INTRODUCTION
Iron is an essential component for virtually all forms of life. This includes bacterial
pathogens that depend on acquiring iron from their hosts in order to replicate and
cause disease (1). A general defensive mechanism of the host is therefore to withhold
iron from invading bacteria to prevent their growth, but this defense is countered
by bacterial pathogens since they possess specific systems to scavenge iron from their
hosts. While the regulation and mechanisms of several of such iron-scavenging systems
are well described (1), not much is known about how the within-host selection pressures
act on the pathogens’ ability to acquire iron. This is especially relevant in relation
to long-term chronic infections in which invading bacteria acquire adaptive mutations
in response to the selective pressures encountered in the host.
The opportunistic pathogen Pseudomonas aeruginosa is a common environmental inhabitant
which is capable of causing long-term chronic infections in the airways of patients
with cystic fibrosis (CF), and P. aeruginosa infections are directly associated with
the morbidity and mortality of CF patients. Chronic infections in CF patients provide
an opportunity for long-term monitoring of the battle between the infecting bacteria
and the host (2
–
6) and thus offer an opportunity for observing evolutionary adaptation of P. aeruginosa
to the human host environment.
Most iron in the human body is bound in hemoglobin, which is an oxygen transport protein
in red blood cells (1). If not bound by essential proteins, such as hemoglobin, iron
is withheld and stored by binding to proteins like transferrin, lactoferrin, and ferritin.
P. aeruginosa is known to scavenge iron from the human host by both siderophore-based
systems and heme acquisition systems (7).
Siderophores are low-molecular-weight molecules secreted by bacteria. The strong association
of iron to siderophores enables them to remove iron from the human iron storage proteins,
whereupon the siderophore-iron complex can be taken up by cognate receptors at the
bacterial surface. The major siderophores secreted by P. aeruginosa are pyoverdine
and pyochelin (7), and iron-loaded pyoverdine and pyochelin are taken up by the outer
membrane receptors FpvA and FptA, respectively (8
–
10).
Alternatively, iron contained in the heme group of hemoglobin can be taken up by either
of two heme uptake systems in P. aeruginosa. The two systems are the Pseudomonas heme
utilization (phu) system and the heme assimilation system (has) (11). The two systems
are different in the sense that the phu system is dependent on the direct uptake of
heme by the outer membrane receptor PhuR, whereas the has system encodes a secreted
hemophore, HasA, that returns heme to an outer membrane receptor, HasR.
Furthermore, P. aeruginosa can take up ferrous iron through the feo system (12) or
ferric citrate through the fec system (13).
It is not clear in which way the different iron uptake systems in P. aeruginosa play
a role for survival in the lungs of CF patients. Detection of pyoverdine in the sputa
of some CF patients has led to the suggestion that pyoverdine plays a key role in
the infection process (14, 15). On the other hand, measurements of the transcription
levels of iron uptake systems in sputum samples have suggested that multiple systems
are active and that siderophore-mediated uptake may not be the dominant iron acquisition
mechanism in all patients (16, 17).
In an effort to understand the genetic adaptation of P. aeruginosa to the CF airways,
we recently mapped all mutational changes in the P. aeruginosa DK2 lineage as it spread
among 21 Danish CF patients by interpatient transmission (2). The study showed that
the selective forces driving the evolution of P. aeruginosa in the CF airways could
be inferred from convergent evolution of DK2 sublineages evolving in parallel in separate
hosts. Here we further analyzed the genomic data, and we provide evidence that within-host
evolution of P. aeruginosa is characterized by adaptation toward iron acquisition
from hemoglobin.
RESULTS AND DISCUSSION
Parallel evolution of mutations in the promoter regions of the phu system.
