Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa

Nazir Barekzi{dagger}, Swati Joshi, Scott Irwin, Todd Ontl and Herbert P. Schweizer

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1658, USA

Correspondence
Herbert P. Schweizer
Herbert.Schweizer{at}colostate.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4'-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.


Abbreviations: ACP, acyl carrier protein; CAS, chrome azurol S; PcpS, P. aeruginosa carrier protein synthase; Ppant, 4'-phosphopantetheine

The GenBank accession number for the sequence reported in this paper is AAG04554.

{dagger}Present address: Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Members of the superfamily of 4'-phosphopantetheine (Ppant)-dependent carrier proteins play central roles in fatty acid synthesis, polyketide synthesis and non-ribosomal peptide synthesis. This family includes the acyl carrier proteins (ACP), aryl carrier proteins and peptidyl carrier proteins, which can exist as distinct proteins or be part of larger polypeptide chains (Lambalot et al., 1996; Walsh et al., 1997). All carrier proteins are synthesized in the inactive apo form and must be converted to the active holo form. This is achieved by enzymic transfer of the Ppant moiety from coenzyme A to a conserved serine residue in the carrier protein by a usually dedicated Ppant transferase (Flugel et al., 2000).

Most bacteria use more than one Ppant transferase pathway. The conversion of ACPs of primary metabolism (fatty acid synthesis) is catalysed by AcpS enzymes. Ppant transferases of this group are ~120 aa in size, act as homotrimers and exhibit a narrow substrate specificity in that they only modify ACPs of fatty acid synthesis and type II polyketide synthesis (Flugel et al., 2000; Mootz et al., 2001). Other Ppant transferases are involved in the synthesis of secondary metabolites. Escherichia coli and Bacillus subtilis, for example, produce the catecholic siderophore enterobactin and the cyclic lipopeptide surfactin via non-ribosomal peptide synthesis pathways, using the associated Sfp-type Ppant transferases EntD and Sfp, respectively (Coderre & Earhardt, 1989; Nakano et al., 1992). EntD cannot substitute for AcpS in primary metabolism (Flugel et al., 2000) but B. subtilis strains from which acpS has been deleted can sustain fatty acid synthesis, presumably because Sfp can accept ACPs of fatty acid synthesis pathways (Mootz et al., 2001). Sfp is about twice the size of AcpS, exhibits a very broad substrate specificity and functions as a monomeric enzyme (Mootz et al., 2001; Reuter et al., 1999).

P. aeruginosa is unique in that it apparently contains only one Ppant transferase enzyme, PcpS, which can modify the carrier proteins of both primary and secondary metabolism (Finking et al., 2002). This bacterium produces two siderophores, pyoverdine and pyochelin, which are involved in iron acquisition and are required for virulence (Meyer & Stintzi, 1998; Takase et al., 2000). Pyoverdine-mediated cell signalling is also involved in virulence factor production and siderophore receptor synthesis (Beare et al., 2003; Lamont et al., 2002). Pyoverdine and pyochelin are produced by non-ribosomal peptide synthases. For pyoverdine, the associated carrier proteins are PvdD (Ackerley et al., 2003), PvdI (Lehoux et al., 2000) and PvdJ (Lamont & Martin, 2003; Ravel & Cornelis, 2003). Similarly, pyochelin synthesis requires the PchE and PchF carrier proteins (Reimmann et al., 2001). Using a yeast two-hybrid system, it has been shown that PcpS interacts with P. aeruginosa PvdD and PchE, and PcpS also acts on purified PchE-aryl carrier protein in vitro (Finking et al., 2002). This implied, but in the absence of a mutant did not prove, that pcpS was essential for siderophore synthesis.

In this study, we show for the first time that pcpS can genetically complement mutations in the E. coli acpS and entD genes, as well as a mutation in B. subtilis sfp. Furthermore, we demonstrate that the previously suggested essentiality of pcpS is most likely due to its requirement for activation of apo-ACP of fatty acid synthesis. A conditional pcpS mutant is non-fluorescent and does not produce pyoverdine and pyochelin, which prevents its growth on minimal medium in the presence of iron chelators.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and media.
Bacterial strains used in this study are listed in Table 1. Bacteria were generally cultivated in Lennox L broth or on L broth agar (Gibco). Low-iron chrome azurol S (CAS) indicator plates were used to assess siderophore production in E. coli and P. aeruginosa (Schwyn & Neilands, 1987). M9 minimal medium (Miller, 1992) supplemented with 10 mM glucose and either 150 µg 2,2'-dipyridyl (DP) ml–1 or 200 µg ethylene-N,N'-diacetic acid (EDDA) ml–1 was used to test the ability of strains to grow in the presence of iron chelators. Casamino acid (CAA) medium contained 5 g CAA l–1, 0·9 g K2HPO4 l–1 and 0·7 g MgSO4.7H2O l–1 (Hoefte et al., 1993). Sfp-complementing activity in B. subtilis was determined on minimal medium (MM) agar plates containing a 5 ml MM soft agar overlay with 10 % (v/v) bovine (ox) blood (Colorado Serum Company) (Nakano et al., 1992). For plasmid maintenance in E. coli, media were supplemented with 100 µg ampicillin ml–1 or 10 µg gentamicin ml–1 unless noted otherwise.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids used in this study

