the same period of time during the transcriptomic analysis .
gfp expression in rpos psf <dig> was higher than in the negative controls under both conditions, but it was significantly lower than that of ks <dig> psf <dig>  in addition, no significant differences were found between rpos psf <dig> in aye broth compared to fraquil.
these findings confirm that expression of bdha in lp is positively regulated by rpos.fig.
<dig>
bdha is highly induced in water and regulated by rpos.
green fluorescence signal of five different strains after 24 h of exposure to aye broth or fraquil.
jr <dig> psf <dig>  rpos psf <dig>  jr <dig> psf <dig> and rpos psf <dig> represent spf <dig>  spf <dig>  spf <dig> and spf <dig>  respectively.
jr <dig> pxdc <dig> was induced with 1 mm iptg.
each sample had three biological replicates and the mean green fluorescence signal of  <dig> cells in each replicate was detected by flow cytometry
discussion
as a bacterium that lives in aqueous environments, lp is frequently exposed to various stresses, such as fluctuations in temperature, ph and levels trace metals, as well as nutrient limitation  <cit> .
nutrient limitation, in particular, is a major concern as the nutrient levels in freshwater rarely fits the growth requirement of lp, which is an auxotroph for at least eight amino acids  <cit> .
in the absence of essential nutrients or suitable hosts, lp cannot grow, but can still persist in water for over one year  <cit> .
previous work has shown that the t2ss is important for the survival of lp in water at low temperature  <cit> .
however, no studies have investigated the global transcriptomic response of lp to freshwater.
in response to this knowledge gap, we performed a transcriptomic analysis to study the changes in gene expression of lp exposed to fraquil, an artificial freshwater medium composed of trace amounts of salts and metals, compared to lp grown in rich medium .
since lp in stationary phase culture is under nutrient limitation and stresses, using it as the control may hinder the opportunity to identify important genes for its survival in water.
therefore, lp in the exponential phase of growth was used as the control.
in fraquil, lp maintained a stable population for at least 35 days and did not show any growth .
nevertheless, a significant reduction in cell size was found within 24 h of water exposure , which might be an adaptative response of lp to nutrient limitation.
in fact, the reduction of cell size is commonly observed in bacteria experiencing environmental stresses, such as nutrient limitation, suboptimal ph and low temperatures .
for example, the cell length of v. parahaemolyticus under starvation decreased drastically from  <dig>  to  <dig>  μm within 24 h  <cit> .
such a morphological change is believed to be a strategy to minimize the metabolic requirements for cell maintenance  <cit> .
the mature intracellular form  of lp, produced after passage through hela cells and tetrahymena tropicalis, also shows a reduction in cell size, suggesting that this morphological change may be a response of lp to nutrient limitation  <cit> .
the transcriptomic study of lp revealed significant changes in gene expression following exposure to fraquil.
since fraquil does not contain any carbon sources that lp can use, it is unable to grow in this medium .
consistent with this observation, expression of several amino acid transporters was induced in water .
therefore, the reduced expression of three cell division proteins, such as ftsa, ftsq and ftsw, was expected .
moreover, bacteria tend to shut down major metabolic pathways when under starvation  <cit> .
following exposure to water, genes involved in the electron transport chain  and eight genes encoding subunits of the atp synthase were significantly repressed .
this suggests a lower level of energy metabolism and thus, reduced metabolic activity in lp, as the down-regulation of atp synthase is usually found in dormant bacteria with a lower cellular atp level  <cit> .
furthermore, compared to the control growing in aye broth, the total rna extracted from lp exposed to water dropped markedly after 2 h and reduced to only one tenth of the control after 24 h .
this finding is consistent with the down-regulation of rpoa, rpob and rpoc, which encode subunits of rna polymerase, potentially resulting in less rna polymerase available to bind to dna and initiate transcription .
in addition, most of the genes encoding 30s ribosomal proteins, 50s ribosomal proteins, translation initiation factors, translation elongation factors as well as trnas were down-regulated in water in two or all three time points tested .
in total, the expression of eight rpm genes,  <dig> rps genes and  <dig> rpl genes was significantly reduced .
in addition, lpg <dig> was strongly induced in water.
this gene encodes a homolog of yhbh, which is a short hibernation promoting factor  with a highly conserved function in γ-proteobacteria  <cit> .
short hpf stabilizes the dimerization of two 70s ribosomes into a translationally inactive 100s ribosome by a ribosome modulation factor, resulting in ribosome hibernation  <cit> .
ribosome hibernation is commonly found in bacteria experiencing nutrient limitation and is rapidly reversible when nutrients become available once again  <cit> .
in e. coli, a mutant unable to form 100s ribosomes survived poorly in the post-exponential phase  <cit> .
therefore, it is possible that the up-regulation of lpg <dig> in lp may allow the formation of 100s ribosomes to maintain long-term survival in water, and the ability to resume growth or intracellular multiplication.
our findings suggest that the translational machinery of lp is shut down following exposure to water, which is a typical characteristic of the bacterial stringent response  <cit> .
the stringent response is the reallocation of cellular resources in bacteria under nutrient limitation, by prohibiting the synthesis of dna, stable rnas, ribosomal proteins and membrane components, and activating the synthesis of factors involved in amino acid synthesis, glycolysis and stress resistance  <cit> .
the massive transcriptomic changes during the stringent response are mediated by the synthesis and accumulation of guanosine tetraphosphate  and guanosine pentaphosphate , which are called ppgpp collectively  <cit> .
the stringent response in lp has been studied in detail.
under amino acid limitation, the binding of uncharged trnas to the a site in ribosomes triggers the ribosomal-associated rela protein to synthesize ppgpp  <cit> .
spot, the second stringent response protein, can also synthesize ppgpp in response to other signals, such as perturbation of fatty acid synthesis  <cit> .
spot possesses a hydrolase activity as well, and therefore, regulates the level of ppgpp and is necessary to terminate the stringent response  <cit> .
