Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK, and Oxford Centre for Molecular Sciences, New Chemistry Building, South Parks Road, Oxford, OX1 3QT, UK1
Author for correspondence: M. Dudley Page. Tel: +31 20 4447183. Fax: +31 20 4447229. e-mail: mpage{at}bio.vu.nl
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ABSTRACT |
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Keywords: Paracoccus denitrificans, c-type cytochrome biogenesis, putative ABC-transporter, siderophores
Abbreviations: ABC, ATP-binding cassette; CAS, chrome azurol sulphonate; HEDS, 2-hydroxyethyldisulphide; TMPD, tetramethylphenylenediamine
The GenBank accession number for the sequence determined in this work is Z71971.
a Present address: Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
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INTRODUCTION |
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We have previously reported the phenotypic consequences of disruption of ccmG in P. denitrificans (Page & Ferguson, 1997 ); one unexpected outcome of this work was that disruption of ccmG led not only to loss of c-type cytochromes but also to absence of cytochrome aa3 and to failure to grow in rich media, indicating that CcmG had a wider role in the metabolism of this organism than had previously been suspected. We now report the phenotypic consequences of disruption of P. denitrificans ccmA, ccmB, ccmC and ccmD. Complementation and phenotypic analyses of the resulting mutant strains have revealed that disruption of ccmC leads to far more severe phenotypic consequences than disruption of either ccmA, ccmB or ccmD. The implications of this result for the existence and organization of the putative ABC-transporter are discussed.
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METHODS |
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General DNA manipulations.
General DNA manipulations were as described by Sambrook et al. (1989) . DNA sequencing was performed using Sequenase version 2.0 (Amersham) and M13 universal primers.
Construction of plasmids for interposon mutagenesis.
Two plasmids containing Pd1222 genomic DNA recovered from the ccmA::Tn5 mutant HN53 were used as the source of DNA for the construction of mutagenic plasmids. These were pDP535, containing a 4·2 kb SalI fragment comprising the 5' region of Tn5 plus 1·7 kb of HN53 genomic DNA, and pDP533, containing a 6·7 kb SalI fragment comprising the 3' region of Tn5 plus 3·7 kb of HN53 genomic DNA (Page et al., 1997 ). Mutagenesis of ccmG has been described previously (Page & Ferguson, 1997
). The SalIHpaI segment from pDP535 was cloned into SalI/SmaI-cut pARO181 to create pDP30A; this was then (i) cut with XhoI and ligated to the
cassette isolated from SalI-cut pUX
to create pDP301M, (ii) cut with EcoRV and ligated to the
cassette isolated from SmaI-cut pHP45
to create pDP302M, and (iii) partially digested with BglII and ligated to the
cassette isolated from BamHI-cut pHP45
to create pDP303M. The BamHIHpaI segment from pDP533 was cloned in pARO181 cut with BamHI and SmaI to create pDP30B; this was then cut with ApaI, ligated to an adaptor formed from the complementary oligonucleotides (5'-CAAGATATCAAGGGCC-3') and (5'-CTTGATATCTTGGGCC-3') to create pDP30BL1, which was cut with EcoRV and ligated to the
cassette isolated from SmaI-cut pHP45
to create pDP304M. pDP30B was partially digested with NcoI and ligated to an adaptor formed from the complementary oligonucleotides (5'-CATGGTTCTCGAGTTC-3') and (5'-CTAGGAACTCGAGAAC-3') to create pDP30BL2, which was cut with XhoI and ligated to the
cassette isolated from SalI-cut pUX
to form pDP305M. The 0·8 kb BamHI fragment from pDP533 was cloned in pARO181 to create pDP30C, which was then cut with MscI and ligated to the
cassette isolated from SmaI-cut pHP45
to create pDP306M. The 1·7 kb BamHISalI fragment from pDP533 was cloned in pARO181 to create pDP30D, which was cut with EcoRV and ligated to the
cassette from SmaI-cut pHP45
to create pDP308M. The ApaIBamHI segment from pDP533 was cloned in pBluescript, excised with BamHI/KpnI and cloned in pARO181 to create pDP30E, which was then cut with BamHI plus SalI and ligated to the 1·7 kb BamHISalI fragment from pDP30D to create pDP30F. This plasmid was cut with BamHI and ligated to the
cassette from BamHI-cut pHP45
to create pDP309M. Mutagenic plasmids were introduced into Pd1222 by conjugation, employing pRK2013 as helper plasmid (Ditta et al., 1980
) and mated cells plated and screened on solid minimal media. In the case of pARO181-based mutagenic plasmids, streptomycin-resistant exconjugants were screened for resistance to kanamycin, a streptomycin-resistant, kanamycin-sensitive phenotype indicating
integration and pARO181 elimination.