It is known that P. aeruginosa undergoes genetic adaptation to CF patients during
long-term chronic infections, and several studies have sequenced the genomes of P. aeruginosa
isolates sampled longitudinally from the airways of CF patients to map the mutations
that accumulate during infection (2
–
6). In one such study, we mapped all the mutations that had occurred in the P. aeruginosa
DK2 lineage during 36 years of infection (2). Whole-genome analysis of 55 DK2 isolates
enabled a fine-grained reconstruction of the evolutionary relationship of the DK2
lineage, and the study identified several genes to be targeted by mutation to optimize
pathogen fitness within the host environment (pathoadaptation). Nonetheless, only
intragenic mutations (i.e., mutations within genes) were examined to identify such
pathoadaptive patterns of mutation. Here, we therefore reanalyzed the data with respect
to intergenic regions, since selection might also act on such sequences due to their
role in regulation and transcription of neighboring genes.
The 6,402,658-bp genome of the P. aeruginosa DK2 strain contains 4,883 intergenic
regions with an average size of 146 bp, and the intergenic regions constitute a total
of 714,368 bp. Marvig et al. (2) found 1,365 intergenic mutations, meaning that one
would expect an average-length intergenic region to be hit by 0.3 mutations (or 0.0019
mutation/bp). Searching for recurrent patterns of mutation of the same genetic loci
makes it possible to identify positive selection for mutations affecting genes important
for host adaptation (2, 18, 19). We therefore focused on the intergenic regions with
the highest densities of mutations and interestingly found the 180-bp intergenic region
containing the promoters of the phu system to be the most frequently mutated, with
a total of 13 mutations (0.072 mutation/bp) (Fig. 1). This number of mutations is
38-fold higher than what would be expected by chance and represents a significant
increase in mutation density [P(X ≥ 13) ~ pois(X; 0.342) = 2.22e-16, where P(X ≥ 13)
is the probability of observing ≥13 mutations given a Poisson distribution with a
mean of 0.342 mutations (0.0019 mutation/bp ∗ 180 bp)].
FIG 1
Maximum-parsimony phylogenetic tree showing the genetic relationship of the 11 DK2
clones included in this study. The phylogenetic tree is a subset of a phylogenetic
tree from the work of Marvig et al. (2), who recently reported the genome sequences
of 55 DK2 isolates. The shown tree depicts the genetic relationship of the 11 DK2
isolates included in this study, and it represents a total of 1,827 mutations (1,486
SNPs and 311 insertion/deletions) identified from whole-genome sequencing. Lengths
of branches are proportional to the numbers of mutations except in the case of the
truncated branch leading to isolate DK2-CF222-2001. For this hypermutator isolate,
the large number of mutations is indicated at the end of the truncated branch. We
searched the genomes for nonsynonymous mutations within genes encoding components
of the pyoverdine, pyochelin, phu, has, feo, and fec iron acquisition systems (7,
11–13), and circles on the evolutionary branches denote that the specified gene is
mutated in the branch. Due to the large number of mutations in the branch leading
to the hypermutable isolate DK2-CF222-2001, only phuR and phuSTUVW intergenic mutations
are specified. *, in addition to the three phuR and phuSTUVW intergenic mutations,
this branch also contains nonsynonymous mutations in pvdS, pvdL, fpvI, the FpvAII
gene, fpvR, phuR, fptA, pchH, pchG, pchF, pchE, and pchD (2).
All of the 13 mutations are located within a narrow region from position −91 to −21
relative to the start codon of phuR, and eight of the mutations are within the annotated
promoter regions of the phu system (Fig. 2). Furthermore, two positions (positions
−35 and −57) were subject to convergent evolution, since they were independently mutated
in parallel evolving DK2 sublineages.
FIG 2
Overview of the intergenic region upstream of phuR. The alignment shows homologue
sequences from different isolates with genetic variants highlighted in bold. Wild-type
sequences of P. aeruginosa strains PAO1, DK1, DK2, and C are shown at the top of the
alignment. Abbreviations of sequence alleles from different isolates are indicated
in parentheses (WT and M1 to M10). Positions of promoters and a Fur box are indicated
with black lines above the alignment (the phuSTUVW promoter is only partially shown).