in lp, the stringent response, and the rela and spot proteins are necessary for replication inside host cells and initiation of the transmissive phase  <cit> .
our results clearly show a repression of the translational machinery, cell replication and changes in metabolic processes, which are consistent with the general transcriptional modulations caused by ppgpp  <cit> .
in addition, we have recently shown that the stringent response is required for the survival of lp in water  <cit> .
due to the imminent shutdown of the translational machinery following water exposure, the important genes should be induced early on to allow the synthesis of their products when lp is still translationally active.
therefore, in order to identify genes essential to maintain long-term survival in water, we examined those that are highly up-regulated within 24 h, a time point when a major metabolic shutdown started to appear.
first, many genes associated with the biosynthesis of flagella, including different assembly, regulatory and structural proteins, are up-regulated in lp exposed to water .
this response is comparable to the response of lp that reaches the late replicative phase inside host cells.
due to exploitation of host cell resources and consequent lack of nutrients, the transmissive phase of lp is triggered, leading to the expression of virulence-associated traits such as flagella formation and other factors to promote release from the host  <cit> .
many genes induced at early time points possess a function related to detoxification and adaptation .
genes involved in resistance to oxidative stress were induced, including sodc, ahpc and ahpd.
we also observed the induction heat shock proteins in water .
in e. coli, heat shock proteins are known to be induced by starvation  <cit> .
they increase bacterial tolerance against various stresses by degrading and reactivating damaged proteins  <cit> .
therefore, it is plausible that the up-regulation of these genes may help lp to persist in water under starvation conditions.
in addition, many genes involved in antibiotic resistance were induced at early time points, including lpg <dig> , lpg <dig> , lpg <dig> , and several efflux pumps .
increased resistance to antibiotics is a hallmark of lp mifs  <cit> .
this phenomenon was also described after incubation of lp in acanthamoeba castellanii buffer at ph  <dig>   <cit> .
antibiotic resistance genes may be important for lp to compete against antibiotic-producing microorganisms residing in water.
in water, lp is more resistant to gentamycin and kanamycin  than when it is cultured in rich medium .
water-exposed lp is also more resistant to erythromycin, but only at a temperature of 37 °c.
since all these antibiotics target the ribosome, it is also possible that the increased resistance is due to a decrease in translational activity, and not solely because of the expression of resistance genes.
differential expression of some genes involved in virulence was also apparent.
despite the repression of  <dig> of the  <dig> structural genes of the icm/dot secretion system , the expression of many icm/dot effectors was increased in water .
in addition, the enhanced-entry genes enha and enhb, and several of the enhc homologs, as well as the rtxa toxin were induced in water at early time points.
on the other hand, the macrophage infectivity potentiator gene, mip, was significantly down regulated in water.
it is not clear at this point if these changes in expression of virulence genes affect the intracellular multiplication of lp in host cells after incubation in water.
we are currently investigating this possibility.
many genes involved in transcriptional regulation were up-regulated, including numerous transcriptional regulators from different families, dna binding proteins, a transcription repair coupling factor and the alternative sigma factor σ <dig>  .
interestingly, rpon is important for the induction of flia and other flagellar genes  <cit> .
consistent with previous observations, our results show up-regulation of both σ <dig> and flagellar genes in water.
several response regulators, sensory proteins and signal transduction proteins involved in “signal transduction/other regulatory functions” were also up-regulated.
among these, many genes encoding proteins harbouring ggdef/eal domains were induced in water .
these proteins are involved in the production and destruction of the second messenger c-di-gmp, and several of them have been shown to play a role in the interaction of lp with host cells  <cit> .
some of them could play a role in sensing exposure to water and regulating the appropriate response.
moreover, expression of the two-component system leta/s was reduced in water, while expression of the small rnas rsmy and rsmz under its control was increased.
these srnas are necessary to relieve csra-mediated repression of transmissive phase traits upon starvation inside host cells  <cit> .
our results suggest that some of the genes repressed by csra could be involved in the survival of lp in water.
interestingly, a link has been made between leta/s, rpos and ppgpp  <cit> .
it is noteworthy that expression of the 6s rna was also induced in water.
6s rna binds to the rna polymerase  holoenzyme and inhibits its binding to promoters  <cit> .
since the affinity of 6s rna to the rnap depends on sigma factors, 6s rna can shut down specific transcriptional programs.
in e. coli, the σ <dig> regulon is turned off in the presence of 6s rna  <cit> .
in lp, deletion of 6s rna reduces fitness during intracellular growth  <cit> .
expression of 6s rna in water could help lp to switch between different transcriptomic programs, but further investigation is needed to confirm its involvement.
there are also many other srnas that are differentially regulated, but since their targets are unknown, it is not yet possible to predict specific functions.
within the highly up-regulated genes in water, we characterized bdha, which is involved in the polyhydroxybutyrate  cycle  <cit> .
phb is an important storage polymer in bacteria, which is synthesized as a carbon and energy reserve in the presence of external nutrients and is consumed during starvation  <cit> .
the bdha gene encodes 3-hydroxybutyrate dehydrogenase, which oxidizes depolymerised phb into acetoacetate and produces reducing power in the form of nadh  <cit> .
acetoacetate can, then, be further processed into acetyl-coa, which enters the tricarboxylic acid cycle  <cit> .
bdha is dispensable for lp growth in the presence of external nutrients, as mutations in this gene do not result in any observable growth defects in aye broth  <cit> .
however, aurass et al.