 
Cloning, overexpression and purification of PcpS.
Using standard methods for PCR amplification of GC-rich templates (including 5 %, v/v, dimethylsulfoxide) and Taq DNA polymerase (Invitrogen) (Hoang et al., 1998), the 728 bp pcpS gene (annotated as PA1165; GenBank accession no. AAG04554) was PCR-amplified from ~100 ng PAO1 genomic DNA using primers PA1165-UP (5'-AAGCGCCACGTGACTGGTGGG-3') and PA1165-DOWN (5'-CCGTCGAGCGCCTGGCGGAAG-3') (all oligonucleotides used in this study were purchased from Invitrogen). Cycle conditions were an initial 5 min of denaturation at 96 °C, followed by 35 cycles of 96 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min and a final extension step for 10 min at 72 °C. The resulting 835 bp fragment was cloned into the TA cloning vector pCR2.1 (Invitrogen) to form pPS1158. The presence of the correct sequence was verified by nucleotide sequencing (University of California-Davis Sequencing Facility).

A vector for expression of a carboxy-terminally tagged PcpS (PcpS-H6) was constructed by PCR amplification of the PcpS coding sequence from pPS1158. The upstream mutagenic primer (5'-AACCGGCcatATGCGCGCCATGAACG-3') contained several mismatches (lower-case letters) and introduced an NdeI site (underlined). The downstream mutagenic primer (5'-GGAACGCCTGAcTCgAgGGCGCCGAC-3') contained mismatches to introduce an XhoI site (underlined). Cycle conditions were an initial 5 min of denaturation at 96 °C, followed by 35 cycles of 96 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min and a final extension step for 10 min at 72 °C. The NdeI+XhoI-digested 734 bp PCR fragment was directly ligated to NdeI+XhoI-digested pET-22b DNA (Novagen) to form pPS1161.

Similarly, a plasmid for expression of PcpS with an amino-terminal H6 tag (H6-PcpS) was constructed by amplification of the PA1165 gene from pPS1158 using the upstream primer and a second downstream mutagenic primer (5'-ACGCCGGGGAtCcCCTGATCAGGCGC-3'), which introduced a BamHI site (underlined) downstream of the PcpS coding sequence. Cycle conditions were an initial 5 min of denaturation at 96 °C, followed by 35 cycles of 96 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min and a final extension step for 10 min at 72 °C. The resulting 756 bp product was cloned into pCR2.1 to yield pPS1159. After digestion of pPS1159 with BamHI+NdeI, the 742 bp fragment was ligated to pET-15b (Novagen) digested with the same enzymes to form pPS1160.

The expression constructs were transformed to E. coli BL21(DE3) (Novagen), and proteins were expressed and purified by metal chelation affinity chromatography on a Ni2+-nitrilotetracetic acid agarose resin (Invitrogen) as described by Hoang et al. (1999). The protein content of the fractions was analysed by 0·1 % (v/v) SDS-10 % (v/v) PAGE (Hoang et al., 1999). Fractions containing H6-tagged PcpS were dialysed against 10 mM Tris/HCl, pH 7·5 containing 10 % (v/v) glycerol and then stored in small aliquots at –70 °C. Protein concentrations were estimated using the Bradford dye-binding assay from Bio-Rad Laboratories with bovine serum albumin as the standard.

AcpS assay.
For assays of AcpS activity, recombinant P. aeruginosa apo-ACP was purified from E. coli as described using growth conditions that favour accumulation of apo-ACP (Kutchma et al., 1999). AcpS reaction mixtures (100 µl) contained 30 µg ACP, 0·25–1 µg PcpS, 0·2 mM coenzyme A in 75 mM Tris/HCl (pH 8·8), 10 mM MgCl2 and 1 mM DTT. The mixtures were incubated at 37 °C for 30 min. Products were analysed at the Colorado State University Macromolecular Resource Facility by matrix-assisted laser desorption ionization (MALDI) mass spectrophotometric analysis using time-of-flight (TOF) detection.

