JB Accepted Manuscript Posted Online 28 August 2017 J. Bacteriol. doi:10.1128/JB.00472-17 Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1
The ferric uptake regulator Fur is conditionally essential in Pseudomonas aeruginosa
2 Martina Pasquaa, Daniela Visaggiob, Alessandra Lo Sciutoa, Shirley Genaha, Ehud Baninc, Paolo
4
Viscab#, Francesco Imperia#
5 6
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Laboratory
7
affiliated to Istituto Pasteur Italia – Fondazione Cenci Bolognetti, Rome, Italya; Department of
8
Sciences, University “Roma Tre”, Rome, Italyb; Institute for Nanotechnology and Advanced Materials,
9
Bar-Ilan University, Ramat Gan, Israelc.
10 11 12
Running head: Pyochelin impairs growth in Fur-depleted P. aeruginosa
13 14 15 16 17
#Address correspondence to Francesco Imperi, [email protected], and/or Paolo Visca,
18
[email protected].
19 20 21 22
Keywords: biofilm, colony formation, infection, iron uptake, oxidative stress, siderophore.
23
1
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
3
Abstract
25
In Pseudomonas aeruginosa the ferric uptake regulator protein (Fur) controls both metabolism and
26
virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion
27
mutants in P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this
28
bacterium. By investigating a P. aeruginosa fur conditional mutant, we demonstrate that Fur is not
29
essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect
30
model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are
31
severely impaired in colony formation. Transposon-mediated random mutagenesis experiments
32
identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the fur
33
conditional mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of
34
pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do
35
not accumulate higher levels of reactive oxygen species (ROS), and are not rescued by antioxidant
36
agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that
37
pyochelin production has no inhibitory effects in a fur deletion mutant of Pseudomonas syringae pv.
38
tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves P. aeruginosa-
39
specific pathway(s).
40 41
Importance
42
Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors
43
which control iron uptake and storage in response to iron availability, thereby playing a crucial role in
44
the maintenance of iron homeostasis. While fur null mutants have been obtained in many bacteria, Fur
45
appears essential in Pseudomonas aeruginosa for still unknown reasons. We obtained Fur-depleted P.
46
aeruginosa cells by conditional mutagenesis, and showed that Fur is dispensable for planktonic growth,
47
while it is required for colony formation. This is because Fur protects P. aeruginosa colonies from 2
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
24
48
toxicity exerted by the pyochelin siderophore. This work provides a functional basis to the essentiality
49
of Fur in P. aeruginosa, and highlights unique properties of the Fur regulon in this species.
50
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
3
Introduction
52
Iron is a critical nutrient for almost all forms of life, due to its fundamental role as a redox cofactor of
53
important enzymes involved in different cellular functions, such as respiration, catabolism, amino acid
54
and nucleoside synthesis, and stress response. Although essential for nutrition, iron can also have
55
deleterious effects if supplied in excess to living organisms. Indeed, intracellular Fe2+catalyses the
56
formation of reactive oxygen species (ROS) through the Fenton reaction, which can damage biological
57
macromolecules, such as proteins and nucleic acids, and lipids (1).
58
To reconcile their nutritional iron requirement with the need to maintain the intracellular iron content
59
below hazardous levels, bacteria have evolved sophisticated strategies to control iron homeostasis.
60
While most bacteria have the genetic potential to express different mechanisms for iron acquisition, as
61
a general rule the expression of iron uptake genes is repressed when sufficient amounts of intracellular
62
iron are available. This negative control occurs through the activity of a transcriptional repressor of the
63
Fur (or DtxR) family (2). When loaded with Fe2+, this repressor forms homodimers that bind the
64
operator sequence (Fur box) within target promoters, thereby blocking transcription of iron uptake
65
genes (3). In addition to this pivotal regulatory mechanism, which is responsible for the restricted
66
expression of iron uptake genes under conditions of iron starvation, iron acquisition is also regulated by
67
additional mechanisms which overall allow optimal expression of a given iron uptake system only
68
when it is effective in delivering iron to the cell (4). In many bacteria Fur also positively controls the
69
expression of non-essential iron-using proteins or ROS detoxifying enzymes, by repressing the
70
transcription of small RNA(s) which in turn inhibits the translation of target mRNAs (3,5). As a result,
71
genes that are involved in iron storage and/or detoxification are induced in the presence of high
72
intracellular iron levels, thus counteracting free iron toxicity as well as decreasing the amount of
73
sequestered (protein-bound) iron under low-iron conditions.