<cit>  demonstrated that a bdha-patd mutant strain of lp is defective in breaking down phb accumulated during growth in a rich medium, resulting in a higher level of cellular phb than the wild-type.
in addition, this mutant has defects in intracellular growth in amoeba and in human macrophages  <cit> .
therefore, we hypothesize that bdha is important for lp to survive in water, enabling the extraction of carbon and reducing power from phb in order to allow cell maintenance in the absence of an external energy source.
our results show that the deletion of bdha reduces the survival of lp in water.
the Δbdha strain lost culturability and seems to have entered the vbnc state earlier, resulting in a higher percentage of mortality after 19 weeks of water exposure at 37 °c compared to the wild-type.
this survival defect was successfully complemented, supporting the hypothesis that lp needs bdha for the complete degradation of phb in order to maintain long-term survival in water.
it is noteworthy that the difference in survival between Δbdha and the wild-type was only observed at 37 °c but not at 25 °c.
since the samples were exhausted after 20 weeks of sampling, we could not determine if the observed difference was eventually be mirrored at 25 °c after a longer period of water exposure.
it is possible that survival at 37 °c requires more energy for cell maintenance than at 25 °c because of a higher metabolic rate, protein turnover and overall damage, and thus, the advantage conferred by bdha is more apparent at the higher temperature.
in addition, we investigated the regulation of bdha expression by rpos when lp is exposed to water.
rpos is a well-known regulator of the response to nutrient limitation  <cit> .
recently, we have shown that the stringent response and the sigma factor rpos are necessary for the survival of lp in water  <cit> .
based on a gfp reporter assay, we found that bdha is positively regulated by rpos following exposure to water .
this is consistent with the findings of hovel-miner et al.
<cit> , which show that, in a rich medium, bdha in lp is positively regulated by rpos in the post-exponential phase.
CONCLUSIONS
our study reveals, for the first time, the global transcriptomic changes of lp in water.
repression of major pathways, such as cell division, transcription and translation, suggests that lp enters a quiescent state in water.
the induction of the enhanced-entry genes  and some icm/dot effectors suggests that lp may be primed to infect a suitable host.
similarly, many genes involved in resistance to antibiotics and oxidative stress, as well as genes involved in the heat shock response were induced.
furthermore, the bdha gene involved in the degradation pathway of the intracellular energy storage compound phb is highly expressed and positively regulated by rpos during short-term exposure to water.
this gene was found to be important for maintaining long-term survival of lp in water.
there is no doubt that many of the genes highly induced upon exposure to water are also necessary for the short and/or long-term survival of lp in water.

 legionella pneumophila is a gram-negative bacterium that inhabits natural freshwater environments and man-made water systems. lp has a broad host range and is able to replicate in different species of amoeba, ciliated protozoa and slime mold. importantly, it is also able to infect humans, resulting in a potentially fatal illness called legionnaires’ disease. human infection occurs through inhalation of aerosols originating from legionella-contaminated sources, such as cooling towers, air conditioning and heating systems, fountains and even showers. upon entry into alveolar macrophages, lp prevents fusion with lysosomes, modulates host cell trafficking, forms a legionella-containing vacuole and then starts intracellular multiplication. in recent years, the occurrence rate of ld has been reported to be on the increase in many countries. in the us, the incidence rate of ld increased by 192 % over the last decade. in europe, france, italy and spain consistently have the highest number of reported cases, with to cases per thousand in. most of these are sporadic cases, either community-acquired, nosocomial or travel-associated. nevertheless, outbreak of ld in which a large population gets exposed to contaminated aerosols from a point-source is of great concern. investigations of previous outbreaks show that the dispersal distance of lp from cooling towers and air scrubbers can be greater than 10 km. many bacteria, such as bacillus megaterium, salmonella enteritidis, staphylococcus aureus and vibrio cholera, die steadily upon exposure to freshwater. chandran et al. showed that the colony-forming units of escherichia coli and vibrio parahaemolyticus decreased by logs after 27 days of exposure to lake water. in contrast, lp is able to survive for a prolonged period of time in freshwater despite the lack of nutrients. in an earlier study, lp exposed to drinking water and creek water had a mere log reduction in cfu counts and still maintained 3 × 106 cfu/ml after an incubation time of  years. the ability of lp to survive in water for long periods is essential for the colonization of water systems, allowing it to persist until optimal conditions and permissive hosts for its growth are encountered. survival in water is, therefore crucial for the transmission of lp to the human host. in turn, it is important to understand the genetic factors of lp that allow its prolonged survival in water. this knowledge may allow future development of strategies that prohibit the survival of lp in water systems and eventually help control legionella outbreaks. lp possesses approximately genes, in which genes are commonly found in all six strains. as a human pathogen, the genes involved in virulence have been extensively studied. two major secretion systems, the lsp type ii secretion system and the icm/dot type ivb secretion system, translocate more than effector proteins into the host cell and are critical for the virulence of lp. moreover, many other genes act as virulence factors that directly contribute to host cell infection or as virulence regulators. apart from the virulence genes, a recent study has showed that at least genes are essential for optimal growth of lp in rich medium. however, the functions of many lp genes still remains unknown. to date, only a few genes are known to be important for the survival of lp in water. söderberg et al. studied the survival of lspd, lspe, lspf and pild mutants in tap water and concluded that the t2ss is important for lp to maintain survival at temperatures between and 17 °c. recently, our group has shown that the sigma factor rpos and the stringent response are required for the survival of lp in water. given the lack of knowledge and the associated risk to public health, it is necessary to identify more lp genes that are required for survival in water. bacteria typically respond to environmental changes through transcriptomic reorganization, where they express genes that are essential for coping with the new condition and repress genes that are no longer required, or that are