Construction of B. subtilis expression vectors.
For expression of H6-PcpS and PcpS-H6 in B. subtilis, the corresponding fragments encoding these proteins were subcloned into pUB19 (Wu & Wong, 1999) on 1130 bp XbaI–HindIII and 2075 bp XbaI–PstI fragments from pPS1160 and pPS1161, respectively. The ligation mixtures were directly transformed into B. subtilis as described by Wu & Wong (1999), followed by selection on L-agar plates containing 10 µg kanamycin ml–1. These procedures yielded the expression vectors pPS1197 (H6-PcpS) and pPS1198 (PcpS-H6).

Construction of a P. aeruginosa strain containing a chromosomally integrated copy of E. coli acpS.
The E. coli acpS coding sequence was excised on a 431 bp XbaI (blunt-ended with T4 DNA polymerase)–NotI fragment from pDPJ (Lambalot & Walsh, 1995) and cloned between the SfiI (blunt-ended)–NotI sites of mini-CTX-lacZ{Delta}M15 (Schweizer et al., 2001). In the resulting construct, acpS is under transcriptional control of the lacUV5 promoter and the Lac repressor encoded by the same plasmid. The resulting vector, mini-CTX-acpS, was integrated into the PAO1 chromosome and unwanted DNA sequences were excised using Flp recombinase utilizing previously described procedures (Hoang et al., 2000). This yielded strain PAO388. Empty mini-CTX-lacZ{Delta}M15 was integrated to derive the control strain PAO387. The presence of the acpS gene was verified by PCR amplification using the primers acpS-UP (5'-GCCTGGCACGCCGCGTATTAAGCG-3') and acpS-DOWN (5'-GGCACAAGCATAGTGCCGCTCATCTG-3'). Cycle conditions were an initial 5 min of denaturation at 96 °C, followed by 30 cycles of 96 °C for 45 s, 70 °C for 45 s, 72 °C for 45 s and a final extension step for 10 min at 72 °C. The same primers were also used to assess expression of acpS in these strains by RT-PCR using previously described procedures (Beinlich et al., 2001).

Construction of a chromosomal pcpS mutant by allelic exchange.
A plasmid-borne pcpS insertional mutant was generated in several steps. First, the pcpS coding sequence was isolated on a 769 bp XbaI–XhoI fragment from the PcpS-H6 expression pPS1161 and cloned between the XbaI–SalI sites of pUCP26 (West et al., 1994) to yield pPS1229. Second, the pcpS gene was then cloned on a 771 bp XbaI–PstI fragment between the same sites of pEX18Ap (Hoang et al., 1998) to form pPS1230. Finally, pPS1231 was derived by insertion of a 853 bp blunt-ended SacI fragment containing a gentamicin resistance (Gmr) cassette from pUCGM (Schweizer, 1993) into the single NcoI site located within the pcpS gene contained on pPS1230. This procedure regenerated NcoI sites at the pcpS–Gmr junctions. Plasmid pPS1231 was conjugally transferred to the PAO388 chromosome and Gmr merodiploids were selected on L medium containing 30 µg gentamicin ml–1 (Hoang et al., 1998). Due to the presence of the sacB gene and the bla gene on pEX18Ap, the merodiploids were also sensitive to sucrose (Sucs) and carbenicillin-resistant (Cbr). Attempts at resolution of merodiploids were performed by selecting Sucr derivatives on L-agar containing 5 % (w/v) sucrose and 30 µg gentamicin ml–1.

For verification of insertional mutagenesis of pcpS, chromosomal DNA was purified from stationary-phase cells of strains PAO1 and PAO391 using a kit from Orca Research. About 100 ng of these DNA preparations were used as templates in PCR reactions primed with PA1165-UP and PA1165-DOWN. Cycle conditions were an initial 5 min of denaturation at 96 °C, followed by 35 cycles of 96 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min and a final extension step for 10 min at 72 °C. Portions (~0·5 µg) of the amplicons were digested for 1 h at 37 °C with 5 U of BglII or NcoI (Invitrogen) and then electrophoresed along with uncut amplicon DNA on 1·5 % (w/v) agarose gels in 1x TAE buffer (Sambrook & Russell, 2001) in the presence of 0·5 µg ethidium bromide ml–1.

