Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens

Christine Baysse1,{dagger}, Sandra Matthijs1, Max Schobert2, Gunhild Layer2, Dieter Jahn2 and Pierre Cornelis1

1 Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Building E, Room 6.6, Pleinlaan 2, B-1050 Brussels, Belgium
2 Institute for Microbiology, Technical University of Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany

Correspondence
Pierre Cornelis
pcornel{at}vub.ac.be


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The cytoplasmic membrane protein CcmC is, together with other Ccm proteins, a component for the maturation of c-type cytochromes in Gram-negative bacteria. A Pseudomonas fluorescens ATCC 17400 ccmC mutant is cytochrome c-deficient and shows considerably reduced production of the two siderophores pyoverdine and quinolobactin, paralleled by a general inability to utilize various iron sources, with the exception of haem. The ccmC mutant accumulates in a 5-aminolevulinic acid-dependent synthesis a reddish, fluorescent pigment identified as protoporphyrin IX. As a consequence a visA phenotype similar to that of a ferrochelatase-deficient hemH mutant characterized by drastically reduced growth upon light exposure was observed for the ccmC mutant. The defect of iron–protoporphyrin formation was further demonstrated by the failure of ccmC cell-free proteinase K-treated extracts to stimulate the growth of a haem auxotrophic hemH indicator strain, compared to similarly prepared wild-type extracts. In addition, the ccmC mutant did not sustain hemH growth in cross-feeding experiments while the wild-type did. Significantly reduced resistance to oxidative stress mediated by haem-containing catalases was observed for the ccmC mutant. A double hemH ccmC mutant could not be obtained in the presence of external haem without the hemH gene in trans, indicating that the combination of the two mutations is lethal. It was concluded that CcmC, apart from its known function in cytochrome c biogenesis, plays a role in haem biosynthesis. A function in the regulatory co-ordination of iron acquisition via siderophores, iron insertion into porphyrin via ferrochelatase and iron–protoporphyrin export for cytochrome c formation is predicted.


Abbreviations: ALA, 5-aminolevulinic acid; CAS, chrome azurol S; PPIX, protoporphyrin IX; PVD, pyoverdine; QB, quinolobactin

{dagger}Present address: Biomerit Research Centre, University College of Cork, Ireland.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Free-living aerobes, such as the fluorescent pseudomonads, need to produce and excrete high-affinity Fe3+-chelating siderophores to satisfy their need for iron (Braun & Killmann, 1999; Braun & Braun, 2002). Under conditions of iron limitation, fluorescent pseudomonads (among others, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens) produce different fluorescent peptidic siderophores, named pyoverdines (PVDs) or pseudobactins (Cornelis & Matthijs, 2002; Ravel & Cornelis, 2003). P. fluorescens ATCC 17400 produces both a PVD and another, non-fluorescent, siderophore, quinolobactin (QB) (Mossialos et al., 2000; Cornelis & Matthijs, 2002). PVDs are composed of a conserved dihydroxyquinoline chromophore, a variable peptide chain, comprising 6–12 amino acids, specific to a producing strain, and a side-chain, generally a dicarboxylic acid or an amide (Ravel & Cornelis, 2003). Both the chromophore and the peptide chain of PVDs are synthesized by non-ribosomal peptide synthetases (Merriman et al., 1995; Lehoux et al., 2000; Mossialos et al., 2002). Ferribactin, the precursor of PVD, is probably exported to the periplasm where it is matured into PVD with a chromophore (Baysse et al., 2002). Recently, we demonstrated that de novo haem biosynthesis is required for the production of PVD in P. fluorescens (Baysse et al., 2001).