Positions are relative to the start codon of phuR.
Correlation between promoter mutations and phu transcription in isolates DK2-CF173-2005
and DK2-CF66-2008.
Using Affymetrix GeneChips, we have previously measured the full transcriptomes of
six of the 11 DK2 isolates listed in Fig. 1 (4), including four early DK2 isolates
without phu promoter mutations and two isolates, DK2-CF173-2005 and DK2-CF66-2008,
with phu promoter mutations. We hypothesized that the mutations, due to their location
immediately upstream of phuR and phuSTUVW, could cause an effect on the transcription
of the phu system. Accordingly, we found the transcription of the phuRSTUVW genes
to be upregulated in both of the mutated isolates (DK2-CF173-2005 and DK2-CF66-2008)
relative to that for their ancestors and a laboratory reference strain PAO1 (Fig. 3).
Most highly upregulated was phuR, showing 116- and 25-fold upregulation, respectively,
but also, the genes of the phuSTUVW operon were on average upregulated 8- and 4-fold,
respectively.
FIG 3
Relative transcriptional levels of genes encoding the phu system. The transcriptomes
of six of the DK2 isolates included in this study have previously been measured at
exponential growth phase in LB medium (4). The expression of the phu genes is shown
for each of the six clinical isolates relative to that for laboratory reference strain
PAO1. Values are averages for three replicates, and the values are normalized relative
to the transcription of the respective gene in strain PAO1.
The phu system is negatively regulated by the ferric uptake regulator (Fur) (11).
As an alternative hypothesis, we therefore speculated that the increased transcription
of the phu system in DK2-CF173-2005 and DK2-CF66-2008 might be due to a decreased
level or activity of the Fur protein. Nonetheless, no mutations or changes in transcription
of the fur gene were found (Table 1) (2).
TABLE 1
Relative transcriptional levels of fur and genes encoding the feo iron acquisition
pathway
a
Gene
Relative transcription in strain:
PAO1
DK2-CF114-1973
DK2-CF43-1973
DK2-CF66-1973
DK2-CF30-1979
DK2-CF173-2005
DK2-CF66-2008
feoA
1
2.9
1.6
16.7
21.2
21.6
28.1
feoB
1
2
1.6
5.1
6
6.8
13.4
feoC
1
1.3
1.5
2.3
2.8
2.4
4.4
fur
1
1.1
1.5
1.4
0.9
1.1
1
a
The transcriptomes of six DK2 isolates included in this study have previously been
measured at exponential growth phage in LB medium (4). We searched the transcriptomes
for genes encoding components of the pyoverdine, pyochelin, phu, has, feo, and fec
iron acquisition systems (7, 11–13), and the table lists the transcription profiles
of those systems in which at least one gene showed differential expression (>3-fold
change) in the post-1973 isolates relative to that in the 1973 isolates or strain
PAO1. Also, the transcription of the fur gene is shown. Values are averages for three
replicates, and the values are normalized relative to the transcription of the respective
gene in reference strain PAO1.
Furthermore, in order to determine if iron acquisition systems in general were subject
to evolutionary changes in transcription, we searched the transcriptomes for other
iron acquisition systems to be differentially transcribed. This search revealed that
the feo operon, encoding a ferrous iron uptake system (12), was upregulated in DK2-CF66-1973
and the four isolates sampled after 1973 (Table 1), indicating that several iron acquisition
systems might play a role in adaptation of P. aeruginosa to the human host airways.
Effect of intergenic mutations on activities of phu system promoters.
To further investigate the effect of the phu promoter mutations on the activity of
the phuR promoter, we cloned the phuR promoter region from six of the mutated DK2
clones in front of a luciferase reporter (luxCDABE) and chromosomally integrated the
transcriptional fusion into P. aeruginosa PAO1 at the attB site by use of the mini-CTX2-derived
plasmid pHK-CTX-lux. The transcriptional fusions enabled us to compare phuR::lux expression
from the mutated promoter regions (M1 to M6) (Fig. 2) relative to the expression from
a construct with a wild type promoter region (WT) (Fig. 2). A construct without an
inserted promoter region was used to correct for background expression from lux gene
cassette integration.