Measurement of pyoverdine and pyochelin production.
For assays of pyoverdine and pyochelin, cells were grown in CAA medium and cell-free supernatants were obtained by centrifugation. The presence of pyoverdine was assessed by obtaining spectra of the supernatants in the 200–1000 nm range using a Multiskan Spectrophotometer (Therma Labsystems). Pyochelin was partially purified from supernatants by ethyl-acetate extraction followed by paper chromatography, and chromatograms were sprayed with an iron or phenolate reagent (Cox & Graham, 1979; Hoefte et al., 1993). On the chromatograms, pyochelin turned red in the presence of the iron reagent, blue in the presence of the phenolate reagent and fluoresced intensely when illuminated with UV light (300 nm).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
PcpS exhibits in vitro Ppant transferase activity
Since we based all of our plasmid constructs used for complementation analyses on plasmids expressing histidine-tagged PcpS, we first wanted to ensure that the pcpS gene amplified from genomic DNA encoded an enzymically active Ppant transferase. To this end, we purified PcpS from E. coli BL21(DE3)/pPS1161 overexpressing PcpS with a carboxy-terminal H6 tag by Ni2+ affinity chromatography. The purified 250 aa and 27 779 Da PcpS-H6 protein exhibited Ppant transferase activity after incubation with CoA and P. aeruginosa apo-ACP in the presence of Mg2+ (Table 2). The results clearly indicated transfer of the 339 atomic mass unit Ppant moiety from CoA onto apo-ACP to form holo-ACP (Table 2) and are therefore in agreement with the results obtained by Finking et al. (2002) using a different Ppant transferase assay. Note that our ACP preparation contained a mixture of ACP, with and without the amino-terminal methionine, which is known to be partially processed (Kutchma et al., 1999). PcpS containing an amino-terminal H6 tag was inactive in this assay using purified P. aeruginosa ACP (not shown), although it complemented a conditional E. coli acpS mutant in vivo (see below). Even though we cannot explain the differences observed with H6-PcpS in vitro and in vivo, we must note that the in vitro assay used P. aeruginosa apo-ACP as the substrate, while the in vivo complementation assay uses E. coli apo-ACP. The observed results might therefore be explained with differential interaction of H6-PcpS with the two different apo-ACP substrates.


View this table:
[in this window]
[in a new window]
 
Table 2. MALDI-TOF data demonstrating the phosphopantetheinylation of P. aeruginosa apo-ACP by recombinant histidine-tagged PcpS

 
Cloned pcpS exhibits in vivo AcpS, EntD and Sfp Ppant transferase activities
Previous studies using in vitro enzymic assays, yeast two-hybrid systems and genetic complementation of a yeast LYS5 Ppant-transferase mutant indicated that PcpS is a multifunctional enzyme involved in multiple Ppant transferase reactions of primary and secondary metabolism (Finking et al., 2002), but no direct complementation analysis of mutations affecting the suggested activities was performed. We therefore set out to probe the range of PcpS Ppant transferase activity by genetic complementation of diverse Ppant transferase defects affecting primary and secondary metabolism in diverse bacteria.

The ability of PcpS to modify apo-ACP in vivo was tested by transformation of the conditional E. coli acpS mutant HT253. This strain exhibits a tetracycline-dependent growth phenotype, unless transformed with an AcpS-expressing plasmid (Flugel et al., 2000; Takiff et al., 1992). Complementation was assessed by plating serial dilutions of HT253 containing various plasmids on L-broth/ampicillin plates with and without 2·5 µg tetracycline ml–1, followed by incubation at 30 °C. H6-PcpS and PcpS-H6 expressed from pPS1160 and pPS1161, respectively, complemented the tetracycline-dependent growth phenotype of HT253, as indicated by the growth of transformants on media without tetracycline (Fig. 1a), whereas HT253 cells containing the vector controls pET-15b or pET-22b only grew in the presence of tetracycline. Although the pcpS genes on pPS1160 and pPS1161 are under transcriptional control of the T7 promoter, and HT253 lacks a source for T7 RNA polymerase, the basal background levels of pcpS transcription, probably originating from a plasmid-borne promoter other than T7 on both plasmids, were sufficient for complementation of the HT253 acpS mutation. A similar observation was made previously using the same strain for complementation with Streptococcus pneumoniae acpS expressed under lac promoter control (McAllister et al., 2000), which is not surprising since it is known that the lac promoter is leaky in L-broth-grown E. coli.



