Different Responses of Pyoverdine Genes to Autoinduction in Pseudomonas aeruginosa and the Group Pseudomonas fluorescens-Pseudomonas putida

Abstract

We investigated the regulation of the psbA and pvdA pyoverdine biosynthesis genes, which encode the l-ornithine N⁵-oxygenase homologues in Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1, respectively. We demonstrate that pyoverdine_B10, as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in Pseudomonas strain B10. In P. aeruginosa PAO1, however, pyoverdine_PAO1 has no apparent role in the positive regulation of the pvdA gene.

The competition for iron is a main determinant for the dynamics of microbial populations in natural ecosystems. Aerobic bacteria living in neutral environments are normally faced with the nutritional iron deficit resulting from the low solubility of iron in its oxidized state (13). Likewise, bacterial parasites, which multiply within higher organisms, must counteract the iron deficiency imposed by the activity of the host's iron binding proteins (15). Iron withholding also affects the interactions between microbial populations in the rhizosphere (14).

Pyoverdines (also referred to as pseudobactins), the fluorescent siderophores produced by the rRNA group I species of genus Pseudomonas, constitute a large family of iron chelators that differ in terms of the length and composition of a hydroxamate-containing oligopeptide joined to a structurally conserved dihydroxyquinoline chromophore (1). Due to their high affinity for ferric iron, pyoverdines can suppress the growth of deleterious rhizomicroorganisms, which lack uptake specificities for ferripyoverdine complexes, thereby contributing to the plant defense from parasitic infection (2, 14).

In Pseudomonas spp. and many other bacteria, regulation of iron transport is primarily controlled by the Fur repressor protein (22). When the level of intracellular iron is high, transcription of iron-repressible genes is blocked through the binding of Fur-Fe(II) complexes to a highly conserved sequence (the Fur box) located within the promoters of these genes (5). However, Fur is not the only regulator of iron uptake in fluorescent pseudomonads (24). In Pseudomonas aeruginosa, the rRNA group I type species, expression of pyoverdine_PAO1 (pvd) genes is controlled by iron through a cascade of negative and positive regulatory proteins (Fig. 1). Briefly, Fur acts as a repressor of the pvdS gene, encoding an extra cytoplasmic function (ECF) sigma factor, called PvdS, which is required for transcription initiation at the promoters of pvd genes (4, 9). A DNA sequence element, the iron starvation box (ISB), has been identified in PvdS-dependent promoters and has been shown to participate in sequence-specific promoter recognition by PvdS, acting as a −35-like sequence element (27). Several Fur-controlled alternative sigma factors, structurally related to PvdS, have been identified in fluorescent Pseudomonas spp. and have been found to be involved in pyoverdine synthesis and/or uptake (7, 20, 23). An additional level of complexity of the siderophore regulatory pathway in these species is inferred by the positive effect of ferric siderophore complexes on the expression of cognate biosynthesis genes. Siderophore-regulated responses have previously been demonstrated for the pyochelin-salicylate operons of P. aeruginosa and for the pyoverdine gene systems of Pseudomonas putida WCS358 and Pseudomonas fluorescens M114 (3, 16, 23, 24).

FIG. 1.

Regulatory models of pyoverdine genes in P. aeruginosa PAO1 and in Pseudomonas strain B10. (A) In iron-replete P. aeruginosa PAO1 cells, transcription of pvdS is prevented by the binding of dimeric Fur-Fe(II) complexes to Fur boxes overlapping the pvdS promoter. In the absence of sufficient iron, release of Fur from DNA allows pvdS to be transcribed. The ECF sigma factor PvdS mediates recognition of the ISB, thereby conferring specificity to core RNA polymerase (cRNAP) for transcription initiation at the pvd promoters. (B) In Pseudomonas strain B10, expression of the psbA gene is similarly regulated by the Fur-controlled ECF sigma factor PsbS. An additional level of regulation is provided by exogenous pyoverdine_B10, which exerts a positive induction effect on expression of the psbA biosynthetic gene.

The pyoverdine_B10 biosynthesis gene psbA, encoding the enzyme l-ornithine (l-Orn) N⁵-oxygenase (PsbA) in the plant growth-promoting Pseudomonas strain B10, has previously been characterized in our laboratory (2). PsbA expression is regulated at the transcriptional level by a Fur-controlled ECF sigma factor, designated PsbS (8). Herein, we provide evidence that pyoverdine_B10 itself plays a crucial role in the expression of the psbA gene. The comparison of autogenous regulation of pyoverdine biosynthesis in Pseudomonas strain B10 and P. aeruginosa PAO1 highlighted relevant differences between the two species.