4
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
51
In spite of the crucial role of Fur in preserving iron homeostasis in bacterial cells, knock-out mutants in
75
the fur gene have been obtained in several bacterial species. These mutants showed variable and
76
species-specific phenotypes, ranging from constitutive expression of iron-uptake systems and/or
77
virulence factors to impaired acid, serum or oxidative stress resistance, defective motility and/or
78
biofilm formation (3). In general, fur deletion has minor effects on bacterial growth in vitro though it
79
can affect pathogenicity, since fur mutants of some pathogenic species appear less virulent and/or
80
invasive than their wild type counterparts (3,6). However, this is not a rule given that some bacterial
81
pathogens, such as pathogenic Escherichia coli strains and Vibrio vulnificus, showed no differences in
82
infectivity between wild type and fur mutant strains in animal models (7-9), suggesting that the
83
relevance of Fur during bacterial infections is species- and, likely, infection-model specific.
84
Notably, fur (or its analogous gene ideR) has been described as an essential gene in few bacterial
85
species, such as Pseudomonas aeruginosa and Mycobacterium tuberculosis, due to the inability to
86
obtain viable fur knock-out mutants (10-12). The relevance of fur for the fitness of the opportunistic
87
human pathogen P. aeruginosa was recently corroborated by two independent transposon sequencing
88
(Tn-seq) studies reporting that fur insertion mutants were outcompeted in transposon mutant pools
89
under several different growth conditions (13-14). In silico predictions and in vitro experiments,
90
including microarray comparison of gene expression between iron-replete and iron-depleted cells and
91
Fur-DNA pull down experiments, were used to define the Fur regulon in P. aeruginosa (15-18).
92
Overall, these and further confirmatory studies revealed that Fur, directly or indirectly via Fur-
93
regulated alternative sigma factors or AraC-like transcriptional regulators, basically represses all iron-
94
uptake genes, including those for biosynthesis and transport of the endogenous siderophores
95
pyoverdine and pyochelin, heme uptake and transport of exogenous iron chelators, and some virulence
96
factors. Moreover, Fur positively regulates iron-storage proteins (bacterioferritins), some enzymes
97
involved in carbon catabolism and respiration, and ROS-scavenging enzymes through Fur-mediated 5
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
74
repression of the two sRNAs PrrF1/2 (reviewed in refs. 19,20). This led to the proposal that Fur
99
essentiality could be related to the inability of P. aeruginosa to grow through fermentation and, thus, its
100
strict requirement to respire, which generates considerable potential for generation of ROS that would
101
not be efficiently detoxified in Fur-deficient cells (20,21). However, while the importance of Fur for
102
resistance to oxidative stress is overall conserved in bacteria (3), it should be noted that fur knock out
103
mutants have been obtained in other non-fermenting Gram-negative bacteria (e.g., Burkholderia
104
multivorans, Moraxella catarrhalis and some Pseudomonas species; 22-26), suggesting that alternative,
105
more specific harmful effect(s) could account for the critical role of Fur in P. aeruginosa physiology.
106
In this work, we used a fur conditional mutant of the reference strain P. aeruginosa PAO1 to
107
investigate the effect of Fur depletion both in vitro and in vivo, and employed a transposon mutagenesis
108
approach to identify genetic determinants affecting growth of Fur-depleted cells. Our results revealed
109
that Fur-depleted P. aeruginosa cells are similar to the wild type with regard to planktonic growth,
110
biofilm formation and pathogenicity in an insect model of infection. In contrast, we found that Fur is
111
essential for colony growth on agar plates, and that biosynthesis of the pyochelin siderophore is
112
partially responsible for this defect, through a mechanism that apparently does not depend on ROS
113
generation.
114 115 116
Results
117
Validation of the fur conditional mutant to investigate the effect of Fur depletion in P.