View larger version (98K):
[in this window]
[in a new window]
 
Fig. 1. Complementation analysis of E. coli acpS and entD, and B. subtilis sfp. (a) E. coli strain HT253 expressing acpS conditionally from a tetracycline-dependent promoter was transformed with the vector controls (pET-15b and pET-22b) and pPS1160 and pPS1161 (pET-22b expressing H6-PcpS and PcpS-H6, respectively). Transformants were plated on L broth/ampicillin medium with 2·5 µg tetracycline ml–1, conditions permissive for growth of HT253, or L broth/ampicillin medium without tetracycline, conditions which are non-permissive for HT253 without expressing a biologically active AcpS Ppant transferase. (b) The E. coli entD+ strain MC4100 or entD mutant AN90-60 were transformed with the indicated plasmids. These included the vector controls (pET-15b and pET-22b), pPS1130 (pET-15b containing E. coli acpS), and pPS1160 and pPS1161. The transformants were patched onto a low-iron CAS indicator plate. Enterobactin production is indicated by halo formation around bacterial colonies. (c) B. subtilis strain 168 was transformed with the vector control pUB19, and pPS1197 and pPS1198 (expressing H6-PcpS and PcpS-H6, respectively). Kmr transformants were patched on blood agar plates. Surfactin production is indicated by halo formation around bacterial colonies.

 
To assess EntD Ppant transferase activities, we used in vivo complementation of E. coli entD strain AN90-60. This strain does not produce enterobactin, a phenotypic trait that can easily be detected on low-iron CAS indicator plates (Schwyn & Neilands, 1987). Wild-type strains produce a yellowish halo around colonies due to siderophore production whereas siderophore-negative strains produce no halo. AN90-60 was transformed with pPS1160 and pPS1161, and transformants were analysed on CAS plates, alongside positive and negative controls (Fig. 1b). Similar to E. coli EntD+ strain MC4100 containing pET-22b, AN90-60 expressing either H6-PcpS (pPS1160) or PcpS-H6 (pPS1161) produced enterobactin, whereas the same strain containing the vector controls pET-15b and pET-22b did not. In accordance with previous observations, E. coli acpS (pPS1130) did not complement the entD mutation.

B. subtilis strain 168 does not produce surfactin because it contains a non-functional sfp gene. To assess sfp complementing activity, transformants of strain 168 expressing either H6-PcpS (pPS1160) or PcpS-H6 (pPS1161) were patched on blood agar plates. Expression of both H6-PcpS and PcpS-H6 restored surfactin production (Fig. 1c). Cells containing the vector control pUB19 did not produce surfactin. These results indicated that PcpS can activate apo-SfrA in vivo.

pcpS is an essential gene
Although a previous study suggested that pcpS may be an essential gene, the evidence provided was circumstantial, i.e. lack of ability to construct a knockout mutant (Finking et al., 2002). No attempts were made to obtain a knockout in the presence of a rescue plasmid or to construct a conditional pcpS mutant. To verify the notion of pcpS being essential, we therefore sought to gather additional evidence, including obtaining a knockout mutant in the presence of a complementing copy of E. coli acpS.

The pcpS gene is located at 1·26 Mbp of the P. aeruginosa chromosome between two genes encoding proteins of unknown function (Fig. 2a). Since pcpS is separated from the upstream gene PA1166 by 226 nt and PA1166 is immediately followed by a possible transcriptional terminator [{Delta}G=–26·8 kcal mol–1 (=–112·1 kJ mol–1)], pcpS seems to be contained in its own transcriptional unit. This is in contrast to Ppant transferases from other bacteria that are usually part of other transcriptional units, e.g. E. coli acpS (Takiff et al., 1992) and entD (Coderre & Earhardt, 1989) or S. pneumoniae acpS (McAllister et al., 2000).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2. Genetic analysis of pcpS. (a) Organization of the pcpS region on the P. aeruginosa chromosome. The pcpS gene lies at 1·26 Mbp of the P. aeruginosa genome sequence (www.pseudomonas.com) and is flanked by two genes of unknown function. Arrows indicate transcription of the respective genes and a lollipop marks the location of a potential transcription terminator downstream of pcpS. The locations of chromosomal transposon insertion sites within PA1164 and PA1166 obtained in a genome-wide transposon analysis (www.genome.washington.edu/UWGC/pseudomonas/index.cfm) are marked with solid triangles. (b) Genomic organization of the pcpS region of two merodiploid strains and a pcpS mutant. A suicide plasmid containing a Gmr determinant in the pcpS gene was integrated via homologous recombination at the pcpS locus of strain PAO1 either harbouring the empty vector (V) or the E. coli acpS gene or at the {phi}CTX attachment site to obtain the two merodiploids, PAO389 and PAO390, respectively. When these merodiploids were plated on L-gentamicin plates in the presence of 5 % sucrose, pcpS strain PAO391 was obtained by deletion of the sequences marked {Delta} from PAO390. These sequences could not be deleted from strain PAO389. (c) Verification of pcpS mutant by PCR analysis. Chromosomal DNAs of strains PAO1 or the pcpS mutant PAO391 were used as templates in PCR reactions primed with PA1165-UP (P1) and PA1165-DOWN (P2) (see Methods for primer sequences). The PCR reactions were electrophoresed on a 1·5 % agarose gel in undigested form (lane 1), or after digestion with or BglII (lane 2) or NcoI (lane 3). Lane M contains molecular size markers (Minnesota Molecular) which are (top to bottom) 2000, 1550, 1400, 1000, 750, 500, 400 and 300 bp.