Confirmation of the positions of cassette integration in DP302DP309.
Genomic DNA was isolated from DP302DP309 and the mutagenized ccmABCDGhisH regions cloned in pUC18 as 7·1 kb SalI fragments, using the spectinomycin resistance of the cassette as a selectable marker, to create the plasmids pDP302RpDP309R. Segments of P. denitrificans genomic DNA upstream and downstream of the
cassette were then subcloned in pUC18 as SalIHindIII and HindIII fragments, respectively (see Fig. 1
); both sets of fragments contained a region of DNA derived from the
cassette, permitting confirmation of the
cassettegenomic DNA junction region by sequencing.
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RESULTS |
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Construction of interposon mutants in the ccmABCDG gene region
A system for interposon mutagenesis of P. denitrificans has been described previously (Page & Ferguson, 1997
). The
interposon cassette was introduced into convenient restriction sites within ccmA, ccmB, ccmC, ccmD, ccmG and hisH, and at two restriction sites within ORF117. A strain in which the DNA region between the BamHI sites in ccmC and ccmG was deleted and replaced by the
cassette was also constructed. The strains generated were named DP301 to DP309 (Fig. 1
, Table 1
).
The exact positions of integration in DP302DP309 were confirmed by marker rescue of the mutagenized ORF117ccmABCDGhisH regions and sequencing of the
cassettegenomic DNA junctions. In the case of DP301, marker rescue was not achieved and the position of
integration was instead confirmed by hybridization.
Growth properties of the interposon mutants
The growth properties of the ccmG:: mutant DP307 have been reported previously (Page & Ferguson, 1997
). Mutants DP302DP307 and DP309 were incapable of growth on methanol or of anaerobic growth with nitrate as terminal electron acceptor, both growth modes of which require the synthesis of c-type cytochromes (Willison & John, 1979
; Baker et al., 1998
), and were unable to oxidize dimethylphenylenediamine (the Nadi reaction; Marrs & Gest, 1973
), a test for the presence of the cytochrome ccytochrome-c oxidase segment of the bacterial electron transport chain. In spite of these defects in electron transport, DP302DP306 grew at similar rates to the parental strain Pd1222 on succinate minimal media (Fig. 2
); mutants of P. denitrificans pleiotropically deficient in c-type cytochromes are able to grow aerobically at a rate similar to that of the parental strain because the bacterium also synthesizes a ubiquinol oxidase (Willison & John, 1979
; Baker et al., 1998
). DP309 (
ccmCDG::
) resembled DP307 (ccmG::
) in that it not only exhibited all these defects in electron transport but also grew at a reduced rate compared to Pd1222 on succinate minimal media (doubling time 2·1 h compared to 1·3 h; Fig. 2
). Like DP307, DP309 exhibited reduced pigmentation compared to Pd1222; colonies formed on solid minimal media were off-white rather than brownish pink. In contrast, DP301 and DP308 were capable of growth on methanol and of anaerobic growth with nitrate as terminal electron acceptor, and were Nadi-positive. Mutant DP308 (hisH::
) was prototrophic, indicating either that the P. denitrificans synthesizes two functional imidazole acetol phosphate transaminase enzymes, or that the gene identified here as hisH by homology does not encode a functional enzyme and that the true hisH gene lies elswhere in the genome, or that there is a HisH-independent route for the transamination of imidazole acetol phosphate to histidinol phosphate in P. denitrificans.
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c-type cytochrome biogenesis in the interposon mutants
Total soluble and membrane fractions of DP301DP309 grown with choline, a gratuitous inducer of c-type cytochromes associated with methylotrophic growth (Harms & van Spanning, 1991 ), were analysed by spectrophotometry. Membranes isolated from DP302DP307 and DP309 contained b-type cytochromes at levels similar to those observed in membranes prepared from choline-grown Pd1222 but were devoid of c-type cytochromes (Fig. 3a
). Cytochromes c were also absent from total soluble fractions from DP302DP307 and DP309 (Fig. 3b
). In contrast, total soluble and membrane fractions from DP301 and DP308 contained wild-type levels of c-type cytochromes. These results were confirmed by SDS-PAGE and haem staining (data not shown) and by measurements of whole-cell TMPD oxidation (Table 3
). While DP301 and DP308 oxidized TMPD at rates comparable to Pd1222, mutants DP302DP307 and DP309 did not oxidize TMPD, reflecting the complete absence of c-type cytochromes in these mutants; both a functional cytochrome oxidase and c-type cytochrome(s) are required for TMPD oxidation (Keilin, 1966
). The presence of c-type cytochromes in DP301 indicates that the ORF117 gene product is not required for c-type cytochrome biosynthesis and suggests that DP302 is deficient in c-type cytochrome assembly because the
interposon blocks expression of one or more ccm genes. Disruption of hisH (in DP308) had no effect on c-type cytochrome biogenesis.