The porphyrin haem in pseudomonads is formed from glutamyl-tRNA via the general precursor molecule 5-aminolevulinic acid (ALA) (Hungerer et al., 1995). Major genetic regulatory points for haem biosynthesis have been found at the initial step of ALA formation via control of hemA expression and the late step of coproporphyrinogen III decarboxylation via hemN and hemF regulation (Rompf et al., 1998; Krieger et al., 2002; Schobert & Jahn, 2002). The final step of haem biosynthesis, the insertion of iron into protoporphyrin IX (PPIX) catalysed by ferrochelatase encoded by hemH, was also proposed to be subject to regulation by iron availability (Qi & O'Brian, 2002). We isolated one mutant of P. fluorescens ATCC 17400 with a transposon insertion in the ccmC gene that showed greatly reduced production of PVD, an impaired maturation of the PVD chromophore, together with a decreased capacity to utilize this particular siderophore (Gaballa et al., 1996, 1998; Baysse et al., 2002). The product of this gene, the cytoplasmic membrane protein CcmC, is primarily involved in the biogenesis of c-type cytochromes (Thöny-Meyer, 1997; Kranz et al., 1998). CcmC binds haem in the periplasm, probably via two conserved histidines in the first and the third periplasmic loops, the interaction being stabilized by hydrophobic amino acids in the second periplasmic loop, mostly composed of tryptophan residues (Goldman et al., 1998; Schulz et al., 2000). It has been established that CcmC further delivers haem to another membrane protein, CcmE, considered to be a haem chaperone that passes haem to a haem lyase complex (comprising the CcmF protein) before covalent binding of the haem prosthetic group to apocytochromes c (Schulz et al., 1998, 1999, 2000). CcmC has been postulated to be a cytoplasm-to-periplasm haem exporter (Kranz et al., 1998; Goldman et al., 1998) and has been demonstrated to interact with the CcmAB ABC transporter (Goldman et al., 1997). Some results, however, seem to indicate that CcmC can sustain cytochrome c biogenesis without CcmA or CcmB (Cook & Poole, 2000; Page & Ferguson, 1999; Schulz et al., 2000). We here present evidence that, in the absence of CcmC, the production of siderophores and their utilization as iron source are impaired. We also demonstrate a reduced production of haem by the ccmC mutant, resulting in increased sensitivity to H2O2. Finally, a model is proposed where the reduced availability of haem is the source of the pleiotropic defects of ccmC mutants.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, vectors and growth conditions.
Strains and plasmids used in this study are listed in Table 1. P. fluorescens ATCC 17400 and the ccmC mutant were maintained in Casamino acids (CAA) medium (Cornelis et al., 1992). Unless otherwise indicated, 50 ml cultures were inoculated from an overnight pre-culture and incubated at 28 °C at 200 r.p.m. (New-Brunswick Innova shaker). Growth was followed in a Bio-Screen apparatus (Life Technologies) using the following parameters: shaking for 10 s every minute, reading every 20 min, temperature 28 °C. When needed, CAA medium was supplemented with ethylenediaminedihydroxyphenylacetic acid [EDDHA (0·5 mg ml-1)], purified PVD (50 µM) or ALA. Antibiotics were added to P. fluorescens strains at the following concentrations: kanamycin, 200 µg ml-1; chloramphenicol, 300 µg ml-1; tetracycline, 100 µg ml-1; gentamicin, 100 µg ml-1. Escherichia coli strains were grown at 37 °C in Luria–Bertani broth (LB) with the appropriate antibiotics: kanamycin, 100 µg ml-1; ampicillin, 100 µg ml-1; chloramphenicol, 25 µg ml-1; tetracycline, 15 µg ml-1.


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Table 1. Strains and plasmids used in this study

 
H2O2 resistance.
The susceptibility of the wild-type P. fluorescens strain and the ccmC mutant to H2O2 was tested as described previously (Baysse et al., 2000).

DNA methodology.
DNA manipulations were done according to standard protocols (Sambrook et al., 1989). Restriction endonucleases, T4 DNA ligase and Klenow fragment (Pharmacia) were used according to the manufacturer's instructions. Sequencing was done by Eurogentec; sequences were analysed using the GENE COMPAR software package (applied maths) and the BLAST server of the NCBI or the Pseudomonas genome project (http://www.pseudomonas.com).

Siderophores detection.
PVD concentration was estimated by fluorescence spectroscopy (Höfte et al., 1993) and normalized by a biomass unit expressed as OD600 of the culture. Siderophore production was measured using the universal chrome azurol S (CAS) detection assay of Schwyn & Neilands (1987).