118
aeruginosa. In an attempt to investigate the possible involvement of Fur in cell aggregation-mediated
119
induction of iron uptake and virulence genes in P. aeruginosa, we recently generated a fur conditional
120
mutant in P. aeruginosa PAO1. This mutant carries an arabinose-inducible copy of the fur coding
121
sequence inserted into a neutral site (attB) of the PAO1 genome and an in-frame deletion in the 6
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
98
endogenous fur gene (27). With the aim of corroborating the suitability of the fur conditional mutant to
123
study the effect of Fur depletion in P. aeruginosa cells, we analyzed this mutant for (i) the effect of
124
arabinose on growth, (ii) Fur intracellular levels and (iii) expression of Fur-repressed genes in response
125
to iron. As shown in Figure 1A, the growth of the fur conditional mutant in microtiter plates in
126
Mueller-Hinton (MH) was impaired with respect to the parental strain PAO1, and was gradually
127
restored by arabinose in a dose dependent manner. In the absence of arabinose Fur was undetectable by
128
Western blot in cells of the fur conditional mutant, while it was detected at wild type levels in mutant
129
cells cultured in the presence of an arabinose concentration (0.5%; Fig. 1B) which completely restored
130
the growth of the fur conditional mutant. Since the inability to immunodetect a given protein is not
131
sufficient to rule out that basal protein levels potentially present in the conditional mutant could still
132
have an effect on Fur-dependent genes, we also monitored pyoverdine production, an easily-detectable
133
phenotype that is promptly repressed by Fur in response to iron (28). Pyoverdine production was about
134
10-fold higher in the fur mutant than in PAO1, but it decreased to wild type levels in the presence of
135
0.5% arabinose (Fig. 1C). More importantly, while addition of 50 µM FeCl3 to the medium shut down
136
pyoverdine production by wild type cells, it had no effect on the pyoverdine levels of the fur mutant
137
grown in the absence of arabinose (Fig. 1C), indicating that iron-mediated repression of Fur-regulated
138
genes is abrogated in the conditional mutant. This was confirmed by the observation that exogenously-
139
added iron was unable to repress transcription from promoters of genes directly repressed by Fur (pvdS
140
and pchR) in the fur conditional mutant (Fig. 1D). Taken together, these preliminary experiments
141
provide evidence that our fur conditional mutant can be used as a tool to investigate the effect of Fur
142
depletion in P. aeruginosa.
143 144
Fur-depleted P. aeruginosa cells show severe growth defects on solid media. In order to investigate
145
more in depth the effect of Fur depletion in P. aeruginosa, we compared planktonic growth of the wild 7
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
122
type strain PAO1 and the fur conditional mutant in flask cultures using different media containing
147
different iron concentrations. The fur conditional mutant was able to grow in the complex medium MH
148
(about 5-10 µM iron concentration; 29), although with lower growth rates and about 50% reduction in
149
growth yields with respect to the wild type PAO1 (Fig. 2A). The differences in growth rates and yields
150
between the two strains were markedly reduced in the iron-deprived complex medium TSBD (ca. 1.5
151
µM iron concentration; 30) (Fig. 2B), while no differences were observed in the iron-depleted minimal
152
medium DCAA, where iron concentration is very low (
1
The ferric uptake regulator Fur is conditionally essential in Pseudomonas aeruginosa
2 Martina Pasquaa, Daniela Visaggiob, Alessandra Lo Sciutoa, Shirley Genaha, Ehud Baninc, Paolo
4
Viscab#, Francesco Imperia#
5 6
Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Laboratory
7
affiliated to Istituto Pasteur Italia – Fondazione Cenci Bolognetti, Rome, Italya; Department of
8
Sciences, University “Roma Tre”, Rome, Italyb; Institute for Nanotechnology and Advanced Materials,
9
Bar-Ilan University, Ramat Gan, Israelc.
10 11 12
Running head: Pyochelin impairs growth in Fur-depleted P. aeruginosa
13 14 15 16 17
#Address correspondence to Francesco Imperi, [email protected], and/or Paolo Visca,
18
[email protected].
19 20 21 22
Keywords: biofilm, colony formation, infection, iron uptake, oxidative stress, siderophore.
23
1
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
3
Abstract
25
In Pseudomonas aeruginosa the ferric uptake regulator protein (Fur) controls both metabolism and
26
virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion
27
mutants in P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this
28
bacterium. By investigating a P. aeruginosa fur conditional mutant, we demonstrate that Fur is not
29
essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect
30
model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are
31
severely impaired in colony formation. Transposon-mediated random mutagenesis experiments
32
identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the fur
33
conditional mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of
34
pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do
35
not accumulate higher levels of reactive oxygen species (ROS), and are not rescued by antioxidant
36
agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that
37
pyochelin production has no inhibitory effects in a fur deletion mutant of Pseudomonas syringae pv.
38
tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves P. aeruginosa-
39
specific pathway(s).
40 41
Importance
42
Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors
43
which control iron uptake and storage in response to iron availability, thereby playing a crucial role in
44
the maintenance of iron homeostasis. While fur null mutants have been obtained in many bacteria, Fur
45
appears essential in Pseudomonas aeruginosa for still unknown reasons. We obtained Fur-depleted P.
46
aeruginosa cells by conditional mutagenesis, and showed that Fur is dispensable for planktonic growth,
47
while it is required for colony formation. This is because Fur protects P. aeruginosa colonies from 2
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
24
48
toxicity exerted by the pyochelin siderophore. This work provides a functional basis to the essentiality
49
of Fur in P. aeruginosa, and highlights unique properties of the Fur regulon in this species.
50
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
3
Introduction
52
Iron is a critical nutrient for almost all forms of life, due to its fundamental role as a redox cofactor of
53
important enzymes involved in different cellular functions, such as respiration, catabolism, amino acid
54
and nucleoside synthesis, and stress response. Although essential for nutrition, iron can also have
55
deleterious effects if supplied in excess to living organisms. Indeed, intracellular Fe2+catalyses the
56
formation of reactive oxygen species (ROS) through the Fenton reaction, which can damage biological
57
macromolecules, such as proteins and nucleic acids, and lipids (1).
58
To reconcile their nutritional iron requirement with the need to maintain the intracellular iron content
59
below hazardous levels, bacteria have evolved sophisticated strategies to control iron homeostasis.
60
While most bacteria have the genetic potential to express different mechanisms for iron acquisition, as
61
a general rule the expression of iron uptake genes is repressed when sufficient amounts of intracellular
62
iron are available. This negative control occurs through the activity of a transcriptional repressor of the
63
Fur (or DtxR) family (2). When loaded with Fe2+, this repressor forms homodimers that bind the
64
operator sequence (Fur box) within target promoters, thereby blocking transcription of iron uptake
65
genes (3). In addition to this pivotal regulatory mechanism, which is responsible for the restricted
66
expression of iron uptake genes under conditions of iron starvation, iron acquisition is also regulated by
67
additional mechanisms which overall allow optimal expression of a given iron uptake system only
68
when it is effective in delivering iron to the cell (4). In many bacteria Fur also positively controls the
69
expression of non-essential iron-using proteins or ROS detoxifying enzymes, by repressing the
70
transcription of small RNA(s) which in turn inhibits the translation of target mRNAs (3,5). As a result,
71
genes that are involved in iron storage and/or detoxification are induced in the presence of high
72
intracellular iron levels, thus counteracting free iron toxicity as well as decreasing the amount of
73
sequestered (protein-bound) iron under low-iron conditions.
4
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
51
In spite of the crucial role of Fur in preserving iron homeostasis in bacterial cells, knock-out mutants in
75
the fur gene have been obtained in several bacterial species. These mutants showed variable and
76
species-specific phenotypes, ranging from constitutive expression of iron-uptake systems and/or
77
virulence factors to impaired acid, serum or oxidative stress resistance, defective motility and/or
78
biofilm formation (3). In general, fur deletion has minor effects on bacterial growth in vitro though it
79
can affect pathogenicity, since fur mutants of some pathogenic species appear less virulent and/or
80
invasive than their wild type counterparts (3,6). However, this is not a rule given that some bacterial
81
pathogens, such as pathogenic Escherichia coli strains and Vibrio vulnificus, showed no differences in
82
infectivity between wild type and fur mutant strains in animal models (7-9), suggesting that the
83
relevance of Fur during bacterial infections is species- and, likely, infection-model specific.
84
Notably, fur (or its analogous gene ideR) has been described as an essential gene in few bacterial
85
species, such as Pseudomonas aeruginosa and Mycobacterium tuberculosis, due to the inability to
86
obtain viable fur knock-out mutants (10-12). The relevance of fur for the fitness of the opportunistic
87
human pathogen P. aeruginosa was recently corroborated by two independent transposon sequencing
88
(Tn-seq) studies reporting that fur insertion mutants were outcompeted in transposon mutant pools
89
under several different growth conditions (13-14). In silico predictions and in vitro experiments,
90
including microarray comparison of gene expression between iron-replete and iron-depleted cells and
91
Fur-DNA pull down experiments, were used to define the Fur regulon in P. aeruginosa (15-18).
92
Overall, these and further confirmatory studies revealed that Fur, directly or indirectly via Fur-
93
regulated alternative sigma factors or AraC-like transcriptional regulators, basically represses all iron-
94
uptake genes, including those for biosynthesis and transport of the endogenous siderophores
95
pyoverdine and pyochelin, heme uptake and transport of exogenous iron chelators, and some virulence
96
factors. Moreover, Fur positively regulates iron-storage proteins (bacterioferritins), some enzymes
97
involved in carbon catabolism and respiration, and ROS-scavenging enzymes through Fur-mediated 5
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
74
repression of the two sRNAs PrrF1/2 (reviewed in refs. 19,20). This led to the proposal that Fur
99
essentiality could be related to the inability of P. aeruginosa to grow through fermentation and, thus, its
100
strict requirement to respire, which generates considerable potential for generation of ROS that would
101
not be efficiently detoxified in Fur-deficient cells (20,21). However, while the importance of Fur for
102
resistance to oxidative stress is overall conserved in bacteria (3), it should be noted that fur knock out
103
mutants have been obtained in other non-fermenting Gram-negative bacteria (e.g., Burkholderia
104
multivorans, Moraxella catarrhalis and some Pseudomonas species; 22-26), suggesting that alternative,
105
more specific harmful effect(s) could account for the critical role of Fur in P. aeruginosa physiology.