Measurements of phuR::lux expression at exponential growth (optical density at 600
nm [OD600] = 0.15) in Luria-Bertani (LB) medium revealed that all six mutant alleles
(M1 to M6) caused a significant increase in promoter activity, with changes in expression
from 5- to 112-fold (Table 2). The largest increases in expressions (93- and 112-fold)
were observed for the alleles M1 and M2, originating with clones DK2-CF66-2008 and
DK2-CF173-2005, respectively. The M1 and M2 alleles contain a 3-bp insertion and a
1-bp deletion, respectively, in the repressor-binding site (Fur box) of the Fur regulator,
known to control the expression of the phuR promoter (11). Since Fur mediates strong
repression of phuR under iron-rich conditions (11), we find it likely that the indels
in the M1- and M2-derived phuR promoters alleviate Fur repression (if there is any
repression from Fur).
TABLE 2
Activities of the phuR and phuS promoters originating with different clinical isolates
of P. aeruginosa
a
Strain
Promoter
Origin of promoter
Allele
Mean luminescence (± SD)
Fold change
P value
PAO1
phuR
PAO1
WT
365 (±1,018)
1
PAO1
phuR
DK2-CF66-2008
M1
34,111 (±3,379)
93
0.00021
PAO1
phuR
DK2-CF173-2005
M2
40,726 (±3,422)
112
0.00004
PAO1
phuR
DK2-CF173-2002
M3
1,879 (±3,422)
5
0.16
PAO1
phuR
DK2-CF240-2002
M4
7,584 (±496)
21
0.00038
PAO1
phuR
DK2-CF222-2001
M5
8,968 (±610)
25
0.00023
PAO1
phuR
DK2-CF180-2002
M6
6,723 (±701)
18
0.00088
PAO1
phuR
DK1-P28F1-1992
M8
13,329 (±1,482)
37
0.00024
PAO1
phuR
DK1-P28F1-2009
M9
12,205 (±603)
33
0.00007
PAO1
phuR
DK1-CF30-2011
M10
9,563 (±1,586)
26
0.0011
PAO1
phuS
PAO1
WT
7,444 (±1,777)
1
PAO1
phuS
DK2-CF173-2005
M2
12,030 (±3,191)
1.6
0.01
a
Luminescence production from laboratory reference strain PAO1 (37) with phuR::lux
reporter fusions was measured at exponential growth (OD600 = 0.15) in Luria-Bertani
(LB) medium and normalized for differences in cell density. Mean luminescence production
and standard deviations (SD) were calculated for three biological replicates. Statistical
analysis concerning the difference between two means was done using a Student t test,
and the P values denote the probability of the mutated alleles having expression equal
to that of the wild type (WT).
Using the same cloning strategy, we tested a phuS::lux reporter fusion to compare
the expression from the mutated promoter region of DK2-CF173-2005 to the expression
from a construct with a wild-type promoter region. Similar to the results for the
phuR promoter, we observed that the mutations also resulted in a significant (P =
0.01) increase in phuS promoter activity (Table 2), albeit the mutations had a larger
effect on the activity of the phuR promoter.
phuR promoter mutations confer a growth advantage in the presence of hemoglobin.
The increased expression from the mutated phu promoters suggested that there has been
positive selection in the CF airways toward iron acquisition from hemoglobin. To test
this hypothesis, we replaced the wild-type phu promoters of isolate DK2-CF30-1979
with the mutated phu promoters of isolate DK2-CF173-2005 by allelic replacement and
tested whether the constructed mutant strain, DK2-CF30-1979-M2, had a growth advantage
relative to the isogenic wild-type strain, DK2-CF30-1979. We chose to test the consequence
of the phu promoter mutations in the genetic background of isolate DK2-CF30-1979 because
this isolate is an immediate ancestor of isolate DK2-CF173-2005 (4). For the growth
experiment, we used FeCl3-free ABTGC minimal medium (which contains glucose and Casamino
Acids), supplemented with hemoglobin and apotransferrin.