detrimental. comparison of transcriptional changes using microarrays can be used to identify candidate genes needed in a particular condition. recently, this technique has been used to identify a new gene, irot, involved in ferrous ion transport based on the transcriptomic profile of lp in an iron restricted condition. in this study, we use a transcriptomic approach to identify genes that are potentially involved in the survival of lp in water. since bacteria tend to have immediate transcriptomic responses within the first few hours upon exposure to stressful conditions, the transcriptomic response of lp to water was studied at an early time point, an intermediate time point and a late time point. genes involved in adaptation and regulatory functions are induced in water, while those involved in energy metabolism and translation are repressed. in particular, our analysis shows that bdha is strongly expressed upon exposure to water, and the deletion of bdha reduces the survival of lp in water at 37 °c.  survival of lp in fraquil in order to ensure reproducibility, an artificial freshwater medium was used to perform the transcriptomic analysis. the freshwater medium fraquil was selected for this purpose since it mimics the composition of freshwater in north america. the survival of the wild-type strain jr in fraquil at 25 °c is shown in fig. 1a. the population of lp in fraquil was stable for at least five weeks.fig. lp survives well in water at 25 °c. a cfu counts of jr during five weeks of exposure to fraquil. dl indicates the detection limit at 100 cfu/ml. b cfu counts of jr cultured in aye broth or exposed to fraquil. all samples were at an od of. data shows the mean +/− sd of three biological replicates. different letters on the bars indicate significant differences between the samples according to tukey's test. c percentage of live, undefined or dead jr cells cultured in aye broth or exposed to fraquil. live/dead staining was used with flow cytometry to determine the status of cells in each sample. d forward scatter of jr cultured in aye broth or exposed to fraquil. each sample had three biological replicates and the mean fsc signal of cells in each replicate was detected by flow cytometry for the transcriptomic analysis, jr was first cultured in aces-buffered yeast extract broth to exponential phase in triplicate and 1 ml samples were harvested to serve as controls. then, the cells were washed three times with fraquil and re-suspended in fraquil at a final od of. the three re-suspensions were transferred to vessels of a biostat® q plus bioreactor to control temperature and dissolved oxygen. samples were taken after and 24 h of exposure and compared to controls grown in aye broth. at each time point, we performed both cfu counts and live/dead staining. although both aye samples and fraquil samples were at the same optical density, the cfu counts of jr exposed to fraquil were consistently lower, albeit by a very small margin, than jr from aye broth. there were no significant differences in cfu counts between the fraquil samples at the different time points tested. flow cytometry analysis of live/dead staining was used to evaluate the percentage of viable cells and showed that there were no differences between samples. taking into account cfu counts and cell staining, lp showed no significant survival defects within the first 24 h of exposure to fraquil. based on the flow cytometry data, a steady reduction in the forward scatter was observed in jr upon exposure to fraquil. the lowest average fsc signal was observed in fraquil samples after 24 h. since the fsc signal is proportional to particle size, this result indicates that lp undergoes a gradual reduction in cell size after exposure to water. transcriptomic response of lp exposed to water in order to understand the genetic regulation of lp during short-term exposure to water, we performed a transcriptomic analysis through dna microarray hybridization. rna was extracted from exponential phase and fraquil-treated samples and the data of each treatment was compared with the control. compared to jr growing in aye broth, the expression of annotated genes and predicted srna encoding sequences changed significantly, in at least one time point after exposure to fraquil. a progressive transcriptomic change over time is clearly seen in the heat map showing the induction and repression of genes. the percentages of significantly up- and down-regulated genes increased from to 6 h of water exposure, while more genes were significantly down-regulated than up-regulated after 24 h. there were  % up-regulated genes and  % down-regulated genes after 2 h of water exposure, demonstrating the rapid transcriptional responses of lp upon exposure to fraquil.fig. progressive transcriptomic changes of lp in water. the global gene expression of jr after and 24 h exposure to fraquil in comparison with the control cultured in aye broth is shown in the heat map. the hierarchical clustering shows the similarities between samples. the genes that were up-regulated are shown in red and those down-regulated are shown in green fig. many lp genes are shut down after 24 h of exposure to water. a percentage of genes significantly up-regulated or down-regulated after and 24 h of exposure to fraquil. the numbers on the top of each bar represent the number of genes differently expressed over a total of annotated genes from the original genome annotation. cluster of orthologous groups analysis of jr genes after 2 h, 6 h and 24 h of exposure to fraquil is shown. up-regulated genes are shown in red and down-regulated genes are shown in green genes that were differentially expressed after water exposure were categorized into orthologous groups. after and 6 h of water exposure, seven of the orthologous groups had a higher percentage of up-regulated genes. these orthologous groups are “chemotaxis/motility/cell division”, “detoxification/adaptation”, “icm/dot effector”, “signal transduction/other regulatory functions”, “transcription”, “unclassified/unknown/hypothetical” and “viral functions/phage/transposases”. in contrast, all orthologous groups showed major down-regulation of genes after 24 h of water exposure. at each time point, “energy metabolism”, “translation” and “transport and binding” remained the top three orthologous groups with the highest percentage of down-regulated genes. examples of genes in these orthologous groups are listed in table  and and will be discussed later.table selected genes significantly down-regulated in water lpg2610 ftsa lpg2611 ftsq lpg2615 ftsw lpg2988 atpb lpg2987 atpe lpg2989 atpi lpg2984 lpg2982 atpd lpg2981 atpc lpg2983 atpg lpg2985 atph lpg2896 lpg2897 lpg2898 lpg2789 nuoa lpg2788 nuob2 lpg2787 nuoc lpg2782 nuoh lpg2781 nuoi lpg0528 sdhc lpg0530 sdha lpg0531 sdhb lpg2704 petb lpg2705 peta lpg1912 lets lpg2646 leta lpg0791 mip lpg0354 rpoa lpg0322 rpob lpg0323 rpoc lpg2667 rpoh lpg0351 rpsm lpg2636 rpst lpg1592 rpsf lpg0325 rps7 lpg0348 rplo lpg0336 rplp lpg0479 rpmb lpg0344 rplf lpg0326 fusa lpg1713 tsf lpg1770 infa lpg2713 infc lpg2292 lpg0797 lpg1929 lpg1866 lpg1658 lpg1691 lpg1917 lpg0970 lpg0228 lpg0049 