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Growth of the interposon mutants on rich media
We have previously reported that DP307 (ccmG::) is incapable of growth on rich media (Page & Ferguson, 1997
). This phenotype is not a consequence of c-type cytochrome deficiency; several other mutants of P. denitrificans that are also pleiotropically deficient in c-type cytochromes grow as well on rich media as the wild-type Pd1222 (Page & Ferguson, 1995
; Pearce et al., 1998
). When the
mutants were tested for their ability to adapt to growth on LB, it was observed that they fell into three groups (Fig. 4
). DP301 and DP308 grew as fast, and to a similar final OD650, as Pd1222. DP302, DP303, DP304 and DP306 also grew on LB, but with slightly reduced growth rates compared to Pd1222; DP306 also exhibited an extended lag phase. DP305 (ccmC::
), DP307 (ccmG::
) and DP309 (
ccmCDG::
) failed to grow on LB. In the light of the cytochrome aa3 biogenesis results (see above), it appears likely that the extended lag phase exhibited by DP306 (ccmD::
) when inoculated into LB was due to attenuated expression of ccmG. Similarly, the slightly reduced growth rates of DP302, DP303 and DP304 could be explained by a weak polarity of
insertions in ccmA, ccmB or the ccmAB promoter region on expression of ccmC. What is clear from the cytochrome aa3 biogenesis data, however, is that the failure of DP305 to grow in LB does not result from polarity of the
cassette in ccmC on either ccmD or ccmG. Whilst some growth of DP307 on LB was observed after extended incubation (2448 h), no growth of DP305 or of DP309 was observed even after incubations of 72 h. This suggests that the rich-media-intolerant phenotype conferred by ccmC disruption is more severe than that conferred by disruption of ccmG. We have reported previously that DP307 will grow on nutrient agar supplemented with 12 mM DTT (Page & Ferguson, 1997
); in contrast, neither DP305 nor DP309 formed colonies on nutrient agar supplemented with DTT at various concentrations between 0·05 mM and 5 mM.
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DISCUSSION |
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We have not previously reported the effects of ccmC or ccmD disruption in P. denitrificans. This series of analyses has shed little light on the role of CcmD. Although insertion in ccmD has some effect on cytochrome aa3 biogenesis, tolerance of oxidized thiol compounds and siderophore biosynthesis/secretion, it appears likely that this is due to a reduction in ccmG expression; in Pseudomonas fluorescens, disruption of the ccmD-homologous gene cytB has no effect on siderophore biosynthesis/secretion (Gaballa et al., 1996
). Previous discussions of the function of CcmC have always focused on its assumed role as a component of the proposed HelA2BC/HelA2BCD ABC-transporter (Thöny-Meyer, 1997
; Kranz et al., 1998
). The finding that ccmC disruption leads to a rich media growth defect, HEDS sensitivity and a defect in siderophore synthesis and/or secretion, whereas disruption of ccmA or ccmB does not, was therefore completely unexpected, indicating that CcmC can carry out at least one of its cellular functions in the absence of CcmA or CcmB. The possibility of a unique role for CcmC was suggested by the finding that the ccmC-homologous gene, cytA, of Pseudomonas fluorescens is required for both siderophore biosynthesis and/or secretion and for c-type cytochrome biogenesis, but the ccmD-homologous gene cytB is required only for the latter function (Gaballa et al., 1996
); however these authors were unable to investigate the effects of the disruption of ccmA- or ccmB-homologous genes in Pseudomonas fluorescens because these were not found upstream of cytA. One possible interpretation of our results is that CcmC might not after all be a component of the putative ABC-transporter, which might instead have the subunit structure CcmA2B2 (like KpsM2T2 of E. coli; Higgins, 1992
) or CcmA2B2D, whilst CcmC functions either independently or as a component of another complex. Indeed, while this manuscript was being revised a paper reporting closely related work (Schulz et al. 1999
) was published. These authors report that haem transfer to the E. coli haem chaperone CcmE, a step required for cytochrome c maturation (Schulz et al. 1998
), was blocked by disruption of ccmC but not by disruption of ccmA or ccmB, and conclude that the putative ABC-transporter comprises only CcmAB. However, the evidence that in R. capsulatus HelA can be co-immunoprecipitated with HelC and that HelC is required for stability of HelA (Goldman et al., 1997
) clearly indicates that that these proteins interact in vivo. Furthermore, this is consistent with a theoretical study showing that both CcmB/HelB and CcmC/HelC contain a conserved sequence motif characteristic of the membrane-integral components of bacterial ABC-transporters and proposed to mediate their interaction with the more hydrophilic ATPase component, in this case CcmA/HelA (Page et al., 1997
). These results suggest that CcmC is perhaps more likely to be bifunctional, acting both as a component of the postulated CcmA2BC/CcmA2BCD-transporter and in another role, either while remaining associated with CcmA and CcmB (±CcmD), or independently, or as a component of another complex.