Confirming our hypothesis, we found that the allelic replacement mutant DK2-CF30-1979-M2
grew significantly faster than its isogenic wild-type counterpart when hemoglobin
was present as the sole iron source (Table 3), while no difference was observed for
rich medium and medium supplemented with Fe3+ as the sole iron source. We suggest
that the growth advantage of the mutant is facilitated by an enhanced uptake of iron
derived from hemoglobin.
TABLE 3
Growth rates of strains DK2-CF30-1979 and DK2-CF30-1979-M2 at exponential growth phase
in different media
a
Growth medium
Doubling time (h)
P value
DK2-CF30-1979
DK2-CF30-1979-M2
LB
1.27 ± 0.05
1.35 ± 0.07
0.16
ABTGC + 10 µM Fe3+
2.74 ± 0.02
2.69 ± 0.03
0.23
ABTGC + 10 µM Fe3+ + 100 µg/ml apo-TF
3.08 ± 0.10
3.07 ± 0.04
0.91
ABTGC + 2.5 µM Hb + 100 µg/ml apo-TF
2.76 ± 0.24
2.13 ± 0.09
0.01
a
The abbreviations Hb and apo-TF are used for hemoglobin and apotransferrin, respectively.
Note that the ABTGC minimal medium standard recipe was modified so that no iron source
other than the one stated in the table was added to the growth medium. Mean doubling
times were calculated from three biological replicates. Statistical analysis concerning
difference between two means was done using a Student t test, and the P values denote
the probability of the two strains having equal means.
Adaptation toward heme utilization is a general adaptive mechanism.
Our results demonstrate parallel adaptation of the DK2 lineage toward hemoglobin utilization
in five different CF patients. This indicates that similar selective conditions for
heme utilization exist across different patients. Next, we speculated on whether the
acquisition of phu promoter mutations is an adaptive mechanism specific to the DK2
lineage or if phuR promoter mutations constitute a general adaptive genetic mechanism
of P. aeruginosa toward heme utilization in the CF airways. To further investigate
the generality, we compared our findings to other lineages of P. aeruginosa isolated
from CF infections.
In addition to the DK2 lineage, our previous investigations have revealed another
distinct clone type, known as the DK1 clone type, which has also spread among Danish
CF patients (21). We sequenced and analyzed the phuR promoter region of five DK1 isolates
sampled in the years 1992 to 2011 in addition to an ancestral DK1 isolate from 1973.
Whereas the sequence of the phuR promoter of the ancestral 1973 isolate (DK1-P33F0-1973)
was identical to the wild-type sequence of strains PAO1 and DK2, all five evolved
DK1 isolates had accumulated 1 to 4 single nucleotide polymorphisms (SNPs) in the
promoter region, and three of the DK1 SNPs were identical to SNPs found in the evolved
DK2 isolates (Fig. 2). We tested the activities of three of the mutated promoters
from the DK1 isolates (M8 to M10) and found that all three mutated promoters resulted
in increased levels of transcription, similar to what has been observed for mutated
DK2 alleles (Table 2). Our results provide strong evidence for convergent adaptive
evolution of different lineages of P. aeruginosa toward iron acquisition from hemoglobin.
To rule out that the adaptive trait was specific for P. aeruginosa CF infections at
the Copenhagen CF Center, we analyzed the available public data for the genomic evolution
of the P. aeruginosa C lineage, which was isolated from a patient attending the CF
clinic at Hannover Medical School, Germany (6). Interestingly, the C lineage, which
has colonized this patient for a period of more than 20 years, also accumulated two
SNPs in the phuR promoter region (Fig. 2). Remarkably, the two SNPs are identical
to SNPs found in the DK1 and DK2 lineages, and this observation suggests that these
mutations were also positively selected for in the host environment.