The bacterial strains and plasmids used in this study are listed in Table 1. The media and growth conditions have been described previously (6, 9, 25).

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Genotype and/or relevant characteristics	Source or reference
Strains
Pseudomonas	Prototroph	12
B10
B10CA1	psbA::cat site-specific insertion mutant	2
P. aeruginosa
PAO1 (ATCC 15692)	Prototroph	American Type Culture Collection
PALS124	pvdA	26
PALS106	pvdC1	26
PALS125	pvdC3	26
Plasmids
pMP220	Broad-host-range, low-copy-number promoter-probe vector, IncP replicon, lacZ Tcr Tra−	21
pMP220::PpvdA	pvdA promoter directionally cloned in pMP220 (formerly designated pPV51)	9
pMP220::PpsbA	psbA promoter from pMP190::PpsbA directionally cloned in pMP220	This study
pMP190	Broad-host-range, low-copy-number promoter-probe vector, IncQ replicon, lacZ Cmr Tra−	21
pMP190::PpsbA	psbA promoter directionally cloned in pMP190	8
pMP190::PpsbS	470-bp PCR-generated fragment encompassing the psbS promoter directionally ligated to the KpnI-SalI sites of pMP190	8
pMP220::PpsbS	DNA fragment of ca. 530 bp excised from pMP190::PpsbS by HindIII digestion and directionally ligated to the HindIII site of pMP220	This study
pMP220::PpvdS	587-bp PCR-generated fragment encompassing the pvdS promoter directionally ligated to the BglII-EcoRI sites of pMP220	This study
pUCP18	Escherichia coli-Pseudomonas shuttle vector derived from pUC18, pMB1, replicon pRO1600 lacZα bla Mob+ Tra− Apr Cbr	19
pCAΔSh	pUCP18-derivative containing a 2.7-kb PstI-SphI fragment encompassing the entire psbA gene	2

Characterization of the psbA promoter in Pseudomonas strain B10.

To define the structure of the psbA promoter (PpsbA), the initiation sites of the psbA transcript were determined by primer extension analysis. The primer PEpsbARV (5′-GGGCGATGCAATTGCCGTTGTCAT-3′) was end labeled with [γ-³²P]ATP and used for reverse transcription of total RNA from iron-deprived Pseudomonas strain B10 cells as described elsewhere (9). The unlabeled primer was used to sequence the DNA region upstream from the psbA gene from plasmid pCAΔSh with a T7 sequencing kit (Pharmacia) and [α-³²P]dATP. The 5′ ends of the psbA mRNA were mapped at four distinct sites (Fig. 2). The most abundant transcripts, T3 and T4, originate at positions −44 and −48 relative to the psbA start codon, respectively, while two minor transcripts, T1 and T2, originate at positions −87 and −89, respectively. No primer extension product was detected with total RNA from bacteria grown in iron-rich medium (data not shown), consistent with the observation that psbA is not transcribed in iron-rich cells (2). The initiation pattern of psbA mRNA is consistent with the presence of multiple transcription start sites in the homologous pvdA promoter (PpvdA) and in P. aeruginosa iron-regulated promoters directly or indirectly controlled by the PvdS sigma factor, including pvdD, regAB, and toxA (9, 17, 22). As shown in Fig. 2, the 158-bp DNA sequence region preceding the psbA start codon is 68% identical to the corresponding PpvdA region (9). Both sequences contain regulatory motifs shared by ECF-dependent promoters, namely the ISB and the TCCTA element, which is also present in the corresponding region of algD and algU promoters of P. aeruginosa (18). It was also noticed that the psbA T2 and T4 transcripts correspond to the pvdA T1 and T2 transcripts, respectively. Interestingly, in Pseudomonas strain B10, the most abundant psbA transcript was T4, while, under the same experimental conditions, the major pvdA transcript was T1 (9). The alignment highlights the presence of a 14-nucleotide (nt) deletion and an 18-nt insertion at positions −137 and −49 upstream of the psbA start codon, respectively. Due to the presence of the 18-nt insertion in PpsbA, the T4 start point is located at a longer distance from the ISB compared with the T2 start point of pvdA (Fig. 2). This insertion is endowed with remarkable features, consisting of two heptameric direct repeats (TCAGGCC), followed by a third partially conserved repeat (cCAGGCt).

FIG. 2.