lpg2658 feoa lpg2657 feob lpg2686 dota lpg2675 dotc lpg2674 dotd lpg0447 lpha lpg0456 icmb lpg0453 icmc lpg0458 icmf lpg0459 icmh lpg0455 icmj lpg0450 icmk lpg0449 icml lpg0708 lpg0383 lpg0448 icmm lpg0446 icmo lpg0444 icmq lpg0443 icmr lpg0442 icms lpg0441 icmt lpg2687 icmv lpg2688 icmw lpg2689 icmx lpg0927 pilm lpg0928 piln lpg0930 pilp aonly values that were significantly different than the control are shown lpg1790 flio lpg1221 flgg lpg1782 flia lpg1788 fliq lpg1784 flhf lpg1219 flge lpg1222 flgh lpg2318 mota lpg1780 motb lpg1781 motc lpg1792 flim lpg1223 flgi lpg1340 flic lpg2349 ahpd lpg2350 ahpc lpg2965 ahpc lpg2024 dnaj lpg2817 yrfi lpg2151 lpg1492 lpg2011 lpg2348 sodc lpg0935 lpg1488 legc5 lpg2144 legau13 lpg0096 ceg4 lpg2591 ceg33 lpg1290 lem8 lpg1496 lem10 lpg0376 sdha lpg0621 sida lpg2157 sdea lpg2830 legu2 lpg1976 legg1 lpg2316 bdha ssrs rsmy rsmz lpg2146 lpg0230 pled lpg2734 lpg0029 rre41 lpg1025 yege lpg0208 pkn5 lpg1206 lpg0156 lpg1336 enha lpg1386 lpg2641 enha lpg0910 enha lpg2640 enhb lpg1172 lpg1356 lpg0645 lpg2441 lpg0477 lpg0954 mfd lpg0586 lpg2723 lpg1486 lpg2167 lpg2524 lpg2138 lpg2140 lpg0453 icmc lpg0472 lpg0454 icmd lpg0446 icmo lpg0445 icmp lpg0441 icmt lpg2687 icmv lpg1616 uup lpg0257 lpg0429 oprm lpg2189 ygjt lpg2563 lpg2120 lpg2363 aonly values that were significantly different than the control are shown validation of microarray results by rt-qpcr to validate the results of the microarray analysis, the expression profiles of ten different genes were confirmed by reverse transcription quantitative pcr using 16 s rrna as an internal control. these ten genes were selected from eight different orthologous groups. according to the microarray data, six of the selected genes and lpg2524) were significantly up-regulated in water at two or all three time points, while the remaining four genes were significantly down-regulated. the rt-qpcr results for these genes are shown in fig. 4b. despite some differences, the general expression profiles were conserved between the microarray and rt-qpcr data, which validates the former.fig. microarray data is validated by rt-qpcr. transcriptomic changes of ten selected jr genes upon water exposure shown by microarray analysis and rt-qpcr. results are shown as the log ratio between the samples exposed to fraquil for or 24 h and the control cultured in aye broth antibiotic resistance of lp subsequent to water exposure many genes involved in detoxification and adaptation were induced in water, including some associated with antibiotic resistance, such as an aminoglycoside 6-adenylyltransferase, a spectinomycin phosphotransferase, an erythromycin transporter and several efflux pumps. the induction of these genes may increase the resistance of water-exposed lp to antibiotics. to investigate this hypothesis, we compared the antibiotic resistance of lp cultured in aye broth to exponential phase and those exposed to fraquil for 24 h. in both cases, we compared the cfu counts with and without the addition of a β-lactam, two aminoglycosides and a macrolide. in order to determine the impact of temperature on antibiotic resistance, lp was grown in aye and exposed to fraquil at both 25 °c and 37 °c prior to testing resistance. there were no significant differences in the cfu counts between aye-grown and fraquil-exposed lp treated with ampicillin. lp grown in aye at 37 °c was more susceptible to erythromycin than lp grown in aye at 25 °c or lp exposed to fraquil at either temperature. lp exposed to fraquil was significantly more resistant to gentamycin than lp grown in aye broth, regardless of the temperature. moreover, exposure to 37 °c seems to slightly increase susceptibility to gentamycin. lp was more resistant to kanamycin after exposure to fraquil than when grown in aye, and this difference was more pronounced at 37 °c. overall, lp is more resistant to aminoglycosides after exposure to fraquil, which is consistent with our prediction based on the transcriptomic data. incubation temperature also seems to affect antibiotic resistance, especially against erythromycin.fig. lp exposed to water is more resistant to antibiotics. cfu changes of lp cultured in aye broth or exposed to fraquil at or 37 °c after antibiotic treatments. the antibiotics used include ampicillin, erythromycin, gentamycin and kanamycin, all at 100 μg/ml. 5x indicates a five times concentration of erythromycin and gentamycin were used as well. the data are expressed as the log transformation of cfus in treated wells divided by the cfus in the control wells without antibiotics. a negative value indicates a cfu reduction upon treatment. data shows the mean +/− sd of three biological replicates. different letters on the bars indicate significant differences between different samples in a particular treatment according to tukey's test. cfu changes between different treatments were not compared importance of bdha for the survival of lp in water genes that are significantly induced upon exposure to fraquil may be important for lp to survive in water. upon this premise, the highly up-regulated bdha gene was selected for further characterization. a deletion mutant of bdha and its complement were constructed. for the complementation, bdha was cloned downstream of the ptac promoter on pmmb207c. the wild-type strain ks Δbdha, spf without isopropyl-β-d-thiogalactopyranoside and spf with 1 mm iptg were exposed to fraquil at and 37 °c. cfu counts were monitored weekly and their survival was also assessed by live/dead staining after 19 weeks of exposure, when the cfus of all strains at 37 °c dropped below the detection limit. no survival defects were observed in the mutant strain compared to the wild-type or the complements in fraquil at 25 °c. however, a minor reduction in the cfu counts was observed in all strains after 20 weeks of water exposure. an overall faster drop in cfus was observed in all strains at 37 °c, compared to 25 °c. the bdha mutant strain showed a more rapid reduction in cfus than the wild-type strain. this phenotype was complemented by expression of bdha from the ptac promoter in the presence of iptg. the cfu count of spf without iptg was similar to the bdha mutant strain. therefore, bdha seems to be required for the survival of lp in water at 37 °c.fig. bdha is important for long-term survival of lp in water. weekly cfu counts of the wild-type ks Δbdha and its complement spf with and without iptg exposed to fraquil at 25 °c and 37 °c. 1 mm iptg was used to induce bdha on the plasmid of the complemented strain. data show the mean +/− sd of three biological replicates. dl: detection limit. c percentage of live, undefined or dead cells of ks Δbdha and its complement spf with and without iptg. live/dead staining analysed by flow cytometry was performed after the strains were exposed to fraquil for 19 weeks at and 37 °c since the cfu counts of all strains exposed to 37 °c decreased below the detection limit after 19 weeks of water exposure, we sought to determine if the cells entered a viable but non-culturable state at this time point. to this end, we used a live/dead staining procedure together with flow cytometry. at 25 °c, no significant differences in the percentage of viable cells were observed between strains, which is consistent with the cfu counts. on the other hand, the wild-type strain showed a small fraction of dead cells at 37 °c, which is not significant compared to 25 °c. in comparison, only 61 % cells of the bdha mutant strain were stained as viable. the complemented strain exposed to water in the presence of iptg had a higher percentage of viable cells but was still lower than that of the wild-type strain. taken together with the cfu counts, our results show that lp enters a vbnc state after 19 weeks of exposure to fraquil at 37 °c. regulation of bdha by rpos hovel-miner et al. showed that the expression of bdha in a rich medium is regulated by rpos when lp reaches the post-exponential phase. moreover, transcriptomic analysis of the rpos mutant in water suggests that the expression of bdha is rpos-dependant. therefore, we investigated this possibility of rpos regulation of bdha by using a green fluorescent protein reporter assay. briefly, a plasmid carrying the pbdha-gfp transcriptional fusion was constructed and transformed into jr and the rpos mutant. jr harbouring the pxdc plasmid, which expresses gfp from the ptac promoter in the presence of iptg, was used as positive control. jr and rpos mutant strains with a plasmid containing no promoter upstream of the gfp encoding sequence served as negative controls. each strain was exposed to aye broth or fraquil for 24 h and the level of gfp in terms of green fluorescence signal was measured by flow cytometry. a significantly higher level of gfp was found to be expressed in ks psf exposed to fraquil than that exposed to aye broth, suggesting that the promoter of bdha was more highly induced after 24 h in water. this induction is consistent with the up-regulation of bdha observed in jr exposed to water for the same period of time during the transcriptomic analysis. gfp expression in rpos psf was higher than in the negative controls under both conditions, but it was significantly lower than that of ks psf in addition, no significant differences were found between rpos psf in aye broth compared to fraquil. these findings confirm that expression of bdha in lp is positively regulated by rpos.fig. bdha is highly induced in water and regulated by rpos. green fluorescence signal of five different strains after 24 h of exposure to aye broth or fraquil. jr psf rpos psf jr psf and rpos psf represent spf spf spf and spf respectively. jr pxdc was induced with 1 mm iptg. each sample had three biological replicates and the mean green fluorescence signal of cells in each replicate was detected by flow cytometry discussion as a bacterium that lives in aqueous environments, lp is frequently exposed to various stresses, such as fluctuations in temperature, ph and levels trace metals, as well as nutrient limitation. nutrient limitation, in particular, is a major concern as the nutrient levels in freshwater rarely fits the growth requirement of lp, which is an auxotroph for at least eight amino acids. in the absence of essential nutrients or suitable hosts, lp cannot grow, but can still persist in water for over one year. previous work has shown that the t2ss is important for the survival of lp in water at low temperature. however, no studies have investigated the global transcriptomic response of lp to freshwater. in response to this knowledge gap, we performed a transcriptomic analysis to study the changes in gene expression of lp exposed to fraquil, an artificial freshwater medium composed of trace amounts of salts and metals, compared to lp grown in rich medium. since lp in stationary phase culture is under nutrient limitation and stresses, using it as the control may hinder the opportunity to identify important genes for its survival in water. therefore, lp in the exponential phase of growth was used as the control. in fraquil, lp maintained a stable population for at least 35 days and did not show any growth. nevertheless, a significant reduction in cell size was found within 24 h of water exposure, which might be an adaptative response of lp to nutrient limitation. in fact, the reduction of cell size is commonly observed in bacteria experiencing environmental stresses, such as nutrient limitation, suboptimal ph and low temperatures. for example, the cell length of v. parahaemolyticus under starvation decreased drastically from to  μm within 24 h. such a morphological change is believed to be a strategy to minimize the metabolic requirements for cell maintenance. the mature intracellular form of lp, produced after passage through hela cells and tetrahymena tropicalis, also shows a reduction in cell size, suggesting that this morphological change may be a response of lp to nutrient limitation. the transcriptomic study of lp revealed significant changes in gene expression following exposure to fraquil. since fraquil does not contain any carbon sources that lp can use, it is unable to grow in this medium. consistent with this observation, expression of several amino acid transporters was induced in water. therefore, the reduced expression of three cell division proteins, such as ftsa, ftsq and ftsw, was expected. moreover, bacteria tend to shut down major metabolic pathways when under starvation. following exposure to water, genes involved in the electron transport chain and eight genes encoding subunits of the atp synthase were significantly repressed. this suggests a lower level of energy metabolism and thus, reduced metabolic activity in lp, as the down-regulation of atp synthase is usually found in dormant bacteria with a lower cellular atp level. furthermore, compared to the control growing in aye broth, the total rna extracted from lp exposed to water dropped markedly after 2 h and reduced to only one tenth of the control after 24 h. this finding is consistent with the down-regulation of rpoa, rpob and rpoc, which encode subunits of rna polymerase, potentially resulting in less rna polymerase available to bind to dna and initiate transcription. in addition, most of the genes encoding 30s ribosomal proteins, 50s ribosomal proteins, translation initiation factors, translation elongation factors as well as trnas were down-regulated in water in two or all three time points tested. in total, the expression of eight rpm genes, rps genes and rpl genes was significantly reduced. in addition, lpg was strongly induced in water. this gene encodes a homolog of yhbh, which is a short hibernation promoting factor with a highly conserved function in γ-proteobacteria. short hpf stabilizes the dimerization of two 70s ribosomes into a translationally inactive 100s ribosome by a ribosome modulation factor, resulting in ribosome hibernation. ribosome hibernation is commonly found in bacteria experiencing nutrient limitation and is rapidly reversible when nutrients become available once again. in e. coli, a mutant unable to form 100s ribosomes survived poorly in the post-exponential phase. therefore, it is possible that the up-regulation of lpg in lp may allow the formation of 100s ribosomes to maintain long-term survival in water, and the