The research team at Hannover Medical School also investigated the microevolution
of a PA14 lineage as it infected a patient over 14 years. Nonetheless, the PA14 lineage
did not accumulate SNPs in any iron acquisition systems. Likewise, a lineage investigated
by Smith et al. (5) over an infection course of 90 months also did not reveal any
mutations in iron acquisition systems, except for a nonsynonymous mutation in pvdS
(which correlated with the loss of pyoverdine production) and an intergenic SNP upstream
of fptA (5). We therefore conclude that despite an apparent selection for phu promoter
mutations in three independent P. aeruginosa lineages, not all lineages accumulate
phu promoter mutations during CF infections.
Selection against pyoverdine secretion might lead to a shift in iron source.
The siderophore pyoverdine has previously been found in sputum of CF patients, and
thus pyoverdine-mediated uptake of iron has been considered important for the survival
of P. aeruginosa in the CF airways (14). Nonetheless, we observed that all three lineages
(DK1, DK2, and C) had accumulated nonsynonymous mutations in the alternative sigma
factor PvdS, which is required for pyoverdine synthesis (Fig. 1 and Fig. 4). Accordingly,
the evolved C clone NN80 was observed to have lost its ability to produce pyoverdine,
in contrast to its predecessors (C clones NN2 and NN11) (6).
FIG 4
Overview of pvdS mutations in the DK1 and C lineages. Mutations that have accumulated
in evolved isolates relative to sequences of their ancestor are shown. The pvdS mutation
found in the DK2 lineage is shown in Fig. 1.
This led us to examine the production of pyoverdine in the DK1 and DK2 isolates, and
we observed a negative correlation between pyoverdine production and mutations in
PvdS (Fig. 5). Accordingly, only the ancestral DK1 and DK2 isolates carrying wild-type
alleles of pvdS were able to produce pyoverdine, whereas all isolates carrying mutated
alleles of pvdS were unable to produce pyoverdine (DK1-CF173F-2002 was not tested).
FIG 5
Pyoverdine production in isolates of P. aeruginosa. The presence of pyoverdine secreted
into the supernatant of bacterial cultures grown in pyoverdine-inducing medium was
quantified by measurement of the absorbance at OD405 and normalized against the cell
density (OD600). The means and standard deviations calculated from three biological
replicates are shown in the bar plot.
Siderophores are generally regarded as highly immunogenic (22), and selection against
pyoverdine production might have driven the accumulation of pvdS mutations, leading
to a loss of pyoverdine production in the evolved isolates. At the same time, we observed
a positive selection for phuR promoter mutations in the CF airways, leading to a bacterial
growth advantage when acquiring iron from hemoglobin. We therefore propose a model
in which the CF airways impose selective pressure on the invading bacteria, forcing
them to adapt toward a shift to hemoglobin as an alternative iron source. This is
of particular interest because inflammation may cause microbleeds, which lead to the
presence of hemoglobin at the delicate CF lung epithelia in the presence of both host
and bacterial proteases (23). Also, hemoglobin is reported to be expressed by alveolar
epithelial cells (24).
Other iron acquisition systems might be affected by mutations.
Several iron acquisition systems and mutations other than the ones that we have investigated
in detail here might play a role in survival of P. aeruginosa in the lungs of CF patients.
Accordingly, we also found nonsynonymous mutations in the FpvAII gene and the genes
fpvI, fpvR, phuR, pchA, pchDEFGH, and fptA when searching for mutations in genes of
the pyoverdine, pyochelin, phu, has, feo, and fec iron acquisition systems (Fig. 1).
We anticipate that the identification of such mutations can facilitate further investigations
of the adaptation of P. aeruginosa to human host airways. For example, it remains
to be elucidated whether the mutations in the pch and fptA genes affect the function
the pyochelin iron uptake system in the DK2 lineage and if isolates with mutations
in the pyoverdine system are unable to cheat on other pyoverdine producers.
Conclusions and implications.