Structural analysis of the psbA promoter. (A) Alignment of the 158-nt region preceding the Pseudomonas strain B10 psbA start codon with the P. aeruginosa pvdA promoter. Identical nucleotides are capitalized and in boldface. The bent arrows indicate the minor (T1 and T2) and major (T3 and T4) transcription start sites of the psbA gene and the direction of transcription. The ISB and the TCCTA conserved motifs, which overlap the −35 and −10 regions, respectively, are underlined. The potential ribosome binding sites (RBS) and the transcriptional start codons (f-Met) of psbA and pvdA are underlined with dots. The locations of the conserved (solid arrows) and partially conserved (dotted arrow) direct repeats are indicated below the psbA sequence. Arrowheads below the pvdA sequence mark the positions of the 5′ ends of the pvdA T1 and T2 transcripts (9). (B) Localization of the transcriptional start sites of psbA. The asterisk indicates primer extension analysis of the psbA mRNA carried out with the 5′-end-labeled oligonucleotide PEpsbARV. Lanes T, C, G, and A represent sequencing ladders of pCAΔSh with the same oligonucleotide. These reactions were run in parallel with primer extension products to determine exactly the 5′ ends of the transcripts. The arrows on the left indicate the origins of psbA transcripts T1, T2, T3, and T4.

Activity of psbA and pvdA promoters in Pseudomonas strain B10 and P. aeruginosa.

The structural similarity between the PpsbA and PpvdA, combined with previous evidence that PsbS and PvdS are functionally interchangeable (8), led us to investigate promoter activity in Pseudomonas strain B10 and P. aeruginosa PAO1. For this purpose, the β-galactosidase levels expressed by pMP220::PpsbA and pMP220::PpvdA fusions were measured in P. aeruginosa PAO1 and Pseudomonas strain B10 cells grown under low- and high-iron conditions. Both promoters showed similar activities in iron-poor cultures of P. aeruginosa PAO1 (Table 2). Comparable levels of β-galactosidase (ca. 10,000 U) were also expressed by iron-limited cultures of Pseudomonas strain B10 carrying the homologous promoter fusion (pMP220::PpsbA), while a dramatic (>90%) reduction in reporter gene activity was observed for the heterologous pMP220::PpvdA fusion (Table 2). These results suggest that the transcriptional apparatus of Pseudomonas strain B10 discriminates between the homologous promoter (PpsbA) and the heterologous promoter (PpvdA), the latter being less efficiently recognized.

TABLE 2.

Activity of PpsbA::lacZ and PpvdA::lacZ transcriptional fusions in wild-type Pseudomonas strain B10 and P. aeruginosa PAO1 and in isogenic pyoverdine-defective mutants

Strain	Plasmid	LacZ activity (Miller units)a
		−Fe(III)	+Fe(III)	−Fe(III) + pyoverdineB10
Pseudomonas strain B10	pMP220	38	31	40
	pMP220::PpsbA	9,828	23	9,614
	pMP220::PpvdA	897	29	941
P. aeruginosa PAO1	pMP220	58	50
	pMP220::PpsbA	10,015	54
	pMP220::PpvdA	10,317	57
B10CA1	pMP220	42	35	36
	pMP220::PpsbA	107	23	6,922
	pMP220::PpvdA	48	30	646
PALS124	pMP220	62	55
	pMP220::PpsbA	9,624	51
	pMP220::PpvdA	10,186	67
PALS106	pMP220	50	43
	pMP220::PpsbA	8,968	31
	pMP220::PpvdA	9,075	57
PALS125	pMP220	47	50
	pMP220::PpsbA	9,920	39
	pMP220::PpvdA	10,080	35

^aDetermined in Pseudomonas culture lysates grown for 12 h in DCAA [−Fe(III)] and DCAA supplemented with 100 μM FeCl₃ [+Fe(III)] (25). LacZ activity is represented by units of β-galactosidase as defined by Miller (11) and correspond to means of four determinations. Standard deviations are <16% of the given value.

^bPyoverdine_B10 was purified from culture supernatants of Pseudomonas strain B10 according to a previously described procedure (26) and added at 50 μM.

Pyoverdine-dependent regulation of pvdA and psbA promoters.