ability to resume growth or intracellular multiplication. our findings suggest that the translational machinery of lp is shut down following exposure to water, which is a typical characteristic of the bacterial stringent response. the stringent response is the reallocation of cellular resources in bacteria under nutrient limitation, by prohibiting the synthesis of dna, stable rnas, ribosomal proteins and membrane components, and activating the synthesis of factors involved in amino acid synthesis, glycolysis and stress resistance. the massive transcriptomic changes during the stringent response are mediated by the synthesis and accumulation of guanosine tetraphosphate and guanosine pentaphosphate, which are called ppgpp collectively. the stringent response in lp has been studied in detail. under amino acid limitation, the binding of uncharged trnas to the a site in ribosomes triggers the ribosomal-associated rela protein to synthesize ppgpp. spot, the second stringent response protein, can also synthesize ppgpp in response to other signals, such as perturbation of fatty acid synthesis. spot possesses a hydrolase activity as well, and therefore, regulates the level of ppgpp and is necessary to terminate the stringent response. in lp, the stringent response, and the rela and spot proteins are necessary for replication inside host cells and initiation of the transmissive phase. our results clearly show a repression of the translational machinery, cell replication and changes in metabolic processes, which are consistent with the general transcriptional modulations caused by ppgpp. in addition, we have recently shown that the stringent response is required for the survival of lp in water. due to the imminent shutdown of the translational machinery following water exposure, the important genes should be induced early on to allow the synthesis of their products when lp is still translationally active. therefore, in order to identify genes essential to maintain long-term survival in water, we examined those that are highly up-regulated within 24 h, a time point when a major metabolic shutdown started to appear. first, many genes associated with the biosynthesis of flagella, including different assembly, regulatory and structural proteins, are up-regulated in lp exposed to water. this response is comparable to the response of lp that reaches the late replicative phase inside host cells. due to exploitation of host cell resources and consequent lack of nutrients, the transmissive phase of lp is triggered, leading to the expression of virulence-associated traits such as flagella formation and other factors to promote release from the host. many genes induced at early time points possess a function related to detoxification and adaptation. genes involved in resistance to oxidative stress were induced, including sodc, ahpc and ahpd. we also observed the induction heat shock proteins in water. in e. coli, heat shock proteins are known to be induced by starvation. they increase bacterial tolerance against various stresses by degrading and reactivating damaged proteins. therefore, it is plausible that the up-regulation of these genes may help lp to persist in water under starvation conditions. in addition, many genes involved in antibiotic resistance were induced at early time points, including lpg, lpg, lpg, and several efflux pumps. increased resistance to antibiotics is a hallmark of lp mifs. this phenomenon was also described after incubation of lp in acanthamoeba castellanii buffer at ph . antibiotic resistance genes may be important for lp to compete against antibiotic-producing microorganisms residing in water. in water, lp is more resistant to gentamycin and kanamycin than when it is cultured in rich medium. water-exposed lp is also more resistant to erythromycin, but only at a temperature of 37 °c. since all these antibiotics target the ribosome, it is also possible that the increased resistance is due to a decrease in translational activity, and not solely because of the expression of resistance genes. differential expression of some genes involved in virulence was also apparent. despite the repression of of the structural genes of the icm/dot secretion system, the expression of many icm/dot effectors was increased in water. in addition, the enhanced-entry genes enha and enhb, and several of the enhc homologs, as well as the rtxa toxin were induced in water at early time points. on the other hand, the macrophage infectivity potentiator gene, mip, was significantly down regulated in water. it is not clear at this point if these changes in expression of virulence genes affect the intracellular multiplication of lp in host cells after incubation in water. we are currently investigating this possibility. many genes involved in transcriptional regulation were up-regulated, including numerous transcriptional regulators from different families, dna binding proteins, a transcription repair coupling factor and the alternative sigma factor σ. interestingly, rpon is important for the induction of flia and other flagellar genes. consistent with previous observations, our results show up-regulation of both σ and flagellar genes in water. several response regulators, sensory proteins and signal transduction proteins involved in “signal transduction/other regulatory functions” were also up-regulated. among these, many genes encoding proteins harbouring ggdef/eal domains were induced in water. these proteins are involved in the production and destruction of the second messenger c-di-gmp, and several of them have been shown to play a role in the interaction of lp with host cells. some of them could play a role in sensing exposure to water and regulating the appropriate response. moreover, expression of the two-component system leta/s was reduced in water, while expression of the small rnas rsmy and rsmz under its control was increased. these srnas are necessary to relieve csra-mediated repression of transmissive phase traits upon starvation inside host cells. our results suggest that some of the genes repressed by csra could be involved in the survival of lp in water. interestingly, a link has been made between leta/s, rpos and ppgpp. it is noteworthy that expression of the 6s rna was also induced in water. 