Our results provide evidence that the selective conditions by which evolution is directed
in the CF airways can result in acquisition of phu promoter mutations in P. aeruginosa
during chronic CF infections and that such mutations provide a growth advantage in
relation to acquisition of iron from hemoglobin. This adaptive trait may be directly
selected for due to an abundance of heme-bound iron in the CF lung. Furthermore, we
also observed that phu promoter mutations coincided with the loss of pyoverdine production,
suggesting that selection for increased heme utilization may be secondary to the loss
of the pyoverdine iron uptake system. Therefore, targeting heme utilization might
be a promising strategy for the treatment of CF infections.
CF patients commonly experience iron deficiency, and P. aeruginosa possibly contributes
to iron deficiency by depletion of the host iron storage and by causing inflammation
(25, 26). In this regard, expanding our knowledge of adaptation of P. aeruginosa to
the CF lung may help to lessen the impact of P. aeruginosa infection and improve the
condition of patients.
MATERIALS AND METHODS
Bacterial strains and media.
Isolates of the P. aeruginosa DK1 and DK2 clone types were sampled from Danish CF
patients attending the Copenhagen Cystic Fibrosis Clinic. Isolation and identification
of P. aeruginosa from sputum were done as previously described (27). The isolates
are named according to their clone type, the patient from whom they were isolated,
and their isolation year (e.g., isolate DK2-CF30-1979). Luria-Bertani (LB) broth was
used for routine preparations of bacterial cultures. ABTGC minimal medium was composed
of 2 g/liter (NH4)2SO4, 6 g/liter Na2HPO4, 3 g/liter KH2PO4, 3 g/liter NaCl, 1 mM
MgCl2, 0.1 mM CaCl2, 0.01 mM FeCl3, 2.5 mg/liter thiamine supplemented with 1% glucose,
and 0.5% Casamino Acids. For the growth rate experiments (Table 3), no FeCl3 was added
to ABTGC minimal medium unless otherwise stated. Human hemoglobin (Sigma-Aldrich)
and human apotransferrin (Sigma-Aldrich) were added to concentrations of 2.5 µM and
100 µg/ml, respectively. Pyoverdine-inducing medium was composed of ABTGC minimal
medium with 50 µM iron chelator 2,2′-dipyridyl (DIPY). Escherichia coli strain CC118(λpir)
was used for maintenance of recombinant plasmids (28) in medium supplemented with
8 µg/ml of tetracycline. Allelic replacement constructs were transferred to P. aeruginosa
by triparental mating using the helper strain E. coli HB101/pRK600 (29). For marker
selection in P. aeruginosa, 50 µg/ml of tetracycline was used. Genetic techniques
were performed using standard methods, and Sanger sequencing was used for verification
of genetic construct and allelic replacement mutants.
Sequencing of phuR promoter region and pvdS gene in DK1 isolates.
Sequencing of DK1 isolates was performed as described earlier (4). Accordingly, genomic
DNA was purified from P. aeruginosa isolates using a Wizard Genomic DNA purification
kit (Promega, Madison, WI) and sequenced on Illumina’s GAIIx or Hiseq2000 platform.
Reads were mapped against the reference genome sequence using the software program
Novoalign (Novocraft Technologies, Selangor, Malaysia) (30), and pileups of read alignments
were produced by the software program SAMtools, release 0.1.7 (31).
Construction of reporter fusions and luminescence measurements.
