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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Biomerit Research Centre, University College of Cork, Ireland.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 HindIIIPstI 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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
).
|
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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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 [2Fe2S] protein, is repressed by iron and the stability of its ironsulphur 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 [FeS] 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 [FeS] 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baysse, C., Matthijs, S., Pattery, T. & Cornelis, P. (2001). Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400. FEMS Microbiol Lett 205, 5763.[CrossRef][Medline]
Baysse, C., Budzikiewicz, H., Uria-Fernandez, D. & Cornelis, P. (2002). Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC. FEBS Lett 523, 2328.[CrossRef][Medline]
Braun, V. & Braun, M. (2002). Iron transport and signalling in Escherichia coli. FEBS Lett 529, 7885.[CrossRef][Medline]
Braun, V. & Killmann, H. (1999). Bacterial solutions to the iron-supply problem. Trends Biochem Sci 24, 104109.[CrossRef][Medline]
Cook, G. M. & Poole, R. K. (2000). Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association. Microbiology 146, 527536.
Cornelis, P. & Matthijs, S. (2002). Diversity of siderophore-mediated iron uptake in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol 4, 787798.[CrossRef][Medline]
Cornelis, P., Anjaiah, V., Koedam, N., Delfosse, P., Jacques, P., Thonart, P. & Neirinckx, L. (1992). Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads. J Gen Microbiol 138, 13371343.[Medline]
de Lorenzo, V., Herrero, M., Jacubzik, U. & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for the insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 65686572.[Medline]
Dennis, J. J. & Zylstra, G. J. (1998). Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 64, 27102715.
Doss, M. O. & Philipp-Dornston, W. K. (1971). Porphyrin and heme biosynthesis from endogenous and exogenous -aminolevulinic acid in Escherichia coli, Pseudomonas aeruginosa and Achromobacter metalcaligenes. Hoppe-Seylers Z Physiol Chem 352, 725733.[Medline]
Gaballa, A., Koedam, N. & Cornelis, P. (1996). A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400. Mol Microbiol 21, 777785.[CrossRef][Medline]
Gaballa, A., Baysse, C., Koedam, N., Muyldermans, S. & Cornelis, P. (1998). Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake. Mol Microbiol 30, 547555.[CrossRef][Medline]
Goldman, B. S., Beckman, D. L., Bali, A., Monika, E. M., Gabbert, K. K. & Kranz, R. G. (1997). Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis. J Mol Biol 268, 724738.[CrossRef][Medline]
Goldman, B. S., Beck, D. L., Monika, E. M. & Kranz, R. G. (1998). Transmembrane heme delivery systems. Proc Natl Acad Sci U S A 95, 50035008.
Höfte, 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, 8591.[Medline]
Hungerer, C., Troup, B., Römling, U. & Jahn, D. (1995). Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa. J Bacteriol 177, 14351443.[Abstract]
Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M. & Peterson, K. M. (1994). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 800801.[Medline]
Kranz, R., Lill, R., Goldman, B., Bonnard, G. & Merchant, S. (1998). Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol Microbiol 29, 383396.[CrossRef][Medline]
Krieger, R., Rompf, A., Schobert, M. & Jahn, D. (2002). The Pseudomonas aeruginosa hemA promoter is regulated by Anr, Dnr, NarL and Integration Host Factor. Mol Genet Genomics 267, 409417.[CrossRef][Medline]
Kwon, S. J., de Boer, A. L., Petri, R. & Schmidt-Dannert, C. (2003). High-level of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis. Appl Environ Microbiol 69, 48754883.
Layer, G., Verfurth, K., Mahlitz, E. & Jahn, D. (2002). Oxygen-independent coproporphyrinogen-III oxidase HemN from Escherichia coli. J Biol Chem 277, 3413634142.
Lehoux, D., Sanschagrin, F. & Levesque, R. (2000). Genomics of the 35-kb locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa. FEMS Microbiol Lett 190, 141146.[CrossRef][Medline]
Létoffé, S., Omori, K. & Wandersman, C. (2000). Functional characterization of the HasA (Pf) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor. J Bacteriol 182, 44014405.
Maciver, I. & Hansen, E. J. (1996). Lack of expression of the global regulator OxyR in Haemophilus influenzae has a profound effect on growth phenotype. Infect Immun 64, 46184629.[Abstract]
Massant, J., Verstreken, P., Durbecq, V., Kholti, A., Legrain, C., Beeckmans, S., Cornelis, P. & Glansdorff, N. (2002). Metabolic channelling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus. J Biol Chem 277, 1851718522.
Merriman, T. R., Merriman, M. E. & Lamont, I. L. (1995). Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases. J Bacteriol 177, 252258.[Abstract]
Miyamoto, K., Nakahigashi, K., Nishimura, K. & Inokuchi, H. (1991). Isolation and characterisation of visible light-sensitive mutants of Escherichia coli K12. J Mol Biol 219, 393398.[Medline]
Miyamoto, K., Nishimura, K., Masuda, T., Tsuji, H. & Inokuchi, H. (1992). Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli. FEBS Lett 310, 246248.[CrossRef][Medline]
Morales, V. M., Backman, A. & Bagdasarian, M. (1991). A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97, 3947.[CrossRef][Medline]
Moser, J., Schubert, W. D., Beier, V., Bringemeier, I., Jahn, D. & Heinz, D. W. (2001). V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis. EMBO J 20, 65836590.
