Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
Correspondence
Robert K. Poole
r.poole{at}sheffield.ac.uk
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ABSTRACT |
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Present address: Equipe AVENIR-INSERM d'Immunite Anti-Microbienne des Muqueuses, E0364, Institut de Biologie de Lille, 1 rue du Professeur Calmette BP 447 59021 Lille, France.
Present address: Department of Microbiology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
Present address: Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3ND, UK.
||Present address: Department of Clinical Neurosciences, Royal Free Hospital, Rowland Hill Street, London NW3 2QG, UK.
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INTRODUCTION |
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In E. coli, two other genes, cydD and cydC, constituting an operon unlinked to cydAB, are necessary for cytochrome bd assembly (Georgiou et al., 1987; Poole et al., 1989
, 1993
; Bebbington & Williams, 1993
; Delaney et al., 1993
). These genes encode the strikingly similar, but distinct, components of an ATP-binding cassette (ABC)-type transporter (Poole et al., 1993
), thought to be the first found in bacteria composed of only two non-identical subunits. ABC transporters are central to many physiological processes, from bacteria to man, being responsible for solute uptake and for secretion of toxins, drugs and substrates that range from small ions to large proteins (for a review, see Schmitt & Tampe, 2002
). The genome of E. coli encodes some 80 ABC transporters (Linton & Higgins, 1998
), many of which, like CydDC, are poorly characterized. It was hypothesized that the substrate (allocrite, i.e. the translocated but not transformed molecule; Holland & Blight, 1999
) of CydDC might be haem (Poole et al., 1993
, 1994
). However, the assembly of haem into heterologous apoproteins (e.g. Ascaris haemoglobin) exported to the periplasm of E. coli does not require cydC (Goldman et al., 1996a
) and, critically, transport studies using inside-out membrane vesicles revealed no discernible differences between wild-type and cydD mutant strains (Cook & Poole, 2000
). Intriguingly though, overexpression of CydDC results in synthesis of a novel haem pigment in anaerobically grown cells (Cook et al., 2002
), suggesting a link with haem metabolism.
An important clue to the function of CydDC was the finding (Goldman et al., 1996a) that the periplasm of a cydC mutant is more oxidizing than that of a wild-type strain. This, and the observation that Cyd defects can be corrected by extracellular provision of reductants (Goldman et al., 1996b
; Pittman et al., 2002
), suggests that CydDC exports a reducing molecule to the periplasm for redox maintenance. Recently, we have demonstrated, using everted membrane vesicles, that CydDC exports cysteine in an ATP-dependent, uncoupler-independent manner (Pittman et al., 2002
). The sensitivity of a cydD mutant to benzylpenicillin and dithiothreitol, and the loss of motility, were all reversed by exogenous cysteine. The amino acid also increases cytochrome c levels in the periplasm of a cydD mutant, but does not restore cytochrome d. Consistent with CydDC being a cysteine exporter, a cydD mutant is hypersensitive to high cysteine concentrations in the medium and accumulates higher cytoplasmic cysteine levels. CydDC overexpression confers resistance to high extracellular cysteine concentrations.
The structure of this novel cysteine transporter is unknown. In an effort to understand its organization within the cytoplasmic membrane, we have determined the topography of each of the subunits. We report here agreement between topological models for these proteins, derived largely from hydrophobicity considerations, with experimental results from protein fusion studies. We also report two mutations in the ATP-binding domain found in the cydD1 chromosomal allele, the first to be isolated (Poole et al., 1989). Site-directed mutagenesis reveals other essential residues in CydD including, in a cytoplasmic loop (CL), a duplicated positively charged heptameric domain in which the positive charge has a key functional role.
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METHODS |
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Construction of fusion proteins.