106
In this work, we used a fur conditional mutant of the reference strain P. aeruginosa PAO1 to
107
investigate the effect of Fur depletion both in vitro and in vivo, and employed a transposon mutagenesis
108
approach to identify genetic determinants affecting growth of Fur-depleted cells. Our results revealed
109
that Fur-depleted P. aeruginosa cells are similar to the wild type with regard to planktonic growth,
110
biofilm formation and pathogenicity in an insect model of infection. In contrast, we found that Fur is
111
essential for colony growth on agar plates, and that biosynthesis of the pyochelin siderophore is
112
partially responsible for this defect, through a mechanism that apparently does not depend on ROS
113
generation.
114 115 116
Results
117
Validation of the fur conditional mutant to investigate the effect of Fur depletion in P.
118
aeruginosa. In an attempt to investigate the possible involvement of Fur in cell aggregation-mediated
119
induction of iron uptake and virulence genes in P. aeruginosa, we recently generated a fur conditional
120
mutant in P. aeruginosa PAO1. This mutant carries an arabinose-inducible copy of the fur coding
121
sequence inserted into a neutral site (attB) of the PAO1 genome and an in-frame deletion in the 6
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
98
endogenous fur gene (27). With the aim of corroborating the suitability of the fur conditional mutant to
123
study the effect of Fur depletion in P. aeruginosa cells, we analyzed this mutant for (i) the effect of
124
arabinose on growth, (ii) Fur intracellular levels and (iii) expression of Fur-repressed genes in response
125
to iron. As shown in Figure 1A, the growth of the fur conditional mutant in microtiter plates in
126
Mueller-Hinton (MH) was impaired with respect to the parental strain PAO1, and was gradually
127
restored by arabinose in a dose dependent manner. In the absence of arabinose Fur was undetectable by
128
Western blot in cells of the fur conditional mutant, while it was detected at wild type levels in mutant
129
cells cultured in the presence of an arabinose concentration (0.5%; Fig. 1B) which completely restored
130
the growth of the fur conditional mutant. Since the inability to immunodetect a given protein is not
131
sufficient to rule out that basal protein levels potentially present in the conditional mutant could still
132
have an effect on Fur-dependent genes, we also monitored pyoverdine production, an easily-detectable
133
phenotype that is promptly repressed by Fur in response to iron (28). Pyoverdine production was about
134
10-fold higher in the fur mutant than in PAO1, but it decreased to wild type levels in the presence of
135
0.5% arabinose (Fig. 1C). More importantly, while addition of 50 µM FeCl3 to the medium shut down
136
pyoverdine production by wild type cells, it had no effect on the pyoverdine levels of the fur mutant
137
grown in the absence of arabinose (Fig. 1C), indicating that iron-mediated repression of Fur-regulated
138
genes is abrogated in the conditional mutant. This was confirmed by the observation that exogenously-
139
added iron was unable to repress transcription from promoters of genes directly repressed by Fur (pvdS
140
and pchR) in the fur conditional mutant (Fig. 1D). Taken together, these preliminary experiments
141
provide evidence that our fur conditional mutant can be used as a tool to investigate the effect of Fur
142
depletion in P. aeruginosa.
143 144
Fur-depleted P. aeruginosa cells show severe growth defects on solid media. In order to investigate
145
more in depth the effect of Fur depletion in P. aeruginosa, we compared planktonic growth of the wild 7
Downloaded from http://jb.asm.org/ on August 28, 2017 by Univ of Texas Austin
122
type strain PAO1 and the fur conditional mutant in flask cultures using different media containing
147
different iron concentrations. The fur conditional mutant was able to grow in the complex medium MH
148
(about 5-10 µM iron concentration; 29), although with lower growth rates and about 50% reduction in
149
growth yields with respect to the wild type PAO1 (Fig. 2A). The differences in growth rates and yields
150
between the two strains were markedly reduced in the iron-deprived complex medium TSBD (ca. 1.5
151
µM iron concentration; 30) (Fig. 2B), while no differences were observed in the iron-depleted minimal
152
medium DCAA, where iron concentration is very low (