 
More convincing evidence for the essentiality of pcpS was recently obtained by genome-wide transposon analysis (www.genome.washington.edu/UWGC/pseudomonas/index.cfm). In this analysis, 30 100 transposon insertions were mapped in strain PAO1. Although PA1164 (270 codons) and PA1166 (262 codons), the two genes flanking pcpS, contained three and four insertions, respectively, no insertions were mapped to the similarly sized pcpS (242 codons).

Based on evidence obtained with other bacteria indicating that Ppant transferases involved in primary (fatty acid) metabolism are essential but those involved in secondary metabolism are not (Finking et al., 2002), we reasoned that pcpS may be essential because of its requirement for apo-ACP activation. If this were the case, then one should be able to obtain pcpS knockout mutants in the presence of a complementing acpS gene. A conditional pcpS mutant would then enable a genetic analysis of non-essential Ppant functions under laboratory conditions. We therefore constructed a P. aeruginosa strain (PAO388) that contained a chromosomally integrated copy of acpS under transcriptional control of the E. coli lac operon promoter. A negative control strain (PAO387) had the empty vector integrated into the chromosome. Next, we derived merodiploids of these two strains by integration of a plasmid carrying an inactivated pcpS gene at the chromosomal pcpS locus. These merodiploids, PAO389 and PAO390, were Gmr, Cbr and Sucs (Fig. 2b).

When we attempted to resolve the merodiploids by plating them on L-broth/gentamicin medium in the presence of 5 % sucrose, we obtained the expected Sucr Gmr Cbs colonies from PAO390, but not from PAO389. Merodiploids of PAO390 could be resolved in the absence or presence of IPTG, indicating that basal levels of acpS transcription caused by leakiness of the lac promoter were sufficient to ensure proper apo-ACP modification. Sucr Gmr Cbs colonies arise from the deletion event depicted by {Delta} in Fig. 2(b). To verify this deletion event, a PCR analysis was performed using primers PA1165-UP and PA1165-DOWN, which were originally employed for amplification of pcpS from PAO1 genomic DNA. When using this primer pair, the expected 836 bp fragment was amplified from PAO1 DNA (Fig. 2c). Its identity was verified by NcoI digestion, which yielded two fragments of 367 and 469 bp, due to the presence of the NcoI site located within pcpS. When genomic DNA from pcpS mutant PAO391 was used as the template, the same primers amplified a 1694 bp fragment (pcpS plus the Gmr fragment). When this fragment was digested with BglII, two fragments of 917 and 777 bp were obtained due to the presence of a single BglII site within the aacC1 gene (Gmr determinant), which is transcribed in the same direction as pcpS. Since insertion of the Gmr cassette generated flanking NcoI sites, digestion with this restriction enzyme yielded the same 367 and 469 bp fragments observed in PAO1 plus the additional 858 bp Gmr fragment.

The pcpS mutant is impaired in siderophore synthesis
In a yeast two-hybrid system, it was previously shown that PcpS interacts with both PvdD and PchE in vivo, implicating that a pcpS mutant should be impaired in pyoverdine and pyochelin synthesis (Finking et al., 2002). However, no proof could be provided for this notion in the absence of a pcpS mutant. If PcpS is indeed the sole Ppant transferase in P. aeruginosa, then PAO391 should be defective in pyoverdine and pyochelin synthesis. Consistent with this hypothesis, PAO391 was non-fluorescent when viewed under UV light (not shown) and did not produce a halo on low-iron CAS plates (Fig. 3a). This observation was further supported by measuring the pyoverdine and pyochelin content in cell-free supernatants. Pyochelin could not be detected in ethyl acetate extracts from PAO391 supernatants that were analysed by paper chromatography and spraying with either an iron or phenolate reagent (data not shown) or by UV illumination (Fig. 3b), and spectral analyses indicated that pyoverdine was absent from supernatants of PAO391 (Fig. 3c). PAO391 did not grow on M9 glucose minimal medium in the presence of the iron chelators dipyridyl (150 µg ml–1) or EDDA (200 µg ml–1), a finding that is consistent with the absence of pyochelin and pyoverdine. It should be noted that P. aeruginosa must contain an iron acquisition mechanism that is independent of Ppant transferase requiring siderophore production since the pyochelin and pyoverdine deficient strain grew normally on M9 glucose minimal medium without iron supplementation.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 3. Siderophore production in the pcpS mutant PAO391. (a) Siderophore production in cells grown on low-iron CAS plates. Freshly grown colonies of PAO1, the merodiploid PAO390 and two different colonies of the pcpS mutant PAO391 were analysed. The plates were incubated at 37 °C for 24 h. (b) Pyochelin content in cell supernatants. Cells were grown in CAA medium and cells were removed by centrifugation. Pyochelin was partially purified from the supernatants by ethyl acetate extraction and aliquots of concentrated preparations were analysed by paper chromatography. Pyochelin (PCH) was visualized by UV illumination and its identity verified by spraying with phenolate and iron reagents. (c) Pyoverdine content in supernatants. Cells of PAO1 ({blacksquare}) and the pcpS mutant PAO391 ({square}) were grown in CAA medium for 24 h. Cells were removed by centrifugation. Spectra were recorded from 200 to 1000 nm. Only the 215–515 nm range is shown in this figure since the absorbance was zero for both supernatants from 515 to 1000 nm. Absorbance values were normalized for cell densities.

 
Conclusions
Although the P. aeruginosa genome encodes many ACPs and non-ribosomal peptide synthases, it apparently contains only one Ppant-transferase-encoding gene, pcpS (Finking et al., 2002; Mootz et al., 2001). This suggests that PcpS must encode a multifunctional Ppant-transferase capable of modifying various phosphopantetheinyl-dependent carrier proteins of primary (fatty acid) and secondary (e.g. siderophore) metabolism. The genetic data presented here for the first time indeed confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism. We were able to knock out pcpS in the presence of the E. coli acpS gene, encoding a Ppant-transferase capable of modifying only apo-ACPs, showing that the in vitro essentiality of pcpS is probably solely due to its requirement for modification of apo-ACP of fatty acid synthesis (primary metabolism). Although not assessed in this study, the in vivo survival (virulence) is probably an altogether different matter. Since pcpS mutants do not produce pyoverdine, and this siderophore is required for P. aeruginosa virulence (Takase et al., 2000), it can be assumed that the mutant is avirulent in animal infection models. This multifactorial essentiality makes PcpS an attractive target for antibacterials.


   ACKNOWLEDGEMENTS
 
This research was supported by NIH grant GM56685. We gratefully acknowledge the gift of bacterial strains and plasmids from C. Walsh, D. Court, M. McIntosh and S.-L. Wong. We also thank S.-L. Wong for providing protocols and advice for working with recombinant plasmids in B. subtilis.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Ackerley, D. F., Caradoc-Davies, T. T. & Lamont, I. L. (2003). Substrate specificity of the nonribosomal peptide synthetase PvdD from Pseudomonas aeruginosa. J Bacteriol 185, 2848–2855.[Abstract/Free Full Text]

Beare, P. A., For, R. J., Martin, L. W. & Lamont, I. L. (2003). Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis. Mol Microbiol 47, 195–207.[CrossRef][Medline]

Beinlich, K. L., Chuanchuen, R. & Schweizer, H. P. (2001). Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa. FEMS Microbiol Lett 198, 129–134.[CrossRef][Medline]

Coderre, P. E. & Earhardt, C. F. (1989). The entD gene of the Escherichia coli K12 enterobactin gene cluster. J Gen Microbiol 135, 3043–3055.[Medline]

Cox, C. D. & Graham, R. (1979). Isolation of an iron-binding compound from Pseudomonas aeruginosa. J Bacteriol 137, 357–364.[Medline]

Finking, R., Solsbacher, J., Konz, D., Schobert, M., Schaefer, A., Jahn, D. & Marahiel, M. A. (2002). Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa. J Biol Chem 277, 50293–50302.[Abstract/Free Full Text]

Flugel, R. S., Hwangbo, Y., Lambalot, R. H., Cronan, J. E. & Walsh, C. T. (2000). Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J Biol Chem 275, 959–960.[Abstract/Free Full Text]

Grossman, T. H., Truckman, M., Ellestad, S. & Osburne, M. S. (1993). Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfp0 and Escherichia coli entD genes. J Bacteriol 175, 6203–6211.[Abstract]

Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. (1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77–86.[CrossRef][Medline]

Hoang, T. T., Ma, Y., Stern, R. J., McNeil, M. R. & Schweizer, H. P. (1999). Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of Mycobacterium tuberculosis RmlD and Pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of RhlI. Gene 237, 361–371.[CrossRef][Medline]

Hoang, T. T., Kutchma, A. J., Becher, A. & Schweizer, H. P. (2000). Integration proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43, 59–72.[CrossRef][Medline]

Hoefte, M., Buysens, S., Koedam, N. & Cornelis, P. (1993). Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2. BioMetals 6, 85–91.[Medline]