Porphyrins detection.
Porphyrins were detected by spectrofluorimetry (Miyamoto et al., 1992). After overnight growth, the cells were recovered by centrifugation (5000 g for 5 min), resuspended in 1/10 volume of acetone/0·1 M NH4OH (9 : 1, v/v) and mixed. Cell extracts were centrifuged and the supernatants collected. Fluorescence emission spectra were recorded in a Shimadzu fluorimeter using an excitation wavelength of 405 nm (emission at 630 nm).

Inactivation of ccmC by allelic exchange.
The gentamicin cassette was amplified with Taq polymerase (Life Technologies) from the pBBR1MCS-5 vector (Kovach et al., 1994) using primers gent1 (5'-ATAAGAATGCGGCCGCACACCGTGGAAACGGA-3') and gent2 (5'-ATAAGAATGCGGCCGCGATCTCGGCTTGAACGA-3'). The 800 bp fragment was then end-blunted using the T4 DNA polymerase.

The cassette was introduced into the blunt end SphI site of the 2 kbp HindIII–PstI fragment containing ccmC, ccmD and ccmE from P. fluorescens and subcloned into the suicide vector pBR325 (Gaballa et al., 1996). After mobilization of the construct into P. fluorescens, ccmC mutants were selected for gentamicin resistance and chloramphenicol sensitivity. The gene replacements were confirmed by PCR using primers ccmc1 (5'-CATCGTCGGCCTGGTATGGA-3') and ccmc2 (5'-GAAAGAGGCGGCGAAACTCATCCA-3').

Inactivation of the hemH gene in the pvsA mutant.
Construction of a hemH mutant was done by transposon mutagenesis using pTnmodOtc (Dennis & Zylstra, 1998). Colonies were screened on LB medium containing 100 µg tetracycline ml-1 and 20 µM of haemin for red fluorescence under UV light after 72 h incubation at 28 °C. One colony showing red fluorescence was isolated. The auxotrophy of this mutant for haemin was confirmed by its inability to growth on CAA medium without externally added haemin. The regions flanking the transposon insertion were isolated as described previously (Baysse et al., 2001) and sequenced. The position of the Tn5 insertion in the hemH gene is described by Baysse et al. (2001). In the double pvsA hemH mutant the insertion occurred at 127 bases from the 5' end of the hemH ORF.

The vector pPAhemH, containing the hemH gene from PAO1 cloned in the expression vector pMMB208 (Baysse et al., 2001), was introduced into the double mutant by conjugation with the helper strain carrying pRK2013. The transconjugants were selected on CAA medium without haemin to confirm the functional complementation.

Cross-feeding of the hemH mutant.
This experiment was performed on CAA medium plates containing 50 µg FeCl3 ml-1 and 0, 20, 40 or 80 µg ALA ml-1. Two vertical lines of the hemH mutant were streaked, followed by two perpendicular lines of the wild-type strain and the ccmC mutant. The plates were incubated at 28 °C for 72 h and the growth of the hemH mutant at the crossing zone was checked. Each experiment was repeated in duplicate.

Proteinase K-treated lysates.
Cultures of wild-type P. fluorescens and ccmC mutant in CAA medium plus 50 µM FeCl3 were harvested by centrifugation and resuspended in 0·1 vol. of 0·01 M Tris/HCl, pH 7·8, 0·01 M EDTA and 0·5 % (w/v) SDS. Cells were broken by sonication (6x30 s with 30 s pauses at 300 W). Cell debris was removed by 3 min centrifugation at 4000 g and the cell-free lysate was incubated for 2 h with 50 µg proteinase K ml-1 at 50 °C. Protease inhibitors (CompleteTM; Roche Diagnostics) were added to the samples before the cross-feeding assays.

Western blot with polyclonal antibodies against P. fluorescens HemH.
Polyclonal antibodies against P. fluorescens HemH were generated as described previously (Baysse et al., 2001). Western blot analysis was performed on cytoplasmic and inner membrane fractions of both wild-type and ccmC mutant using the BM Chromogenic Western Blotting Kit from Roche.

HPLC analysis of porphyrins in P. fluorescens.
P. fluorescens was grown aerobically on M9 minimal medium (Sambrook et al., 1989) containing 40 mM glucose as carbon source to the stationary phase in the presence or absence of 50 µg ALA ml-1. Cells were harvested by centrifugation, resuspended in 100 µl H2O and disrupted by brief sonication. Porphyrins were extracted by addition of 100 µl acetone/concentrated HCl (97·5/2·5, v/v). After centrifugation, 20 µl of the resulting supernatant was loaded directly onto a 4·6x250 mm ODS Hypersil C18 reversed-phase column (Techlab) with a pore width of 120 Å. Separation and identification of porphyrins were performed at a flow rate of 0·5 ml min-1 as described previously (Layer et al., 2002). Coproporphyrin III (Porphyrin Products) and PPIX (Sigma) were used as porphyrin standards.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Defect in the haem exporter CcmC or the ferrochelatase HemH leads to PVD and QB deficiency
Previously, we demonstrated that a P. fluorescens ATCC 17400 ccmC mutant had a greatly reduced PVD production and was impaired in PVD utilization as iron source, but not at the level of the uptake (Gaballa et al., 1996, 1998; Baysse et al., 2002). To investigate whether the absence of CcmC also affects other siderophore-mediated iron uptake systems, besides PVD, we decided to introduce the ccmC mutation into a mutant of P. fluorescens ATCC 17400 deficient for the non-ribosomal peptide synthetase PvsA, which is needed for the PVD chromophore synthesis (Mossialos et al., 2002). The pvsA mutant still produces high levels of another siderophore, QB (Mossialos et al., 2000). The ccmC gene in the pvsA mutant was disrupted by double recombination. The single and the double mutants grew similarly in liquid Casamino acid (CAA) medium (data not shown). The effects of the mutation on the production of QB were evaluated by examining the diameter of the halos of discoloration around colonies of the pvsA and pvsA ccmC mutants on a CAS agarose plate (Fig. 1a). A decrease in the diameter of the clear halos around the colony of the pvsA ccmC mutant was observed, indicating that QB production was strongly reduced.



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Fig. 1. (a) QB production by P. fluorescens mutant pvsA (left) and pvsA ccmC (right) as visualized by the CAS assay. (b) QB production by P. fluorescens mutant pvsA (left), pvsA hemH (centre) and pvsA hemH carrying pPAhemH (right) as visualized by the CAS assay. (c) Growth stimulation of P. fluorescens mutant pvsA (black bars) and pvsA ccmC (bars with squares) by desferrioxamine B (Df), haem (Hm), P. aeruginosa PVDs type II (PvdII) and type I (PvdI), and by cognate PVD (Pvdpf). The bars represent the diameter (in cm) of the growth stimulation zone around a disc impregnated with the iron source (see Methods for details). No value is shown for Df and pvsA ccmC because of the total lack of growth stimulation.

 
Likewise, inactivation of hemH affects QB production since a double pvsA hemH mutant did not produce a detectable CAS discoloration. The production of the siderophore was restored, although weakly, by complementation of the mutant with the hemH gene from P. aeruginosa PAO1 (Fig. 1b), but not by externally added haemin present in the medium (data not shown). This observation might provide a first indication for the importance of a common factor that is limiting for the biosynthesis of both siderophores (PVD and QB) in the ccmC and the hemH mutants. One likely candidate is iron–PPIX, since de novo haem biosynthesis has been shown to be important for the biosynthesis of both siderophores (Baysse et al., 2001; S. Matthijs, unpublished results, see further in Discussion). Recent data showed that overexpressing a heterologous ferrochelatase in E. coli did not lead to significant accumulation of haem, although the enzyme activity could be demonstrated in vitro (Kwon et al., 2003). Indeed, the three last enzymes of the haem biosynthetic pathway are supposed to work in an interactive manner that requires a compatible set of enzymes. Using the hemH gene from P. aeruginosa PAO1 to complement a mutant of P. fluorescens may affect the in vivo activity due to insufficient enzyme compatibility.