This uncertainty is reflected in the physical organization of the P. denitrificans ccmABCDG gene region. While ccmA and ccmB appear to be translationally coupled via a small open reading frame, ORF5, ccmC is separated from ccmB by a 60 bp untranslated region and appears instead to be translationally coupled to ccmD and ccmG (Page et al., 1997 ). A similar organization of ccmA-, ccmB- and ccmC-homologous genes is found in other organisms, for example R. capsulatus, Bradyrhizobium japonicum and other
-proteobacteria (Thöny-Meyer, 1997
), whilst in at least one
-proteobacterium, Pseudomonas fluorescens ATCC17400 (Gaballa et al., 1996
), a number of bacteria from other divisions, and mitochondrial and chloroplast genomes (Kranz et al., 1998
), genes homologous to ccmC are found without adjacent ccmA- or ccmB-homologous genes.
The putative CcmA2BC/CcmA2BCD-transporter has been postulated to translocate haem (Thöny-Meyer, 1997 ; Kranz et al., 1998
). We have argued on the basis of sequence analysis that this is unlikely to be the case (Page et al., 1997
), and the observation that mutants of E. coli carrying in-frame deletions in ccmA and ccmB can assemble the periplasmic b-type cytochrome b562 (Throne-Holst et al., 1997
) would appear to support this argument. However, the results of Schulz et al. (1999)
suggest that the CcmC proteins (and the related proteins CcmF and CcsA; Xie & Merchant, 1998
) may indeed bind haem. In the light of these new data, the involvement of P. denitrificans CcmC and the CcmC-homologous CytA of Pseudomonas fluorescens ATCC 17400 (Gaballa et al., 1996
) in both cytochrome c assembly and the biosynthesis and/or secretion of siderophores suggests that the CcmC homologues, at least, may have a general affinity for iron chelates. In the case of the Pseudomonas fluorescens CytA, these two activities have been resolved; some site-directed mutants of CytA function in c-type cytochrome assembly but cannot promote the biosynthesis and/or secretion of siderophores, whilst others promote the biosynthesis/secretion of siderophores but cannot function in c-type cytochrome biosynthesis (Gaballa et al., 1998
). The challenge now is to integrate these various observations into a scheme for CcmC function.
Intriguingly, the phenotypes resulting from disruption of ccmC (DP305) and ccmG (DP307) are rather similar. We have previously suggested, based partly on the sensitivity of DP307 to oxidized thiol compounds (HEDS, oxidized DTT and one or more components of rich media), that CcmG is a protein-disulphide reductase interacting with a range of periplasmic proteins in vivo (Page & Ferguson, 1997 ). It is presumed that exposure to oxidized thiol compounds causes the formation of aberrant protein disulphide bonds within or between proteins and that when CcmG is present it confers a degree of protection by reducing these bonds when they occur in or between periplasmic proteins; DTT can substitute for CcmG. Our observation that disruption of ccmC also results in sensitivity to growth inhibition by HEDS and failure to grow on rich media suggests that CcmC may contribute in some way to disulphide bond reduction by CcmG, but how is not obvious. CcmC cannot simply be acting to stabilize CcmG, because ccmC disruption does not lead to the loss of cytochrome aa3 as is observed on disruption of ccmG, and because the rich media growth defect of DP305 (ccmC::
) cannot be overcome by supplementation with DTT. A role for CcmC in the provision of reducing power to CcmG can be ruled out for the same reasons. One hypothesis we are currently considering is that CcmC may bind and (perhaps partially or locally) unfold certain target proteins such that buried disulphide bonds are made accessible for reduction by CcmG or by added DTT; in the absence of CcmC these bonds would remain buried and thus inaccessible to reduction by either CcmG or DTT. Whether this is the case, and if so whether this group of target proteins includes the apocytochromes c, remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 1 April 1999;
revised 6 July 1999;
accepted 22 July 1999.