Preliminary reports from independent laboratories have related the down-regulation of pyoverdine promoters in pyoverdine biosynthetic mutants to pyoverdine-mediated induction of cognate siderophore genes (3, 23). On this basis, we compared the pyoverdine-regulated responses in wild-type Pseudomonas strain B10 and P. aeruginosa PAO1 and in their isogenic pyoverdine-defective derivatives, B10CA1 (psbA::cat) and PALS124 (pvdA) (2, 26). Under low-iron conditions, the activities of both PpsbA::lacZ and PpvdA::lacZ fusions were strongly reduced in Pseudomonas strain B10CA1 compared with those in the parental strain, and this effect was reversed by addition of 50 μM pyoverdine_B10 to the culture medium (Table 2). In contrast, the activities of both promoter fusions were similar in P. aeruginosa PAO1 and in the PALS124 mutant. The pyoverdine-insensitive behavior of the PpvdA promoter was confirmed in P. aeruginosa mutants blocked at later steps of pyoverdine biogenesis, including PALS106 (pvdC1) and PALS125 (pvdC3) (Table 2). These results were supported by Western blot analysis of PvdA expression in the pvd mutants (Fig. 3). Under conditions of iron deficiency, comparable levels of PvdA were synthesized by P. aeruginosa PAO1, PALS106, and PALS125, irrespective of the presence of exogenous pyoverdine_PAO1 in the culture medium (Fig. 3). As expected, addition of 100 μM FeCl₃ to the iron-poor medium completely repressed PpvdA::lacZ activity (Table 2) and PvdA expression (Fig. 3). Therefore, it can be deduced that homologous pyoverdine is involved in the activation of the psbA promoter in the Pseudomonas strain B10 system, while it plays no apparent role in positive regulation of the pvdA biosynthesis gene in P. aeruginosa PAO1. This effect cannot be ascribed to pyoverdine_B10-mediated induction of the psbS gene, since both pvdS::lacZ and psbS::lacZ promoter fusions (as in plasmids pMP220::PpvdS and pMP220::PpsbS) were found to be equally expressed and iron regulated in P. aeruginosa PAO1 and Pseudomonas strain B10 wild-type strains and in their l-Orn N⁵-oxygenase-defective mutants (data not shown).

FIG. 3.

Western blot analysis of iron-regulated PvdA expression in wild-type and pvd mutants of P. aeruginosa PAO1. Immunodetection of the 50-kDa PvdA protein in crude lysates (≅20 μg of proteins per lane) of exponentially grown bacteria (A₆₀₀ ≅ 0.5) probed with an anti-PvdA mouse antiserum was performed as previously described (2). Cultures were performed in KB medium supplemented or not with either 100 μM FeCl₃ [Fe(III)] or 50 μM pyoverdine_PAO1 (pvd_PAO1), as indicated below each lane. Lanes: 1 to 3, P. aeruginosa PAO1 (wild type); 4 to 6, PALS106 (pvdC1); 7 to 9, PALS125 (pvdC3). The position of PvdA is indicated on the left.

Conclusions.

The data just presented demonstrate that pyoverdine_B10, as the end product of its biosynthesis pathway, is a key participant in the control circuit regulating its own production in Pseudomonas strain B10. In P. aeruginosa PAO1, however, extracellular pyoverdine_PAO1 has no apparent role in the positive regulation of the homologous pvdA gene see Fig. 1 for a comparison between the two systems). The lack of pvdA response to exogenous pyoverdine is consistent with the observation that pvd mutants blocked in late steps of pyoverdine_PAO1 biogenesis produce wild-type levels of hydroxamate nitrogen in iron-poor medium (26). This implies that PvdA is equally expressed in the pyoverdine_PAO1-proficient and -deficient backgrounds, and immunoblot analysis of PvdA expression in the wild-type and pvd mutants corroborates this conclusion.

Despite the differences in individual activity between PpsbA and PpvdA, which are likely to reflect their structural diversity, both promoters were found to respond positively to homologous pyoverdine in the Pseudomonas strain B10 system, but not in P. aeruginosa. The mechanism by which pyoverdine_B10 increases psbA expression does not involve up-regulation of psbS transcription, implying that sensing of pyoverdine_B10 could result in posttranscriptional activation of the PsbS sigma factor. Siderophore-dependent induction has been elucidated in the pupIR-pupB system of P. putida WCS358. The Fur-controlled pupIR operon encodes a surface-signaling system. PupI is an ECF sigma factor required for the expression of pupB, encoding the receptor for heterologous pyoverdine_BN8. PupR up-regulates pupB expression through activation of PupI in the presence of ferripyoverdine_BN8, but it prevents the PupI-dependent transcription of pupB in its absence (7). Sequencing of the complete genomes of P. fluorescens and P. putida (available at http://www.jgi.doe.gov/and http://www.ncbi.nlm.nih.gov/, respectively) disclosed the presence in these species of multiple genes encoding putative PupR-like proteins. Whether a similar regulatory device also exists in Pseudomonas strain B10, it would account for modulation of PsbS activity through signaling of ferripyoverdine_B10 binding to its outer membrane receptor (10). This would ensure up-regulation of psbA expression under conditions in which pyoverdine_B10 is effective in delivering iron to the cell: namely, when ferripyoverdine_B10 is engaged with its receptor.