Haem-complexed iron, but not siderophore-bound iron, sustains growth of the pvsA ccmC double mutant
Since the pvsA mutant cannot grow in media supplemented with the strong iron(III) chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA), it is possible to use this mutant as an indicator for the use of high-affinity heterologous siderophores as a source of iron. Fig. 1(c) shows that the growth of the single pvsA mutant was stimulated by both the cognate and two heterologous PVDs (from P. aeruginosa) and by desferrioxamine B. The pvsA ccmC double mutant on the other hand showed a reduced capacity to utilize the three PVDs and desferrioxamine B (Fig. 1c) could not stimulate its growth at all. Interestingly, the growth of this double mutant was still stimulated by haemin, to the same extent as the pvsA mutant, indicating that the ccmC mutation only affects siderophore-mediated iron acquisition but not haem-mediated iron uptake (Fig. 1c).

The ccmC mutant shows reduced haem production and accumulates PPIX
Colonies of the ccmC mutant showed a reddish fluorescence under UV, especially on iron-supplemented medium. Porphyrinogens and porphyrins accumulated by the ccmC mutant grown in the presence and absence of ALA were determined by HPLC analysis. ALA was added to growth media to overcome the initial rate-limiting step of tetrapyrrole biosynthesis and to allow significant detectable tetrapyrrole accumulation (Doss & Philipp-Dornston, 1971; Philipp-Dornston & Doss, 1973). The data presented in Fig. 2 indicate that there is a general decrease in the production of tetrapyrroles by the cells of the ccmC mutant in the absence of added ALA, an indication that the haem content might be decreased (Fig. 2c versus Fig. 2a). As expected, and in agreement with earlier observations for various other bacteria, the wild-type and mutant strains grown in the absence of ALA did not contain significantly overproduced tetrapyrroles (Doss & Philipp-Dornston, 1971; Philipp-Dornston & Doss, 1973).



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Fig. 2. Separation by HPLC of porphyrins from wild-type P. fluorescens (a and b) and ccmC mutant (c and d). In (a) and (c), the cultures were grown in the absence of ALA while, in (b) and (d), the cells were grown in the presence of ALA. See Methods for details. Copro III, coproporphyrinogen III; Proto IX, PPIX.

 
When grown in the presence of ALA, the mutant accumulated higher amounts of PPIX compared to the wild-type, indicative of a defective ferrochelatase activity (Fig. 2d versus Fig. 2b).

To distinguish between enzymically generated PPIX and oxidized protoporphyrinogen IX chemically formed during the isolation procedure, tetrapyrrole preparation was performed anaerobically. One half of the resulting preparation was analysed immediately, while the other half was completely oxidized using H2O2 treatment. No significant difference between the differentially treated tetrapyrrole preparations was observed. These experiments clearly indicate that PPIX accumulation was caused by the ccmC mutation. The parallel observed increase in the cellular coproporphyrinogen III concentration of the ccmC mutant (Fig. 2d) is in agreement with previous investigations of the physiological effects of an E. coli hemH mutant. Due to the rate-limiting step of coproporphyrinogen III oxidation on the late haem biosynthetic pathway, mutation of late genes always results in a minor coproporphyrinogen III accumulation (Schobert & Jahn, 2002).

Further in vivo evidence for PPIX accumulation is provided by the light-sensitive vis phenotype of the ccmC mutant. Similar to observations made for a hemH mutant of E. coli, the PPIX-accumulating mutant fails to grow in bright light due to the light-depending formation of detrimental radicals from the porphyrin ring system (Miyamoto et al., 1991, 1992) (results not shown).

The ccmC mutant fails to rescue a haem-auxotrophic hemH mutant via cross-feeding
The product of the hemH gene, the enzyme ferrochelatase, is responsible for the insertion of Fe2+ into the protoporphyrin ring. Consequently, a mutant devoid of ferrochelatase activity accumulates large quantities of PPIX (Nakahigashi et al., 1991; Nakayashiki & Inokuchi, 1997; Yang et al., 1995). The accumulation of PPIX results in reddish-orange colonies that give a typical red fluorescence under UV and in light sensitivity (Nakahigashi et al., 1991). As expected, the ferrochelatase-deficient mutant of P. fluorescens is auxotrophic for haem (Baysse et al., 2001). Therefore, we decided to use this mutant as an indicator either for the presence of haem in cell extracts after proteinase K treatment or for the presence of free haem released by the cells. Fig. 3 shows that the wild-type P. fluorescens cells excrete enough haem to promote the growth of the hemH mutant in their vicinity. This growth promotion was dependent on the presence of both iron and ALA in the medium. Clearly this phenotype was not observed in the vicinity of the ccmC mutant (Fig. 3a). Growth promotion was again observed when the ccmC mutant was complemented with the plasmid pPYOV35 containing a wild-type copy of the ccmC gene (Gaballa et al., 1996). Similar results were obtained when proteinaseK-treated cell extracts were used. In this case, again, no stimulation of the growth of the hemH mutant was observed when the extracts from the ccmC mutant were used, as shown in Fig. 3(b): under UV exposure, no growth of red-fluorescent bacteria, indicative of the PPIX-accumulating hemH mutant, is observed around the well containing cell-free extract from the ccmC mutant. These results confirm that in vivo the ccmC mutant produces and exports fewer tetrapyrroles, both functions being complemented by ccmC in trans.



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Fig. 3. (a) Cross-feeding of a hemH mutant (vertical streak) by wild-type P. fluorescens (left), ccmC (middle) and ccmC complemented with ccmC in trans (right). (b) Growth stimulation of the hemH mutant by proteinase K-treated cellular extracts from wild-type P. fluorescens (left) and ccmC mutant (right). The conditions are described in Methods. After overnight incubation, the plate was exposed to UV to detect the PPIX-accumulating hemH mutant.

 
The ccmC mutant shows reduced resistance to oxidative stress
Decreased haem content and increase in PPIX are likely to affect the response of the ccmC mutant to oxidative stress. Indeed, catalases are enzymes that employ haem as co-factor; therefore, a decrease in the supply of haem, as observed for the ccmC mutant, is expected to result in increased sensitivity to H2O2. However, PPIX is known to be toxic for the cells, especially because it is a photo-reactive molecule that can be easily degraded, a process resulting in the production of reactive oxygen species (Yang et al., 1995).

The inhibition caused by H2O2 on the growth of the wild-type and the ccmC mutant was tested. The ccmC mutant displayed an increased sensitivity to H2O2 (zone of inhibition 5·85±0·15 cm compared to 3·9±0·1 cm for the wild-type strain; n=3). Addition of external haem in the form of haemin (final concentration, 40 µM) restored an almost normal level of sensitivity to H2O2 to the ccmC mutant (zone of inhibition 4·15±0·05 cm compared to 3·9±0·1 cm for the wild-type strain), another confirmation of a haem shortage in the ccmC cells.

Lethality of a double hemH ccmC mutation
A double hemH ccmC mutant was obtained, but only when the P. aeruginosa hemH gene was present in trans. Furthermore, it was impossible to cure this hemH ccmC mutant from the plasmid bearing the hemH gene (results not shown). These results indicate that the combination of hemH and ccmC mutations is lethal.

Ferrochelatase expression is not influenced by the ccmC mutation
By performing a Western blot using antibodies against P. fluorescens HemH (Baysse et al., 2001), we detected the protein in the membrane fractions of both the wild-type and the ccmC mutant. No significant difference in the HemH level was observed (data not shown).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The importance of the cytoplasmic membrane protein CcmC in the biogenesis of c-type cytochromes in Gram-negative bacteria has been clearly established, but its role is still controversial (Thöny-Meyer, 1997; Kranz et al., 1998; Page & Ferguson, 1999; Cook & Poole, 2000; Schulz et al., 2000). Obviously, absence of CcmC causes a range of defects which are not all due to the absence of c-type cytochromes (Gaballa et al., 1998).