The lux gene cassette (luxCDABE) was subcloned from the plasmid pUC18-mini-Tn7T-Gm-lux
(32) fragment into mini-CTX2 (33) using the restriction sites XhoI and PstI to produce
pHK-CTX2-lux, used for the transcriptional fusion experiments. For phuR::lux reporter
fusions, a 220-bp fragment containing the intergenic region upstream of phuR was amplified
from genomic DNA using Phusion polymerase (Thermo Scientific) with the primers PhuR_F-PstI
(5′ GAGACTGCAGAGGCTGGGAGTGCTGCTCAT 3′) and PhuR_R-XhoI (5′ ACATCTCGAGAAGGGCGGGGAGAGCGGCAT 3′)
and ligated with T4 DNA ligase into pHK-CTX2-lux after double digestion of the PCR
fragment and vector with the restriction enzymes XhoI and PstI. For phuS::lux reporter
fusions, a 220-bp fragment containing the intergenic region upstream of phuS was amplified
with the primers PhuS_F-XhoI (5′ ACATCTCGAGAGGCTGGGAGTGCTGCTCAT 3′) and PhuS_R-PstI
(5′ GAGACTGCAGAAGGGCGGGGAGAGCGGCAT 3′) and ligated into pHK-CTX2-lux after double
digestion of the PCR fragment and vector with the restriction enzymes XhoI and PstI.
The resulting plasmids were introduced into P. aeruginosa strain PAO1 by transformation
as previously described (32).
Allelic replacement of phuR promoter region in DK2-CF30-1979.
A 1,296-bp fragment containing the intergenic region upstream of phuR was amplified
from genomic DNA of DK2-CF173-2005 using Phusion polymerase (Thermo Scientific) with
the primers PhuSi_F-XbaI (5′-ACATTCTAGACGGACGTCGCTGGCCTCG-‘3) and PhuRi_R-SacI (5′-GAGAGAGCTCTCTCGTGGCCCTGGCGGTAG-3′).
The PCR fragment was ligated into the vector pNJ1 (34) after digestion with the restriction
enzymes XbaI and SacI. The allelic replacement construct was transferred into strain
DK2-CF30-1979 by triparental mating, and merodiploid mutants were selected by plating
the conjugation mixture on LB agar plates with tetracycline. Colonies were restreaked
on selective plates before being streaked on 8% (wt/vol) sucrose-LB plates without
NaCl. Sucrose-resistant and tetracycline-sensitive colonies were restreaked on sucrose-LB
plates and screened for the presence of mutated alleles by PCR followed by restriction
fragment length polymorphism (RFLP) analysis. Positive mutants were finally sequenced
by Sanger sequencing at LGC genomics (Germany).
Measurement of growth and luminescence in reporter fusion strains.
Overnight cultures of the reporter fusion strains were diluted 40 times in fresh LB,
and aliquots of 100 µl were transferred to a black (clear-bottom) 96-well microtiter
plate (Nunc). Three technical replicates were used for each strain, and measurements
of growth (OD600) and luminescence were recorded in a Synergy Hybrid H1 reader (Bio-Tek)
with 6-min intervals for 10 h and under shaking conditions (200 rpm) at 37°C. Data
were analyzed using a custom-made script in the R software environment, version 2.15.2
(35). The experiment was repeated three times to obtain biological replicates.
Growth rate measurements.
Growth rate experiments were carried out in 250 ml baffled shake flasks containing
50 ml of growth medium under shaking (200 rpm) at 37°C. Culture flasks were inoculated
to a starting OD600 of 0.005 in 50-ml minimal medium, and measurements of OD600 were
started 9 h after the inoculation and recorded every 30 min. In the experiment where
the cells were cultivated in LB, the measurements were started after 2 h. The experiment
was stopped when the cells reached stationary growth phase, typically after around
23 h of growth in minimal medium. Growth experiments were repeated three times for
each strain under each condition to obtain biological replicates.
Pyoverdine quantification assay.
Pyoverdine concentrations were quantified as previously described (36). All strains
were grown in pyoverdine inducing medium for up to an OD600 of >1.5. Cultures were
moved into 2-ml microcentrifuge tubes and centrifuged at 16,000 × g for 2 min. The
supernatants were diluted in 100 mM Tris-HCl buffer (pH 8), and pyoverdine concentrations
were quantified by measurement of the absorbance at OD405. Finally, the values of
absorbance at OD405 were normalized against the cell densities (OD600) for each strain.
The procedure was repeated for three independent biological replicates.