Insertions of TnphoA/in and TnlacZ/in were made as previously described by Manoil & Bailey (1997). Strain CC118 (Manoil, 1990
), harbouring plasmid pRP33, was grown for 23 h (OD600=1·0) and aliquots (0·2 ml) were infected with
TnphoA/in or
TnlacZ/in at a m.o.i. of 0·10·3. The mixture was incubated at 37 °C for 10 min without aeration followed by the addition of 0·8 ml LB medium (Miller, 1972
). Cells were aerobically grown at 37 °C for 12 h followed by plating of dilutions on LB supplemented with tetracycline (10 µg ml1) and kanamycin (50 µg ml1). The resulting colonies were then replica-plated onto LB agar containing the chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (X-P; 40 µg ml1) or 5-bromo-4-chloro-3-indolyl
-D-galactoside (X-G; 40 µg ml1), tetracycline, and an increased concentration of kanamycin (300 µg ml1) to select for transposition of TnphoA/in or TnlacZ/in into plasmids. Plasmid DNA was isolated by alkaline-SDS extraction of pooled lysogenic colonies that had grown after 12 days of incubation at 37 °C. The pooled plasmid DNA was used to transform strain CC118 and selection was on LB agar containing tetracycline, kanamycin (300 µg ml1) and X-P or X-G. Individual blue colonies were purified and plasmids were isolated from these clones. Transpositions were mapped with restriction digests and were localized precisely by DNA sequencing using the primers TnphoA-I or TnlacZ-II (Table 1
). To construct specific cydDphoA fusions, the phoA gene was amplified by PCR using
TnphoA/in as template (GenBank accession no. U25548) and primers RP253 and RP254, both including an AgeI site, or primers RP161 and RP162, both including an MfeI site (Table 1
). Plasmid pRKP1602 was cut by AgeI restriction enzyme and ligated to the PCR-generated phoA gene to construct a protein fusion at residue G284. The 1·06 kb MfeI fragment of plasmids pRKP1602 and derivatives (R210G/R216G, R238G/R244G, R210K/R216K and R238H/R244H) was exchanged with a 1·38 kb PCR-generated MfeI fragment containing the phoA gene. The cydDphoA constructs have an in-frame fusion after the I261 residue of CydD and the mature alkaline phosphatase (AP). The lacZ gene was amplified by PCR using pRS414 as template. Specific cydDlacZ gene fusions were constructed in pRKP1602 as follows. The lacZ gene amplified with primers RP249 and RP250 (with NsiI sites) or RP251 and RP252 (with Bst98I sites; Table 1
), was inserted either in NsiI or Bst98I unique sites of the plasmid to construct protein fusions at residues H134 and S202, respectively. The orientations of the fusions were determined by restriction analysis. Sequences and reading frames were checked by DNA sequencing, using primers TnphoA-I (Manoil & Bailey, 1997
) or RP140 (Table 1
, for cydDlacZ gene fusions). The constructs were transformed into E. coli CC118 and transformants were screened on nutrient agar containing ampicillin (100 µg ml1) and X-P or X-G as before.
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Analysis of the cydD1 allele.
The cydD1 mutation was found by screening survivors of nitrosoguanidine mutagenesis for loss of spectroscopically detectable cytochrome d (Poole et al., 1989). The mutation in strain AN801 was transduced to other strains as described by Poole et al. (1989)
. A mutant allele in strain AN2343 (cydD1) was obtained and amplified from chromosomal DNA by PCR using primers flanking the entire gene sequence. The double-stranded product was used as template for another series of reactions using each primer separately to generate single-stranded DNA. Single-stranded template was purified by agarose gel electrophoresis, phenol/chloroform and Centricon 100 (Amicon) microconcentration, prior to sequencing using the ABI protocol for PCR products.
Construction of mutant CydD proteins.
Site-directed mutagenesis of the cydD gene was performed using the QuickChange method (Stratagene) or the M13-based Sculptor (Amersham) method. Table 1 shows the details for QuickChange mutagenesis of pRKP1604, namely the residue substitutions, mutagenic oligonucleotide sequences, changed nucleotides, altered codons, restriction sequences and newly introduced or ablated restriction sites. pRKP1604 derivatives carrying the mutated sequences were amplified in E. coli XL-1 Blue. Mutations from these plasmids were transferred to pRKP1602 for complementation analysis by replacing restriction fragments containing the altered sequences.
For the M13-based Eckstein method (Olsen et al., 1993), a 1·8 kb DdeI fragment was excised from pRP39 and cloned into the SmaI site of pBluescript SK. Single-stranded DNA was prepared by superinfection with helper phage M13K07 (Vieira & Messing, 1987
) and annealed with the mutagenic primers shown and described in Table 1
. A mutant heteroduplex was generated using Klenow polymerase and T4 DNA ligase and the non-mutant strand removed by virtue of incorporation of a thionucleotide in the mutant strand during in vitro synthesis. Homoduplex mutant plasmids were reconstructed in vitro according to the Amersham protocol and used to transform E. coli TG1. Mutated plasmids were detected by endonuclease restriction analysis or sequencing and all changes were confirmed by sequencing. Complementation of AN2343 (cydD) was performed using the constructs in pBluescript, or after cloning the mutated cydD gene into pBR328 by replacement of an EcoRVBamHI fragment.
Immunoblot analysis.
Detection of the CydD protein in cell membrane fractions was performed by immunoblot analysis with an antibody raised to a peptide comprising amino acids Q302 to V318 in the cytoplasmic carboxy-terminus (Cook et al., 2002). Microaerobic cultures were grown with shaking in Erlenmeyer flasks containing 40 % of their volume of Luria medium supplemented with D-glucose (0·5 %) and ampicillin (100 µg ml1) with shaking. Membrane fractions prepared from shear-disrupted cells (Poole & Haddock, 1974
) were washed and resuspended in buffer (50 mM Tris/HCl pH 7·4, 2 mM MgCl2, 1 mM EGTA). Protein was estimated by the method of Markwell et al. (1978)
using bovine serum albumen as standard. After SDS-PAGE, gels were electroblotted onto Hybond-C nitrocellulose (Amersham Biosciences). Blots were developed by using anti-CydD serum (diluted 1 : 200), monoclonal anti-rabbit IgG peroxidase conjugate (Sigma, diluted 1 : 2000) and the ECL Western blotting detection system (Amersham Biosciences).
Assay of the Cyd phenotype.
Strains were challenged on zinc-azide medium (ZnSO4-NaN3, 0·15 mM each) supplemented with Casamino acids (ZABC plates; Poole et al., 1989) and on nutrient agar containing 0·1 mM of the metal-chelating agent ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) (Cook et al., 1998
).
Spectrophotometric analysis and protein assays for determining the cytochrome bd levels of the mutants were performed on cells from microaerobic cultures, grown as described above. Cells were washed and resuspended in Tris/HCl buffer (pH 7·5). A few grains of sodium dithionite were added to reduce the sample, and a few grains of ammonium persulfate and potassium ferricyanide were used with shaking to oxidize the sample. The reduced sample was bubbled with a steady stream of CO gas for 2 min. The reduced minus oxidized and CO-reduced minus reduced spectra were recorded on a Johnson Research Foundation SDB4 dual-wavelength scanning spectrophotometer (Kalnenieks et al., 1998).
The H2O2 sensitivity of strains was examined on solid and in liquid media. Bacterial growth inhibition was measured in a disk diffusion assay as follows. A sample (500 µl) of liquid culture was plated in top agar on NA plates and a sterile mini-disc containing 20 µl of a 3 % (w/v) H2O2 solution was placed on top. Plates were incubated overnight at 37 °C and the diameter of growth inhibition was measured. Second, the viability method of Gort et al. (1999) was used. LB cultures with ampicillin (100 µg ml1) were inoculated at a starting OD600
0·02. After cultures reached mid-exponential phase (OD600
0·6), aliquots were removed and diluted 1 : 10 000 in PBS, pH 7·4, before adding 2 mM H2O2. Surviving cells were enumerated by plating in top agar on NA plates with appropriate antibiotic, after overnight incubation at 37 °C.
All complementation tests were done by transforming strain AN2343 (cydD1 recA+) with plasmids bearing wild-type or mutant copies of cydDC. We have not formally ruled out the possibility of intragenic complementation, specifically the energization by ATPase activity in one polypeptide, of solute transport via the transmembrane segments of another polypeptide. However, we are not aware of such activity and, in the case of CydDC, mutations in one ABC region are sufficient to inactivate all cysteine transport by the heterodimer (Pittman et al., 2002).
In silico analysis of CydDC sequences.
Sequence searches were performed with the BLAST 2.0 and WU-BLAST 2.0 programs accessible at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), and the Genome Sequencing Center (http://genome.wustl.edu/blast/client.pl), respectively. Searches were also made using the Bordetella BLAST Server from the Sanger Centre (http://www.sanger.ac.uk/Projects/B_pertussis/blast_server.shtml). Protein sequences were obtained from GenBank, the Comprehensive Microbial Resource of the Institute for Genomic Research (http://www.tigr.org), the Colibri and SubtiList databases (http://genolist.pasteur.fr/Colibri/ and http://genolist.pasteur.fr/SubtiList/, respectively). The program MEME was employed to detect conserved (repeated) motifs in protein sequences.
To predict the topography of CydD and CydC, TMHMM 2.0 (Sonnhammer et al., 1998; Krogh et al., 2001
) was used. Multiple sequence alignments and additional analyses of transmembrane segments in the CydDC sequences were performed using the program CLUSTALW and both the von Heijne Helix (transmembrane) and KyteDoolittle hydrophilicity algorithms in MacVector 7.0 software. Gap creation and extension penalties used were 3·0 and 1·0, respectively. In certain regions, the alignments had to be adjusted manually.
The sequence of CydDC was compared with the sequences of all known protein structures (the pdb sequence database at http://www.ncbi.nlm.nih.gov) using BLAST (Altschul et al., 1997). Very significant similarities were found to the MsbA proteins from Vibrio cholerae and from E. coli. The former was the more significant and includes the whole sequence of CydDC except for the short N-terminal helix and the first transmembrane helix (25 % sequence identity over amino acids 66577), whilst the latter (26 % identity, but only over residues 154580) omits the first three transmembrane sequences. Threading using 3D-PSSM (Kelley et al., 2000
) also yields the V. cholerae MsbA as having the most significant similarity over the whole sequence range of CydD. The structure of V. cholerae MsbA (Chang, 2003
) therefore provides a good structural model for CydDC. The two point mutations were modelled and their immediate environments in the structure examined using TurboFrodo (Roussel & Cambillau, 1991
).
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RESULTS AND DISCUSSION |
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However, experimental evidence in support of topological models has been lacking. To assess which of these regions actually span the membrane, fusions between random amino acid residues in CydD and CydC with both AP and BG proteins were generated using TnphoA/in and TnlacZ/in transposition into pRP33, resulting in plasmids bearing in-frame cydD, cydCphoA and cydD, cydClacZ fusions. A total of 26 fusions were well distributed, covering both protein sequences (Fig. 2a, b; Table 2
). To obtain each hydrophobic segment flanked by either phoA or lacZ fusions, we also constructed fusion proteins with specific residues in CydD (i.e. H134, S202 and G284) as described in Methods.
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Problems can arise from the use of these fusions through instability of the fusion proteins. For example, when a LacZ fusion protein is cleaved during synthesis, it is released into the cytoplasm giving a false positive. However, PhoA fusion proteins must be translocated to the periplasm to be measurable, so that stability is of less concern. The results from the LacZ and PhoA fusions are complementary and internally consistent and have been used to construct the transmembrane topological models of CydD and CydC shown in Figs 2(a) and (b), respectively. Both polypeptides span the membrane six times (TM IVI) and each protein has two large CLs (labelled CL III in Fig. 2a, b
) and three small PLs (PL IIII in Fig. 2
). The positions of the helices predicted from TMHMM lie within the regions defined by protein fusion activity. von Heijne transmembrane and KyteDoolittle hydrophilicity plots also reveal hydrophobic clusters in CydD and CydC sequences similar to those shown in Fig. 2
(results not shown). Although the cytoplasmic location of the ATPase domains cannot be deduced from the positive LacZ fusions obtained alone (see above), there is ample evidence for a cytoplasmic location for the nucleotide-binding domain in related proteins (e.g. MsbA; Chang & Roth, 2001
) and this location is consistent with the exclusively intracellular availability of ATP. Our experimental findings are also consistent with the topography of other prokaryotic and eukaryotic ABC transporters. For example, the Drosophila melanogaster pigment precursor transporter is also a heterodimer, in which both the White and Brown subunits possess six transmembrane domains with both N and C termini in the cytoplasm (Ewart et al., 1994
). Also, in the crystal structure of E. coli MsbA the lipid A flippase the transmembrane region is composed of six tilted
-helices per monomer, with contacts between helices from different monomers (Chang & Roth, 2001
).
Mutations in the cydD1 allele and the nucleotide-binding domain
The first mutant allele of cydD was isolated after random mutagenesis and screening spectroscopically for loss of the distinctive absorbance properties of cytochrome bd (Poole et al., 1989). Despite use of this strain over many years, during which time the stability of this allele has been noted, the mutated residue(s) have not been determined. Here we report two point mutations, G319D and G429E in the cydD1 mutant allele. Both are in the C-terminal, cytoplasmic portion of the CydD protein, flanking the Walker A-motif (Fig. 2a
). To test the importance of each mutation in conferring a Cyd phenotype, we individually engineered these changes into the cydD gene cloned into pBluescript and tested the ability of the mutant constructs to complement the cydD1 allele in strain AN2343. The G319D mutation alone was sufficient to eliminate complementation as judged by the absence of spectroscopically detectable cytochrome bd. The G429E mutation in pBluescript reduced cytochrome d content to less than one-third the level of that afforded by the wild-type cydD+ gene cloned into either pBluescript or pBR328. Thus, either mutation affects cytochrome bd assembly, perhaps explaining the failure to find spontaneous revertants at this locus. Fig. 3
shows a model of the CydD protein based on the solved structure of MsbA in Vibrio cholera (Chang, 2003
). The cytoplasmic domain comprises the N terminus, two large CLs, and the C terminus bearing the ATP-binding cassette, including Walker motifs A and B, as demonstrated in the fusion analysis. The upper half of the structure in Fig. 3
comprises the six membrane-spanning helices and small PLs, again as demonstrated in this work. G429 (in E. coli; Ser423 in Vibrio) is close to a conserved Asp residue. Substitution with Glu, a large negatively charged residue, next to a conserved Asp (510 in E. coli; 505 in Vibrio) may be responsible for disruption of structure near the Walker B region. G319 is buried in a pocket and may make van der Waals contact with Leu104 at the extreme end of a transmembrane helix, although the reason for the Cyd phenotype is unclear at this time. Mutation of G319 might slow down conformational changes that are required during the catalytic cycle. This region of the ABC domain may serve an intramolecular signalling function coupling the hydrolysis of ATP with the membrane-spanning components (Ames & Lecar, 1992
). Glycines are expected to effect transmission of conformational changes within proteins, since they lack a lateral chain and appear crucial in dynamic functionality. Glycine residues have been shown to interact with the phosphoryl groups of ATP. A G313A mutation within the G box of HPK was observed to diminish greatly the formation of a covalent adduct with ATP upon UV irradiation (Ninfa et al., 1993
).
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Sequence-informed targeting of residues for mutagenesis in the CydD protein
A search for similarities between two transmembrane regions of both CydDC proteins (TM III and TM IV) with other proteins in GenBank has shown numerous hits with mitochondrial b-type cytochrome sequences from both invertebrates and vertebrates (Cook et al., 2002). Moreover, TM IV of CydD has a pair of glycines (G171 and G184; Fig. 2a
) separated by 12 residues, which, with a similar pair in TM I of CydC (G21 and G34; Fig. 2b
) resemble the quartet of conserved glycines in cytochromes b involved in configuration of the haem pocket (Esposti et al., 1993
). Inspection of putative CydD and CydC sequences in other bacteria is confounded by a lack of information on features that distinguish CydDC transporters (i.e. that export Cys and GSH) from other members of the ABC transporter family. However, CLUSTALW alignments show that the Salmonella typhimurium LT2 CydD and CydC proteins exhibit precisely the same quartet of glycines as in E. coli, and similar groupings of glycines occur in the presumed CydD and CydC proteins of other Gram-negative bacteria. Thus, in both Vibrio vulnificus YJ016 (accession NP_934247) and Photobacterium profundum (accession CAG19569) a pair of glycines is found in CydD, separated by 17 residues, the second of the pair aligning with E. coli G184. In Brucella melitensis 16M (accession NP_459933), the alignment and spacing are as in E. coli and Salmonella. In all these bacteria, glycine pairs also occur in CydC, the first of each pair aligning with E. coli G21: in V. vulnificus, there are 12 residues between the glycines, whereas in Photobacterium profundum and Brucella melitensis, 10 residues separate the glycines. Interestingly, no such glycine pairs occur in the putative CydD and CydC proteins of the Gram-positive bacteria Mycobacterium tuberculosis, Bacillus cereus or Listeria monocytogenes. Another glycine pair (G274 and G284) is seen in CydD of E. coli, Salmonella and Photorhabdus luminescens (accession NP_928889). In this preliminary examination of the possible roles of these paired glycines, we targeted glycine residues G171, G274 and G284 in CydD TM IV, PL III and TM VI, respectively (Fig. 2a
), for mutagenesis.
Statistical analysis of transmembrane segments of ion channels (Brandl & Deber, 1986) reveals a frequent distribution of prolines. Furthermore, in light of the recent demonstration that CydDC transports cysteine (Pittman et al., 2002
), it is notable that most prolines in the TMs of prokaryote amino acid transporters are highly conserved (Pi et al., 1998
). Therefore, we decided to change the proline residues in CydD TM III (P142 and P151, Fig. 2a
) for leucines whose relatively large size may change the local structure. These two prolines are strongly conserved in bacterial CydD-like proteins (data not shown).
When CL II of the CydD protein was analysed using the MEME program, we noticed two positively charged heptameric motifs separated by 21 residues. These are shown in Fig. 4; the first starts at residue 210 in E. coli CydD and the second at residue 238. Each of these is well conserved in CydD proteins of other species. In the first heptamer, the second position is G in 17 of the 19 cases examined, whereas the second position in the second heptamer is T in 17 of 19 cases. We therefore describe the consensus' at this position as G/T. The third residue in the first heptamer is usually L (12L/19) and the third residue in the second heptamer is usually M (17M/19). We therefore describe the consensus' at this position as L/M. Overall, we suggest that the consensus is R-G/T-L/M-X-T/V-L-R, where / separates the most probable occupants of certain positions (Fig. 4
). The R210, R216 and R244 residues are frequently preceded by another positively charged residue, i.e. lysine. Thus, we decided to introduce the R210G/R216G and R238G/R244G changes into the CydD protein to annul the positive charges of these regions. Fig. 3
shows the heptameric repeat region. Residues 207246 are disordered in the structures of both the V. cholerae and the E. coli MsbA; the approximate location of this region is indicated by a blue ellipse. Residues 238246, which are observed, are coloured in blue.
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To assess the functional importance of the duplicated heptameric motif in CydD CL II, we screened arginine-to-glycine substitutions. pRKP1602 (carrying wild-type cydD+) complemented strain AN2343 (cydD1) as shown by the high level of cytochrome d, lack of inhibition by EDDHA, zinc or azide, and small zones of inhibition around disks impregnated with H2O2 (Fig. 5). However, plasmids encoding the R210G/R216G and R238G/R244G mutant forms of CydD exhibited significantly lower levels of cytochrome d than the wild-type (2·3- and 1·4-fold less, respectively; Fig. 5
) and slower growth when challenged with the above agents.
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Arg to Gly mutations are overcome by replacement of positive charges
We constructed plasmids including the R210K/R216K and R238H/R244H changes in CydD, and introduced them into the cydD1 background. These substitutions prevented the growth inhibition in challenging conditions, and the cytochrome d levels were increased close to those from the wild-type strain (Fig. 5). Thus, regardless of the different residue side-chains, lysine and histidine are functional substitutions for arginines in the duplicated heptameric motif of CydD CL II, and are able to restore the wild-type phenotype by the sole supply of a positive charge. The function of this region is unclear but it may be involved in cysteine binding.
Evidence from sequence analysis for conservation, but shuffling, of cydDC genes
In E. coli, the genes encoding the ATP transporter are denoted cydDC because the second gene of the operon was discovered first (Georgiou et al., 1987; Poole et al., 1989
). Gene nomenclature in Bacillus subtilis follows an alphabetical order and, in contrast with E. coli, the genes are transcribed as a cydABCD polycistronic message (Winstedt et al., 1998
). The E. coli nomenclature has been used in mycobacteria as well as in Gram-negative bacteria, whereas the Bacillus subtilis nomenclature has been reserved for Gram-positive bacteria. A further 17 and 18 complete orthologue sequences for cydD and cydC, respectively, were analysed. Interestingly, the cyd locus of Streptomyces coelicolor A3(2) encodes three proteins, namely CydA, CydB and a protein (GI:3928723; 1172 amino acids) that has two ABC transporter family signatures, one at the middle (ABC1) and the second at its C terminus (ABC2). This apparent fusion protein (CydDC) resembles, therefore, some eukaryotic ABC transport systems where a tandemly duplicated molecule yields a single protein with two ABC domains. In this case, we considered separately the N-terminal ABC1 (575 residues, here named CydD) and the C-terminal ABC2 (remaining 597 residues, named CydC here) segments, and used these truncated proteins as supplementary sequences for CydD and CydC analyses. An alignment of the CydDC sequences was performed. Alignment and analysis of these sequences using the von Heijne Helix (transmembrane) and KyteDoolittle hydrophilicity algorithms revealed similar topological features, namely six TMs, two major CLs and three minor PLs with both N and C termini located in the cytoplasm. Almost all the CLs from CydDC sequences showed a net positive charge. We also investigated the organization in bacterial genomes of the cyd genes and found four different types of genetic organization. The cydAB and cydDC genes can constitute (i) independent clusters, (ii) neighbouring divergent clusters (
cydCD cydAB
), or show a tandem cydABDC arrangement encoding (iii) three or (iv) four proteins. In the first case, we traced the presence of the trxB gene that is upstream of the E. coli cydD gene. It appears that across short phylogenetic distances this gene organization is conserved but vanishes in organisms such as Streptomyces violacea and Gram-positive bacteria. A similar analysis of the aat gene downstream of E. coli cydC in these organisms showed no conservation. In this context, it is interesting to mention that the Aeromonas jandaei locus (GI:1903398) displays the trxB and aat genes separated not by the cydDC cluster but by a totally unrelated gene encoding a response regulator protein, RrpX. A search of the upstream genes of the cydAB clusters from a few genomes showed weak conservation of gene ordering. Intriguingly, Lactococcus lactis exhibits the tra983B gene encoding the transposase of IS983B. Immediately upstream of the cydAB operon of Brucella melitensis biovar Abortus, two genes encoding ABC-type transporters of 331 and 322 residues were found (Endley et al., 2001
). Tentatively, these genes were named cydC and cydD by the authors although no functional evidence was provided.
Conservation of the cydD and cydC genes in an operon possibly facilitates interactions of the encoded proteins (Dandekar et al., 1998), may favour lateral gene transfer (Lawrence & Roth, 1996
) or allow co-localization of the mRNAs in the same region of the cell (Danchin et al., 2000
). The conservation of gene order is obvious between closely related species, but rapidly becomes less conspicuous among more distantly related organisms (Tamames, 2001
). Nevertheless, cydDC gene ordering in Staphylococcus aureus, Bacillus halodurans or Streptococcus pyogenes resembles more the E. coli organization (except for the upstream trxB gene) than the corresponding one found in Gram-positive bacteria. On the basis of the presence of a small number of insertion sequences among 11 complete genome sequences, Bacillus subtilis was found to have the most stable genome retaining the ancestral genome structure (Itoh et al., 1999
). Gene translocation events may be responsible for loss of gene order in some bacteria, as Itoh et al. (1999)
have proposed for the elimination of operons.
Conclusions
We propose a heterodimeric structure for the ABC-type transporter CydDC that is absolutely required for cytochrome bd assembly in E. coli. The two constituent polypeptides are strikingly similar and each is shown here to comprise six N-terminal transmembrane segments and a C-terminal, cytoplasmic ATP-binding cassette. The function of this transporter appears to be the export of reductant, specifically cysteine (Pittman et al., 2002), to the periplasm. Although cydD- and cydC-like genes have been reported in most bacteria that synthesize cytochrome bd, it is possible that the heterodimeric structure proposed may not in all cases have the same transport function. Thus, in D. melanogaster, the White and Scarlet proteins that constitute a heterodimeric ABC transporter form a tryptophan transporter, whereas the White and Brown proteins form a guanine transporter (Tearle et al., 1989
; Mackenzie et al., 1999
). Different pairings of CydD- and/or CydC-like proteins might export diverse substrate molecules.
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ACKNOWLEDGEMENTS |
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Received 25 March 2004;
revised 6 July 2004;
accepted 7 July 2004.
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