Absence of CcmC affects haem biosynthesis at the level of iron chelation
In this study, using both direct (detection of tetrapyrroles by HPLC) and indirect methods, we came to the conclusion that in a ccmC mutant haem production is reduced, while PPIX accumulates when haem biosynthesis is boosted by ALA. Wild-type P. fluorescens, but not a ccmC mutant, was able to cross-feed the hemH mutant on solid medium, in an iron- and ALA-dependent fashion. This result strongly suggests that wild-type cells can excrete free haem when its synthesis is boosted. We therefore propose a model (Fig. 4) where CcmC (probably in association with the other Ccm proteins) is a transporter for haem, and is associated with other haem biosynthesis proteins, including ferrochelatase, to form a metabolon. Inactivation of ccmC would directly or indirectly down-regulate the haem biosynthetic pathway to prevent formation of toxic free haem. It is interesting to mention that metabolite channelling has already been proposed for several steps in the pathway of haem biosynthesis (Moser et al., 2001; Olsson et al., 2002). Channelling is observed when precursors in a metabolic pathway are known to be potentially harmful for the cell (Massant et al., 2002). This model also explains why different mutations in periplasmic loops of P. fluorescens CcmC affect differently the ability to produce and use PVD, on the one hand, and cytochrome c biogenesis, on the other (Gaballa et al., 1998). Mutations affecting the binding of haem by CcmC in the periplasm preferentially affect cytochrome c biogenesis since only the transfer of haem from CcmC to the haem chaperone CcmE would be impaired (Schulz et al., 2000). On the other hand, mutations affecting to a different extent the transport function of CcmC would only partially affect cytochrome c biogenesis if sufficient amounts of haem still reach the periplasmic space. These mutations seem to have more negative consequences at the level of haem biosynthesis, more probably by inactivating the last step of haem biosynthesis, resulting in accumulation of PPIX. We have evidence that this regulation is not at the level of the ferrochelatase expression (results not shown). However, absence of CcmC may affect the activity of the enzyme.



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Fig. 4. Model explaining the different phenotypes observed in the ccmC mutant. (a) In the wild-type, the complex of Ccm proteins in the inner membrane (shown as a grey box) is associated with haem biosynthesis enzymes, including ferrochelatase (HemH). Haem (represented as a white cross with Fe2+) is either channelled via CcmC to the periplasm (where it is transferred to CcmE and CcmF for the biogenesis of c-type cytochromes) or incorporated into cytoplasmic haemoproteins including some siderophore biosynthetic enzymes. Haem can also be transported or diffuse to the periplasm where it can be incorporated into cytochromes other than the c-type. Our results also indicate that haem can be exported. Iron(III) is released from ferrisiderophores by a [Fe–S] reductase. (b) In the absence of CcmC, the haem metabolon is destabilized, resulting in decreased haem production (shown in dotted lines) and accumulation of PPIX (white crosses), which can result in the accumulation of reactive oxygen species (ROS). As a consequence of the oxidative stress, the [Fe–S] centre(s) of the ferrisiderophore reductase(s) are destroyed (shown by the cross), resulting in an inability to release iron from them. The transport of ferrisiderophores is, however, unaffected. Lack of haem results in decreased siderophore production and reduced synthesis of catalase. Haem transport to the periplasm is decreased (dotted line). In the absence of the Ccm complex, no cytochrome c is produced.

 
Absence of CcmC induces a state of oxidative stress
Accumulation of porphyrins is known to cause an oxidative stress, as demonstrated for ferrochelatase-deficient E. coli mutants (Yang et al., 1995). PPIX is known to be toxic for the cells, especially because it is a photo-reactive molecule that can be easily degraded, a process resulting in the production of reactive oxygen species (Yang et al., 1995; Maciver & Hansen, 1996). Indeed, we demonstrated that the growth of the ccmC mutant that accumulates PPIX is totally impaired when the cells are exposed to light (results not shown). In a double hemH ccmC mutant, the high levels of PPIX could result in cell death due to oxidative damage, explaining why such a double mutant could not be obtained in the absence of complementation by hemH in trans (Yang et al., 1995). Similarly, catalase, which is a haemoprotein, shows a reduced activity in the ccmC mutant, as judged by the increased sensitivity of this mutant to H2O2. Interestingly, externally added haemin restores almost wild-type levels of resistance to H2O2, but does not increase the production of siderophores. Haemin is probably taken up via a TonB-dependent receptor by P. fluorescens, as is the case in P. aeruginosa (Létoffé et al., 2000; Ochsner et al., 2000; Wandersman & Stojilkovic, 2000). It therefore seems that haemin taken up via TonB-dependent receptors can restore catalase activity.