6s rna binds to the rna polymerase holoenzyme and inhibits its binding to promoters. since the affinity of 6s rna to the rnap depends on sigma factors, 6s rna can shut down specific transcriptional programs. in e. coli, the σ regulon is turned off in the presence of 6s rna. in lp, deletion of 6s rna reduces fitness during intracellular growth. expression of 6s rna in water could help lp to switch between different transcriptomic programs, but further investigation is needed to confirm its involvement. there are also many other srnas that are differentially regulated, but since their targets are unknown, it is not yet possible to predict specific functions. within the highly up-regulated genes in water, we characterized bdha, which is involved in the polyhydroxybutyrate cycle. phb is an important storage polymer in bacteria, which is synthesized as a carbon and energy reserve in the presence of external nutrients and is consumed during starvation. the bdha gene encodes 3-hydroxybutyrate dehydrogenase, which oxidizes depolymerised phb into acetoacetate and produces reducing power in the form of nadh. acetoacetate can, then, be further processed into acetyl-coa, which enters the tricarboxylic acid cycle. bdha is dispensable for lp growth in the presence of external nutrients, as mutations in this gene do not result in any observable growth defects in aye broth. however, aurass et al. demonstrated that a bdha-patd mutant strain of lp is defective in breaking down phb accumulated during growth in a rich medium, resulting in a higher level of cellular phb than the wild-type. in addition, this mutant has defects in intracellular growth in amoeba and in human macrophages. therefore, we hypothesize that bdha is important for lp to survive in water, enabling the extraction of carbon and reducing power from phb in order to allow cell maintenance in the absence of an external energy source. our results show that the deletion of bdha reduces the survival of lp in water. the Δbdha strain lost culturability and seems to have entered the vbnc state earlier, resulting in a higher percentage of mortality after 19 weeks of water exposure at 37 °c compared to the wild-type. this survival defect was successfully complemented, supporting the hypothesis that lp needs bdha for the complete degradation of phb in order to maintain long-term survival in water. it is noteworthy that the difference in survival between Δbdha and the wild-type was only observed at 37 °c but not at 25 °c. since the samples were exhausted after 20 weeks of sampling, we could not determine if the observed difference was eventually be mirrored at 25 °c after a longer period of water exposure. it is possible that survival at 37 °c requires more energy for cell maintenance than at 25 °c because of a higher metabolic rate, protein turnover and overall damage, and thus, the advantage conferred by bdha is more apparent at the higher temperature. in addition, we investigated the regulation of bdha expression by rpos when lp is exposed to water. rpos is a well-known regulator of the response to nutrient limitation. recently, we have shown that the stringent response and the sigma factor rpos are necessary for the survival of lp in water. based on a gfp reporter assay, we found that bdha is positively regulated by rpos following exposure to water. this is consistent with the findings of hovel-miner et al., which show that, in a rich medium, bdha in lp is positively regulated by rpos in the post-exponential phase.  our study reveals, for the first time, the global transcriptomic changes of lp in water. repression of major pathways, such as cell division, transcription and translation, suggests that lp enters a quiescent state in water. the induction of the enhanced-entry genes and some icm/dot effectors suggests that lp may be primed to infect a suitable host. similarly, many genes involved in resistance to antibiotics and oxidative stress, as well as genes involved in the heat shock response were induced. furthermore, the bdha gene involved in the degradation pathway of the intracellular energy storage compound phb is highly expressed and positively regulated by rpos during short-term exposure to water. this gene was found to be important for maintaining long-term survival of lp in water. there is no doubt that many of the genes highly induced upon exposure to water are also necessary for the short and/or long-term survival of lp in water. 
we compared the global transcriptomic response of lp grown in a rich medium to that of lp exposed to an artificial freshwater medium for and 24 hours. indeed, lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than lp cultured in rich medium. the increase of antibiotic resistance and the importance of bdha to the survival of lp in water seem consistent with the observed induction of these genes when lp is exposed to water. legionella pneumophilasurvivalfreshwaterstringent responsetranscriptomeantibiotic resistancebdhaissue-copyright-statement© the author 2015 the induction of flagella associated genes, enhanced-entry genes and some icm/dot effector genes suggests that lp is primed to invade a suitable host in response to water exposure. the repression of major pathways involved in cell division, transcription and translation, suggests that lp enters a quiescent state in water. deletion of bdha reduces the survival of lp in water at 37 °c. in addition, the bdha gene, involved in the degradation pathway of the intracellular energy storage compound polyhydroxybutyrate, is also highly expressed in water. further characterization show that expression of bdha during short-term water exposure is dependent upon rpos, which is required for the survival of lp in water. moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that lp may be more tolerant to these stresses in water. we uncovered successive changes in gene expression required for the successful adaptation to a nutrient-limited water environment. legionella pneumophila is a water-borne opportunistic pathogen.
 legionella pneumophila is a water-borne opportunistic pathogen. in water, lp can survive for an extended period of time until it encounters a permissive host. therefore, identifying genes that are required for survival in water may help develop strategies to prevent legionella outbreaks.  we compared the global transcriptomic response of lp grown in a rich medium to that of lp exposed to an artificial freshwater medium for and 24 hours. we uncovered successive changes in gene expression required for the successful adaptation to a nutrient-limited water environment. the repression of major pathways involved in cell division, transcription and translation, suggests that lp enters a quiescent state in water. the induction of flagella associated genes, enhanced-entry genes and some icm/dot effector genes suggests that lp is primed to invade a suitable host in response to water exposure. moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that lp may be more tolerant to these stresses in water. indeed, lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than lp cultured in rich medium. in addition, the bdha gene, involved in the degradation pathway of the intracellular energy storage compound polyhydroxybutyrate, is also highly expressed in water. further characterization show that expression of bdha during short-term water exposure is dependent upon rpos, which is required for the survival of lp in water. deletion of bdha reduces the survival of lp in water at 37 °c.  the increase of antibiotic resistance and the importance of bdha to the survival of lp in water seem consistent with the observed induction of these genes when lp is exposed to water. other genes that are highly induced upon exposure to water could also be necessary for lp to maintain viability in the water environment. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords legionella pneumophilasurvivalfreshwaterstringent responsetranscriptomeantibiotic resistancebdhaissue-copyright-statement© the author 2015 