Kutchma, A. J., Hoang, T. T. & Schweizer, H. P. (1999). Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A : ACP transacylase (FabD). J Bacteriol 181, 5498–5504.[Abstract/Free Full Text]

Lambalot, R. H. & Walsh, C. T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J Biol Chem 270, 24658–24661.[Abstract/Free Full Text]

Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A., Reid, R., Khosla, C. & Walsh, C. T. (1996). A new enzyme superfamily – the phosphopantetheinyl transferases. Chem Biol 3, 923–936.[Medline]

Lamont, I. L. & Martin, L. W. (2003). Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa. Microbiology 149, 833–842.[Abstract/Free Full Text]

Lamont, I. L., Beare, P. A., Ochsner, U., Vasil, A. I. & Vasil, M. L. (2002). Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 99, 7072–7077.[Abstract/Free Full Text]

Lehoux, D. E., Sanschagrin, F. & Levesque, R. C. (2000). Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa. FEMS Microbiol Lett 190, 141–146.[CrossRef][Medline]

McAllister, K. A., Peery, R. B., Meier, T. I., Fischl, A. S. & Zhao, G. (2000). Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis. J Biol Chem 275, 30864–30872.[Abstract/Free Full Text]

Meyer, J.-M. & Stintzi, A. (1998). Iron metabolism and siderophores in Pseudomonas and related species. In Biotechnology Handbooks 10 – Pseudomonas, pp. 201–243. Edited by T. C. Montie. New York: Plenum.

Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Mootz, H. D., Finking, R. & Marahiel, M. A. (2001). 4'-Phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis. J Biol Chem 276, 37289–37298.[Abstract/Free Full Text]

Nakano, M. M., Corbell, N., Besson, J. & Zuber, P. (1992). Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide surfactant, surfactin, in Bacillus subtilis. Mol Gen Genet 232, 313–321.[Medline]

Peters, J. E., Thate, T. E. & Craig, N. L. (2003). Definition of the Escherichia coli MC4100 genome by use of a DNA array. J Bacteriol 185, 2017–2021.[Abstract/Free Full Text]

Ravel, J. & Cornelis, P. (2003). Genomics of pyoverdine-mediated iron uptake in pseudomonads. Trends Microbiol 11, 195–200.[Medline]

Reimmann, C., Patel, H. M., Serino, L., Barone, M., Walsh, C. T. & Haas, D. (2001). Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa. J Bacteriol 183, 813–820.[Abstract/Free Full Text]

Reuter, K., Mofid, M. R., Marahiel, M. A. & Ficner, R. (1999). Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily. EMBO J 18, 6823–6831.[Abstract/Free Full Text]

Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schweizer, H. P. (1993). Small broad-host-range gentamycin resistance cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15, 831–833.[Medline]

Schweizer, H. P., Hoang, T. T., Propst, K. L., Ornelas, H. R. & Karkhoff-Schweizer, R. R. (2001). Vector design and development of host systems for Pseudomonas. In Genetic Engineering, pp. 69–81. Edited by J. K. Setlow. New York: Kluwer Academic/Plenum.

Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 47–56.[Medline]

Takase, H., Nitanai, H., Hoshino, K. & Otani, T. (2000). Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice. Infect Immun 68, 1834–1839.[Abstract/Free Full Text]

Takiff, H. E., Baker, T., Copeland, T., Chen, S.-M. & Court, D. L. (1992). Locating essential genes by using mini-Tn10 transposons: the pdxJ operon. J Bacteriol 174, 1544–1553.[Abstract]

Walsh, C. T., Gehring, A. M., Weinreb, P. H., Quadri, L. E. & Flugel, R. S. (1997). Post-translational modification of polyketide and nonribosomal peptide synthases. Curr Opin Chem Biol 1, 309–315.[CrossRef][Medline]

Watson, J. M. & Holloway, B. W. (1978). Chromosome mapping in Pseudomonas aeruginosa. J Bacteriol 133, 1113–1125.[Medline]

West, S. E. H., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J. (1994). Construction of improved EscherichiaPseudomonas shuttle vectors derived from pUC18/19 and the sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 128, 81–86.

Wu, S.-C. & Wong, S.-L. (1999). Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis. J Biotechnol 72, 185–195.[CrossRef][Medline]

Received 8 October 2003; revised 2 December 2003; accepted 9 December 2003.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Barekzi, N.
Articles by Schweizer, H. P.
Articles citing this Article
PubMed
PubMed Citation
Articles by Barekzi, N.
Articles by Schweizer, H. P.
Agricola
Articles by Barekzi, N.
Articles by Schweizer, H. P.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.