Absence of CcmC affects the production of siderophores
One obvious phenotype of mutants affected in the biogenesis of c-type cytochromes is their reduced capacity to grow under conditions of iron limitation (Gaballa et al., 1996, 1998; Yeoman et al., 1997; Pearce et al., 1998; Polesky et al., 2001; Baysse et al., 2002, Viswanathan et al., 2002). The decrease in haem production can explain some of the observed phenotypes of the ccmC mutant, such as reduced production of siderophores PVD and QB. We have already demonstrated that de novo haem biosynthesis was needed for the production of PVD (Baysse et al., 2001). Haem is probably a necessary component for the biosynthesis of QB as well, since the enzyme tryptophan-2,3-dioxygenase, a haemoprotein, is needed for its production (S. Matthijs, unpublished results). This is supported by the fact that a hemH mutant does not produce QB.

Absence of CcmC affects the utilization of ferrisiderophores as iron source
The cells of the ccmC mutant are also characterized by a general inability to use different ferrisiderophores as iron source. In the case of PVD, we have already demonstrated that the defect was not due to an impaired uptake or absence of production of siderophore receptors (Gaballa et al., 1996). Haemin utilization, on the other hand, was not affected. For the release of iron from ferrisiderophores in the cytoplasm, a reduction mechanism is probably needed. The E. coli FhuF protein is likely to be the reductase for the release of iron from desferrioxamine B (Patzer & Hantke, 1999). The production of FhuF, a [2Fe–2S] protein, is repressed by iron and the stability of its iron–sulphur centre is maintained by different enzymes encoded by the sufABCDSE operon (Patzer & Hantke, 1999). In a recent article, Nachin et al. (2003) showed that SufC from Erwinia chrysanthemi (also part of the same iron-regulated operon) is needed for the biogenesis of [Fe–S] centres under conditions of oxidative stress (Nachin et al., 2001, 2003). Interestingly, the same authors observed that a sufC mutant is unable to use the siderophore chrysobactin as iron source, even though the uptake of the ferrisiderophore was not impaired. This phenotype is very similar to the one we describe here. In fact, we can predict that the hemH mutation should give the same phenotype, but this is difficult to demonstrate experimentally since it needs haemin (itself a source of iron) to grow.

A model explaining the pleiotropic phenotype of the ccmC mutant
We propose a model (Fig. 4) where in the absence of a functional CcmC protein (and/or other Ccm proteins) the biosynthesis of haem is impaired, leading to a reduction in haem content and in an accumulation of PPIX. Since some haemoproteins are probably involved in the biosynthesis of PVD (Baysse et al., 2001) and QB (S. Matthijs, unpublished results), this would explain why fewer siderophores are produced by the ccmC mutant. As mentioned above, PPIX can induce the production of active oxygen radicals, which in turn can destroy the [Fe–S] centre(s) of ferrisiderophore reductase(s), resulting in a general incapacity to use ferrisiderophores as iron source without affecting the capacity to utilize haemin.

Future research should help to get a better insight into the function of CcmC and other proteins of the Ccm complex, by using a proteomic approach.


   ACKNOWLEDGEMENTS
 
We wish to thank the Flemish Fund for Scientific Research (FWO), and the Alphonse & Jean Forton fund against Cystic Fibrosis for their financial support. S. Matthijs was a recipient of a FWO fellowship. Many thanks to Willy Verheulpen for computer assistance.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 June 2003; revised 22 August 2003; accepted 1 September 2003.



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