Mossialos, D., Meyer, J. M., Budzikiewicz, H., Wolff, U., Koedam, N., Baysse, C., Anjaiah, V. & Cornelis, P. (2000). Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine. Appl Environ Microbiol 66, 487492.
Mossialos, D., Ochsner, U., Baysse, C. & 8 other authors (2002). Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine. Mol Microbiol 45, 16731685.[CrossRef][Medline]
Nachin, L., El Hassouni, M., Loiseau, L., Expert, D. & Barras, D. (2001). SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase. Mol Microbiol 39, 960972.[CrossRef][Medline]
Nachin, L., Loiseau, L., Expert, D. & Barras, F. (2003). SufC: an unorthodox cytoplasmic ABC/ATPase required for [FeS] biogenesis under oxidative stress. EMBO J 22, 427437.
Nakayashiki, T. & Inokuchi, H. (1997). Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli. Mol Gen Genet 255, 376381.[CrossRef][Medline]
Nakahigashi, K., Nishimura, K., Miyamoto, K. & Inokuchi, H. (1991). Photosensitivity of a protoporphyrin-accumulating, light sensitive mutant (visA) of Escherichia coli K12. Proc Natl Acad Sci U S A 88, 1052010524.[Abstract]
Ochsner, U. A., Johnson, Z. & Vasil, M. L. (2000). Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146, 185198.
Olsson, U., Billberg, A., Sjovall, S., Al-Karadaghi, S. & Hansson, M. (2002). In vivo and in vitro studies of Bacillus subtilis ferrochelatase mutants suggest substrate channeling in the heme biosynthesis pathway. J Bacteriol 184, 40184024.
Page, M. D. & Ferguson, S. J. (1999). Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB. Microbiology 145, 30473057.
Patzer, S. I. & Hantke, K. (1999). SufS is a NifS-like protein, and SufD is necessary for stability of the [2Fe2S] FhuF protein in Escherichia coli. J Bacteriol 181, 33073309.
Pearce, D. A., Page, M. D., Norris, H. A., Tomlinson, E. J. & Ferguson, S. J. (1998). Identification of the contiguous Paracoccus denitrificans ccmF and ccmH genes: disruption of ccmF, encoding a putative transporter, results in formation of an unstable apocytochrome c and deficiency in siderophore production. Microbiology 144, 467477.[Abstract]
Philipp-Dornston, W. K. & Doss, M. O. (1973). Comparison of porphyrin and heme in various heterotrophic bacteria. Enzyme 16, 5764.[Medline]
Polesky, A. H., Ross, J. T., Falkow, S. & Tompkins, L. S. (2001). Identification of Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection. Infect Immun 69, 977987.
Qi, Z. & O'Brian, M. R. (2002). Interaction between the bacterial iron response regulator and ferrochelatase mediates genetic control of heme biosynthesis. Mol Cell 9, 155162.[Medline]
Ravel, J. & Cornelis, P. (2003). Genomics of pyoverdine-mediated iron uptake in pseudomonads. Trends Microbiol 11, 195200.[Medline]
Rompf, A., Hungerer, C., Hoffmann, T. & 7 other authors (1998). Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr. Mol Microbiol 29, 985997.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schobert, M. & Jahn, D. (2002). Regulation of heme biosynthesis in non-phototrophic bacteria. J Mol Microbiol Biotechnol 4, 287294.[Medline]
Schulz, H., Hennecke, H. & Thöny-Meyer, L. (1998). Prototype of a heme chaperone essential for cytochrome c maturation. Science 281, 11971200.
Schulz, H., Hennecke, H. & Thöny-Meyer, L. (1999). Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC transporter CcmAB. Proc Natl Acad Sci U S A 96, 64626467.
Schulz, H., Pellicioli, E. C. & Thöny-Meyer, L. (2000). New insights into the role of CcmC, CcmD, and CcmE in the heme delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC. Mol Microbiol 37, 13791388.[CrossRef][Medline]
Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 4756.[Medline]
Thöny-Meyer, L. (1997). Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev 61, 337376.[Abstract]
Viswanathan, V. K., Kurtz, S., Pedersen, L. L., Abu-Kwaik, Y., Krcmarik, K., Mody, S. & Cianciotto, N. P. (2002). The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element. Infect Immun 70, 18421852.
Wandersman, C. & Stojilkovic, I. (2000). Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores. Curr Opin Microbiol 3, 215220.[CrossRef][Medline]
Yang, H., Inokuchi, H. & Adler, J. (1995). Phototaxis away from blue light by an Escherichia coli mutant accumulating protoporphyrin IX. Proc Natl Acad Sci U S A 92, 73327336.[Abstract]
Yeoman, K. H., Delgado, M. J., Wexler, M., Downie, J. A. & Johnston, A. W. (1997). High affinity iron acquisition in Rhizobium leguminosarum requires the cycHJKL operon and the feuPQ gene products, which belong to the family of two-component transcriptional regulators. Microbiology 143, 127134.[Abstract]
Received 13 June 2003;
revised 22 August 2003;
accepted 1 September 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |