Universität Osnabrück, FB Biologie/Chemie, Barbarastraße 11, D-49069 Osnabrück, Germany
Correspondence
Darío Ortiz de Orué Lucana
ortiz{at}biologie.uni-osnabrueck.de
Hildgund Schrempf
schrempf{at}biologie.uni-osnabrueck.de
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ABSTRACT |
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INTRODUCTION |
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Haem is the prosthetic group of numerous proteins involved in a wide variety of biological processes, including oxygen carriers, redox enzymes and regulatory proteins. Haem-containing enzymes are abundant in many micro-organisms and include cytochrome c oxidase, catalases, different types of peroxidases and catalase-peroxidases (Woloszczuk et al., 1980; Zou et al., 1999
). During early stages of growth, the Gram-positive bacterium Streptomyces reticuli was found to produce a mycelia-associated, haem-containing enzyme (CpeB), which exhibits a catalase-peroxidase activity with broad substrate specificity and manganese-peroxidase activity (Zou & Schrempf, 2000
). The cpeB gene and the regulator gene furS form an operon which is transcribed under the control of a promoter(s) located upstream of the furS gene. Thus FurS also acts as autoregulator in a redox-dependent fashion (Ortiz de Orué Lucana & Schrempf, 2000
). The thiol form of FurS contains one zinc ion per monomer and binds in this state to its cognate operator upstream of the furS gene. Oxidation of -SH groups within FurS induces Zn2+ release (Ortiz de Orué Lucana et al., 2003
).
CpeB can use H2O2 to oxidize a number of substrates in dependence on an attached haem group (ferric-protophorpyrin) or in a haem-independent reaction which is coupled to Mn(II)/(III) peroxidation (Zou & Schrempf, 2000). The additional haem-dependent catalase activity of the enzyme leads to a disproportionation of H2O2 to O2. Thus the mycelia-associated enzyme also plays an important part in detoxifying H2O2 and in minimizing reactions caused by highly reactive oxygen species arising from the interaction of H2O2 with certain divalent metal ions (Fe2+ and others). Therefore S. reticuli is well equipped to minimize the Fenton reaction, the reaction products of which are hazardous to every organism (Fridovich, 1986
).
In this report we describe the identification of a gene whose product has been characterized as a novel haem-binding protein from Streptomyces (HbpS). Using biochemical studies, we showed that the secretion of HbpS depends on the presence of twin arginine residues within its signal peptide. Chromosomal mutants carrying a disruption of the hbpS gene were designed and analysed to gain insights as to the physiological role of HbpS.
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METHODS |
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Chemicals and enzymes.
Chemicals for SDS-gel electrophoresis were obtained from Serva. Molecular mass markers, nitrophenyl, o-dianisidine, 4-chloro-1-naphthol, haemin and 3,3',5,5'-tetramethylbenzidine were supplied by Sigma. Hydrogen peroxide (30 %, w/v) was bought from Merck.
Test for peroxidase activity.
CpeB was released from the mycelium using acetate buffer (pH 5·5) containing 0·1 % Triton X-100 (Zou et al., 1999), and aliquots (30 µl) were tested for activity. Samples were loaded onto a native 10 % polyacrylamide gel. After the run, the gel was washed twice with acetate buffer (20 mM, pH 5·5) and activity staining was carried out with 4-chloro-1-naphthol and 5 mM H2O2 (Conyers & Kidwell, 1991
).
Cleavage of DNA, ligation and agarose gel electrophoresis.
DNA was cleaved with various restriction enzymes according to the suppliers' instructions. Ligation (Sambrook et al., 1989) was performed with T4 ligase (New England Biolabs). Gel electrophoresis was carried out in 0·82 % agarose gels using TBE buffer.
Transformation and isolation of plasmids.
E. coli was transformed with plasmid DNA by electroporation (Dower et al., 1988). Plasmids were isolated from E. coli with the aid of a mini plasmid kit (Qiagen).
Hybridization experiments.
The transfer of DNA fragments of the restricted genomic S. reticuli DNA onto nylon membranes was performed as described by Sambrook et al. (1989). The hybridization probes were labelled using Klenow enzyme and digoxigenin-11-dUTP (Roche). Hybridization and immunological detection were carried out according to standard procedures (Sambrook et al., 1989
).
RNA isolation.
To obtain well-grown mycelia, S. reticuli (wild-type and hbpS mutant) spores [20 µl in 80 %, (v/v) glycerol] were inoculated in 10 ml complete medium (Schlochtermeier et al., 1992b) and grown as a standing culture at 30 °C for 16 h. The culture was diluted (1 : 10) in the same medium, and cultivation was continued on a rotary shaker at 30 °C for 12 h. For further scaling up, the culture was diluted to 1 l, and cultivation was continued (16 h). The cultures were washed twice in minimal medium (MM) without supplement, and kept shaking after resuspension in the same medium. The mycelia were suspended in 1 l MM, divided into 100 ml portions and supplemented with Avicel (1 % final concentration). Samples (100 ml) were taken for enzyme activity tests, immunoblotting and RNA isolation. Genomic RNA was isolated as previously described (Ortiz de Orué Lucana & Schrempf, 2000
).
Analysis of transcripts.
Total RNA was electrophoretically separated on a 2 % agarose gel containing 2 % formamide and 1x MOPS buffer and was transferred over 3 h to a positively charged nylon membrane under vacuum using 20x SSC. RNA size marker I (0·36·9 kb; Roche) was used for size determination. The membrane was dried at 80 °C for 30 min and subsequently exposed to UV radiation for 3 min to fix the RNA. Hybridization was performed in a solution containing 5x SSC, 0·1 % SDS, 100 µg salmon sperm DNA ml1 and 5x Denhardt's reagent (Sambrook et al., 1989) at 64 °C for 2 h. The 32P-labelled probe was added and hybridization was continued for another 20 h.
The cpeB probe was radioactively labelled with the Rediprime DNA labelling system (Amersham Biosciences) using Klenow polymerase and [32P]dCTP. Firstly, the DNA region of cpeB was amplified by PCR. The following primers were used: CD, 5'-GAGTTCCGCACAGTTCGGAAGG-3', and CE, 5'-CGTTGGCACCGCCGCGCTTGTCGC-3'.
The membrane was washed twice with 2x SSC containing 0·1 % SDS at room temperature for 20 min and subsequently with 0·1x SSC supplemented with 0·1 % SDS at 64 °C for 45 min, and subjected to autoradiography at 70 °C.
Cloning of the hbpS gene in E. coli.
The hbpS-encoding region of the previously described construct pWKS10 (Zou et al., 1999; Table 1
), harbouring S. reticuli furS, cpeB and the newly identified hbpS genes, was amplified by PCR using the following primers: HBP1, 5'-CCTGAGCATGCCCAGCCGCAAGAAGCCGTCCC-3' consisting of an SphI restriction site, followed by the sequence encoding N-terminal amino acids of HbpS; and HBP2, 5'-GCCGGATCCTGGCCGAGCACGGCCGCGCC-3', determining the C-terminal amino acids of HbpS, followed by a BamHI restriction site. The PCR product was digested with SphI and BamHI. After digestion with BamHI the DNA was treated with the Klenow polymerase to get a blunt-ended fragment which was ligated with SphI/SmaI-digested pQE32 (Table 1
). The resulting plasmid pQH1 (Table 1
) was transformed into E. coli M15(pREP4). The plasmid was sequenced and the correctness of the designed hbpS gene and its in-frame fusion with the His-tag codons were confirmed.
Cloning of a hbpS fusion gene in S. lividans.
The downstream region of the hbpS gene was amplified using the oligonucleotides HBP3, 5'-GTCGGTGGCCGTCGTCGACCGCAACGGCAACACCCTGGTCACCCTG-3', annealing upstream of the BsiWI restriction site in the middle of hbpS, and HBP4, 5'-CTCGGCCCGGGCGGGTCAGTGGTGGTGGTGGTGGTGGCCGAGCACGG-3', containing an SrfI restriction site, a stop codon and six histidine codons. Following the PCRs, the generated product was cleaved with BsiWI and SrfI and ligated with the large BsiWISrfI fragment of the plasmid pUKS10 (Zou et al., 1999; Table 1
). The 4·6 kb EcoRIHindIII fragment of the resulting plasmid pUKS15 (Table 1
) was ligated with EcoRI/HindIII-digested pWHM3 vector. The resulting plasmid pWKS15 (Table 1
) carried a hbpS gene with six histidine codons in front of its stop codon. The correctness of the in-frame insertion was confirmed by restriction and sequencing. The plasmid pWKS15 (Table 1
) was transformed into S. lividans protoplasts, which were generated as described by Hopwood et al. (1985)
. Transformants were selected using an overlay of 0·4 % agarose containing 200 µg thiostrepton ml1 (Hopwood et al., 1985
).
Site-directed mutagenesis of hbpS.
The codons for arginines (R9 and R10) from the identified twin arginines within the signal sequence of HbpS (Fig. 1b) were replaced by two lysine-encoding codons. Lysines and arginines are highly basic with a long side-chain and possess similar hydrophobicity coefficients; thus, it is less likely that the substitutions induce unfavourable steric interactions in the resulting protein. For this purpose the upstream region of hbpS was amplified using the following oligonucleotides: HBP5, 5'-CTTCCTCAGCATGTCCAGCCLGCAAGAAGCCGTCCAAGAAGACCCGCGTCCTCGT-3', containing a BbvCI restriction site and two lysine codons replacing the two arginine codons of the hbpS wild-type gene, and HBP4 (see above). For the PCR and further ligation the plasmid pUKS10 (Zou et al., 1999
; Table 1
) was used. The PCR product was digested with BbvCI and BsiWI, resulting in a 0·328 kb mutagenized hbpS fragment which was ligated with the large BbvCIBsiWI fragment of plasmid pUKS10 (Table 1
). The 4·6 kb EcoRIHindIII fragment of the resulting plasmid pUKS16 (Table 1
) was ligated with the EcoRI/HindIII-digested pWHM3. The resulting plasmid pWKS16 (Table 1
) carried an hbpS gene containing mutations in the codons encoding arginines 9 and 10 within HbpS. The correctness of the in-frame replacement was checked by restriction and sequencing. The plasmid pWKS16 was transformed into S. lividans protoplasts as described above.
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Localization of HbpS in Streptomyces.
S. reticuli (wild-type), S. reticuli hbpS disruption mutant, S. lividans pWKS10 (Zou et al., 1999), S. lividans pWKS17 (derivative of pWKS10 lacking hbpS, see below and Table 1
) and S. lividans pWHM3 (control strain with vector) were grown in complete medium. The pWKS17 (Table 1
) plasmid was generated by self-ligation of the longer SrfIBbvCI (Fig. 1a
) restriction fragment of pWKS10. Thus, pWKS17 lacks the hbpS gene. The cultures were transferred to a minimal medium containing 1 % Avicel (for S. reticuli) or 0·5 % yeast extract (for S. lividans) and cultivated on a rotary shaker at 30 °C. S. reticuli was incubated for 6 h, S. lividans for 14 h. Mycelia were centrifuged (4000 g). The extracellular proteins from the culture filtrate were precipitated by adding (NH4)2SO4 (90 % saturation). The precipitated proteins were tested for immuno-reactivity with anti-HbpS antibodies.
Purification of HbpSHis-tag from Streptomyces.
S. lividans transformants carrying the plasmid pWKS15 (Table 1) or the construct pWKS17 (derivative of pWKS10 lacking hbpS) were grown as described above. After precipitation [using (NH4)2SO4, 90 % saturation] of proteins from the culture filtrate, the protein solutions were dialysed against solution A (see above). Afterwards, the HbpSHis-tag protein was purified using NiNTA-affinity chromatography as described previously (Ortiz de Orué Lucana & Schrempf, 2000
).
Detection of haem binding by HbpS.
Haem binding in HbpS was identified in SDS-polyacrylamide gels as described by Moore et al. (1978) and Smalley et al. (2001)
using tetramethylbenzidine/H2O2, which detects the presence of haem in proteins. For the preparation of gels, loading buffer and electrophoresis buffer with 0·1 % SDS was used. The HbpSHis-tag-containing sample was free from DTT and was not boiled prior to electrophoresis, which was carried out at 4 °C in the dark. Then, the gel was incubated for 30 min in 20 mM Tris/HCl (pH 7·3) buffer containing 50 % methanol to fix the protein and to lower the concentration of SDS within the gel. This was consecutively stained for 45 min using the following solutions: 0·25 mM sodium acetate, pH 5·3; 0·25 % (w/v) 3,3',5,5'-tetramethylbenzidine, 25 % (v/v) methanol, 0·75 % H2O2 [2·5 % (v/v) of a commercial 30 % solution]. H2O2 was added immediately prior to use. After staining, the gel was incubated for 10 min in 25 % (v/v) methanol to remove excess of tetramethylbenzidine. The gel was then stored in 0·1 M Tris/HCl, pH 7·3.
Generation of disruption mutant.
The plasmid pUKS10 (Zou et al., 1999) was digested with EcoRI. The linearized DNA was then partially digested with SalI to obtain a 4·6 kb DNA fragment containing part of cpeB, the hbpS gene and its downstream region. After treatment with the Klenow enzyme, the DNA was circularized by self-ligation. The resulting construct was named pUCH1 (Table 1
). Using a pBR322 derivative with a hygromycin-resistance gene containing terminator sequences (hyg) (Blondelet-Rouault et al., 1997
), a HindIII fragment (2·3 kb) containing this hyg was generated. This fragment was blunt-ended using the Klenow enzyme and ligated with the BsiWI-digested pUCH1 which was also blunt-ended. The resulting plasmid pUCH2 (Table 1
) contains hyg, which is flanked on the left by 0·87 kb and on the right by 1·15 kb of the above-described plasmid pUCH1. The ligation mixture was added to electrocompetent E. coli DH5
. Hygromycin-resistant E. coli transformants were selected, and the correctness of their plasmid constructs was analysed by restriction. One of the correct constructs was isolated. Ten micrograms thereof was denatured (0·2 M NaOH, 10 min, 37 °C), chilled on ice and neutralized by rapid addition of HCl. Then the DNA was used to transform 50 µl protoplasts (
109 ml1) generated from S. reticuli, which were spread onto osmotically stabilized medium, as described by Hopwood et al. (1985)
and incubated at 30 °C for 19 h. The plates were overlaid with 2 ml molten agarose (40 °C) containing hygromycin (1 mg ml1). Hygromycin-resistant colonies were restreaked several times, and their genomic DNA was analysed as to the size of fragments carrying the hygromycin gene.
Growth assays.
The sensitivity of S. reticuli wild-type and S. reticuli hbpS mutant to haemin was determined using a disc inhibition assay. A sample of 100 µl spores (5x108) was added to 3 ml soft agar (Sambrook et al., 1989) poured onto the respective R2 plates and allowed to solidify. Sterile 6 mm-diameter blank papers discs (Schleicher & Schuell) were added to the bacteria-overlaid plates and saturated with 20 µl of different concentrations (100, 200, 300 and 400 µM) of haemin. Plates were incubated overnight at 30 °C before zones of inhibition were measured.
SDS-PAGE and Western blotting.
SDS-PAGE was performed in the presence of 0·1 % SDS (Laemmli, 1970). Proteins were separated by 12·5 % SDS-PAGE and transferred to a fluorotrans membrane (Sambrook et al., 1989
). The membrane was blocked for 1 h at room temperature with PBS (40 mM Na2HPO4, 8 mM NaH2PO4, 150 mM NaCl, pH 7·4) containing 5 % skimmed milk powder, and subsequently incubated overnight at 4 °C with the generated anti-CpeB or anti-HbpS antibodies. After treatment with secondary anti-rabbit or anti-guinea pig antibodies, respectively, conjugated with alkaline phosphatase, the membrane was stained with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (Blake et al., 1984
).
PCR, DNA sequencing and computer analysis.
PCR was performed using Pfu DNA polymerase (Invitrogen). To test the correctness of cloned genes, sequencing was done using the Ready Reaction mix and ABI PRISM equipment (PE Biosystems) by the departmental sequence service (U. Coja, FB Biologie, University of Osnabrück). Sequence entry, primary analysis and ORF searches were performed using Clone Manager 5.0. Database searches using the PAM120 scoring matrix were carried out with BLAST algorithms (BLASTX, BLASTP and TBLASTN) on the NCBI file server (BLAST{at}ncbi.nlm.nih.gov) (Altschul et al., 1997). Multiple sequence alignments were generated by means of the CLUSTAL W (1.74) program (Higgins et al., 1992
). Putative ShineDalgarno (ribosome-binding) sites (Gold et al., 1981
; Strohl, 1992
) and signal peptide cleavage sites were predicted as described by Nielsen et al. (1997)
.
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RESULTS |
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Comparative analysis revealed that the amino acid sequence of HbpS shows the highest identity (69·9 %) to an ORF from Streptomyces coelicolor A3(2). HbpS also shares a limited number of identical amino acids with a few other deduced proteins with so far uncharacterized features, including Atu1117 from Agrobacterium tumefaciens C58 (38·3 %) (Wood et al., 2001), CpmX from Sphingomonas aromaticivorans (36·6 %) (Yrjala et al., 1997
) and NahX from Pseudomonas putida G7 (33 %) (Grimm & Harwood, 1999
) (Fig. 1b
). The genes cpmX and nahX are each located in an operon, which is responsible for degradation of chlorinated or methylated aromatic compounds via the meta-cleavage pathway. The N-terminal region of Atu1117 shows a predictable signal peptide cleavage site, however without twin arginines.
The following investigations were focused on analysing the function of the protein encoded by the hbpS gene.
Secretion of HbpS requires a twin arginine in its signal peptide
A S. lividans transformant was generated previously which has the plasmid pWKS10 (Table 1); this containing the furScpeB operon and hbpS (Zou et al., 1999
). As outlined in Methods, the hbpS fusion gene with six histidine codons was cloned and overexpressed in E. coli. The subsequently purified His-tagHbpS protein molecules consist predominantly of the monomeric form, including its signal peptide (25 kDa), and smaller quantities of the dimeric form (50 kDa) (Fig. 2
). After generation of antibodies, immunological studies revealed that the culture filtrate of S. lividans pWKS10 contained the mature monomeric (apparent molecular mass 16·5 kDa) and dimeric (apparent molecular mass 33 kDa) forms of HbpS (Fig. 3
b, lanes 3 and 6). The control strains S. lividans pWKS17 (without hbpS) and S. lividans pWHM3 (vector alone) lacked any extracellular protein immuno-reacting with anti-HbpS antibodies (Fig. 3b
, lanes 4 and 5). N-terminal sequencing of HbpS (secreted by S. lividans pWKS15) showed that the cleavage site in the signal peptide could be located between the residues Asp37 and Thr38 and not between the alanine residues (positions 35 and 36) which would have been predicted for a signal peptidase cleavage site (Nielsen et al., 1997
). Thus, it has to be assumed that the N-terminus of the mature HbpS was further modified via action of an extracellular peptidase. The predicted mature secreted protein has a molecular mass of 15·1 kDa and a pI of 5·9.
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To facilitate purification of larger amounts of HbpS for further analysis the hbpS gene was extended by six histidine codons (corresponding to the codon usage of Streptomyces). This was cloned in such a way that it replaced the original hbpS gene in pWKS10 and resulted in the plasmid pWKS15 (Table 1). The HbpS fusion protein with the His-tag predicted at the C-terminus could be concentrated by affinity chromatography from the culture filtrate of S. lividans pWKS15 (Fig. 3a
, lane 4), but not from the control strain S. lividans pWKS17 (lacking the hbpS gene) (Fig. 3a
, lane 3). The isolated protein has an apparent molecular mass of 17·2 kDa corresponding to the secreted form of the His-tag protein. Small quantities of its dimeric form (34 kDa) were also observed (Fig. 3a
). Proteins corresponding to the non-mature form (20 kDa) and to the mature form (17·2 kDa) were purified from the cytoplasm of S. lividans pWKS15 (Fig. 4
a, lane 3).
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To investigate haem-binding properties of the non-mature HbpSHis-tag protein, it was purified from the cytoplasmic fraction of S. lividans pWKS15 (Fig. 4a, lane 3). Tetramethylbenzidine/H2O2 staining showed that the non-mature (20 kDa) form of HbpS also contained haem (Fig. 4a
, lane 4).
Analysis of HbpS production in S. reticuli wild-type and hbpS disruption mutant
Using antibodies, S. reticuli was found to secrete small amounts of HbpS (Fig. 3b, lane 1). As expected, the levels were considerably lower than those from S. lividans pWKS10 carrying the hbpS gene on the multicopy plasmid. As a basis for further investigations, the hbpS gene within the S. reticuli genome was disrupted according to the strategy outlined in Methods. Southern hybridizations using hbpS- and hyg-probes (data not shown) revealed that a double crossover between the genomic S. reticuli hbpS gene and the residual hbpS portions flanking the hyg in pUCH2 (Table 1
) had occurred as desired (Fig. 1a
, hbpS mutant). Immunological studies (Western blot analysis) revealed that the culture filtrate of the chromosomal S. reticuli hbpS disruption mutant lacked HbpS (Fig. 3b
, lane 2).
Resistance to haemin correlates with HbpS production
It is well known that haemin, as a natural porphyrin, possesses significant antibacterial activity that is augmented by the presence of physiological concentrations of hydrogen peroxide or a reducing agent (Stojiljkovic et al., 2001). Increasing concentrations (100400 µM) of haemin (the Fe3+ oxidation product of haem) in the culture medium led to a higher growth-inhibition of the S. reticuli hbpS mutant strain (Table 2
) than of the S. reticuli wild-type strain. S. lividans pWKS10 also produced considerably greater amounts of HbpS in the presence of haemin (050 µg ml1) than in the absence of haemin (Fig. 4d
). The data imply that HbpS plays an important role in defence against the high toxicity of haemin, which catalyses free radical formation.
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DISCUSSION |
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Whereas some haem-binding proteins (e.g. those of the NapC/NirT cytochrome c family) have the CXXCH motif, which is necessary for attaching of haem, others lack this motif. Albumin can bind several haem groups per molecule due to the presence of a hydrophobic region (Shin et al., 1994). Similarly, the mouse p22 haem-binding protein contains a hydrophobic region, which is speculated to bind haem (Taketani et al., 1998
). The chaperone CcmE interacts with haem transiently in the periplasm of E. coli and delivers it to newly synthesized and exported c-type cytochromes. Alanine scanning mutagenesis of conserved amino acids revealed that only H130 is strictly required for haem-binding and delivery. Mutation of the hydrophobic amino acids (F37, F103, L127 and Y134) to alanine affected the interaction with haem of CcmE more than the mutation of other charged and polar amino acids. The data suggest that haem is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond (Enggist et al., 2003
). In this context, it is important to mention that HbpS is rich in hydrophobic residues and contains three histidine residues (H60, H83 and H187) surrounded by leucines and valines (Fig. 1b
). The crystal structure of yeast cytochrome c peroxidase (CCP) shows that the histidine (H) residue at position 181 interacts with haem (Finzel et al., 1984
). This residue is located within the motif LX2THLX10AA, which exhibits a similarity to the region LX3THLX10AA including the histidine residue at position 60 within HbpS (Fig. 1c
).
The S. reticuli hbpS mutant strain was found to be more sensitive than the wild-type strain to higher concentrations of haemin (Fe3+-oxidized form of haem). It is known that haemin at higher concentrations is highly toxic because of its ability to catalyse free radical formation (Baker et al., 2003). The enhanced sensitivity to haemin in the mutant strain correlated with the lack of HbpS. This result suggested that within the wild-type strain haemin is titrated by HbpS, leading to a reduction of free haemin.
Haem has been found to be necessary for the accumulation and assembly of cytochrome c oxidase in Saccharomyces cerevisiae, which is able to synthesize haem (Woloszczuk et al., 1980). In addition, it was shown that haem is important not only for the formation of active catalases but also for the synthesis, or at least accumulation, of the apoproteins of catalases (A and T) in yeast. During these processes, haem could either act as a positive regulator of the synthesis of apocatalases or it could prevent rapid degradation of the enzyme precursors (Howe & Merchant, 1994
).
Physiological studies showed that the amount of the catalase-peroxidase CpeB in the S. reticuli hbpS disruption mutant in comparison to S. reticuli wild-type was considerably lower. Within the mutant strain the amount of cpeB mRNA is reduced, suggesting that HbpS influences the expression of cpeB in a positive manner. These data lead to the following explanations: HbpS could act as chaperone that binds haem and delivers it to the mycelium-associated CpeB, or HbpS could be involved in a signal transduction cascade regulating the expression of cpeB. As HbpS is an extracellular protein, it could interact with extracellular or membrane-associated proteins involved in such signal transduction. Interestingly, downstream of hbpS two ORFs are located, which are divergently transcribed from hbpS and encode a predicted sensor kinase and a putative response regulator (data not shown). These proteins could be the components for the corresponding signal transduction system.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Baker, H. M., Anderson, B. F. & Baker, E. N. (2003). Dealing with iron: common structural principles in proteins that transport iron and heme. Proc Natl Acad Sci U S A 100, 35793583.
Bateman, A., Birney, E., Cerruti, L. & 7 other authors (2002). The Pfam protein families database. Nucleic Acids Res 30, 276280.
Berks, B. C., Sargent, F. & Palmer, T. (2000). The Tat protein export pathway. Mol Microbiol 35, 260274.[CrossRef][Medline]
Blake, M. S., Johnston, K. H., Russel-Jones, G. J. & Gotschlich, E. C. (1984). A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 136, 175179.[Medline]
Blondelet-Rouault, M. H., Weiser, J., Lebrihi, A., Branny, P. & Pernodet, J. L. (1997). Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces. Gene 190, 315317.[CrossRef][Medline]
Bogsch, E. G., Sargent, F., Stanley, N. R., Berks, B. C., Robinson, C. & Palmer, T. (1998). An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J Biol Chem 273, 1800318006.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Braun, V. & Braun, M. (2002). Iron transport and signaling in Escherichia coli. FEBS Lett 529, 7885.[CrossRef][Medline]
Brink, S., Bogsch, E. G., Edwards, W. R., Hynds, P. J. & Robinson, C. (1998). Targeting of thylakoid proteins by the pH-driven twin-arginine translocation pathway requires a specific signal in the hydrophobic domain in conjunction with the twin-arginine motif. FEBS Lett 434, 425430.[CrossRef][Medline]
Cartron, M. L., Roldan, M. D., Ferguson, S. J., Berks, B. C. & Richardson, D. J. (2002). Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases. Biochem J 368, 425432.[CrossRef][Medline]
Conyers, S. M. & Kidwell, D. A. (1991). Chromogenic substrates for horseradish peroxidase. Anal Biochem 192, 207211.[Medline]
Dower, W. J., Miller, J. F. & Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16, 61276145.[Abstract]
Enggist, E., Schneider, M. J., Schulz, H. & Thony-Meyer, L. (2003). Biochemical and mutational characterization of the heme chaperone CcmE reveals a heme binding site. J Bacteriol 185, 175183.
Finzel, B. C., Poulos, T. L. & Kraut, J. (1984). Crystal structure of yeast cytochrome c peroxidase refined at 1·7-Å resolution. J Biol Chem 259, 1302713036.
Fridovich, I. (1986). Biological effects of the superoxide radical. Arch Biochem Biophys 247, 111.[Medline]
Gold, L., Pribnow, D., Schneider, T., Shinedling, S., Singer, B. S. & Stormo, G. (1981). Translational initiation in prokaryotes. Annu Rev Microbiol 35, 365403.[CrossRef][Medline]
Grimm, A. C. & Harwood, C. S. (1999). NahY, a catabolic plasmid-encoded receptor required for chemotaxis of Pseudomonas putida to the aromatic hydrocarbon naphthalene. J Bacteriol 181, 33103316.
Higgins, D. G., Bleasby, A. J. & Fuchs, R. (1992). CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci 8, 189191.[Abstract]
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
Howe, G. & Merchant, S. (1994). Role of heme in the biosynthesis of cytochrome c6. J Biol Chem 269, 58245832.
Johansson, P. & Hederstedt, L. (1999). Organization of genes for tetrapyrrole biosynthesis in Gram-positive bacteria. Microbiology 145, 529538.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Moore, R. W., Welton, A. F. & Aust, S. D. (1978). Detection of hemoproteins in SDS-polyacrylamide gels. Methods Enzymol 52, 324331.[Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 16.[CrossRef]
Ortiz de Orué Lucana, D. & Schrempf, H. (2000). The DNA-binding characteristics of the Streptomyces reticuli regulator FurS depend on the redox state of its cysteine residues. Mol Gen Genet 264, 341353.[CrossRef][Medline]
Ortiz de Orué Lucana, D., Troller, M. & Schrempf, H. (2003). Amino acid residues involved in reversible thiol formation and zinc ion binding in the Streptomyces reticuli redox regulator FurS. Mol Genet Genomics 268, 618627.[Medline]
Panek, H. & O'Brian, M. R. (2002). A whole genome view of prokaryotic haem biosynthesis. Microbiology 148, 22732282.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sargent, F., Bogsch, E. G., Stanley, N. R., Wexler, M., Robinson, C., Berks, B. C. & Palmer, T. (1998). Overlapping functions of components of a bacterial Sec-independent protein export pathway. EMBO J 17, 36403650.
Schaerlaekens, K., Schierova, M., Lammertyn, E., Geukens, N., Anné, J. & van Mellaert, L. (2001). Twin-arginine translocation pathway in Streptomyces lividans. J Bacteriol 183, 67276732.
Schaerlaekens, K., van Mellaert, L., Lammertyn, E., Geukens, N. & Anné, J. (2004). The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis. Microbiology 150, 2131.[CrossRef][Medline]
Schlochtermeier, A., Niemeyer, F. & Schrempf, H. (1992a). Biochemical and electron microscopic studies of the Streptomyces reticuli cellulase (Avicelase) in its mycelium-associated and extracellular forms. Appl Environ Microbiol 58, 32403248.[Abstract]
Schlochtermeier, A., Walter, S., Schröder, J., Moorman, M. & Schrempf, H. (1992b). The gene encoding the cellulase (Avicelase) Cel1 from Streptomyces reticuli and analysis of protein domains. Mol Microbiol 6, 36113621.[Medline]
Shin, W. S., Yamashita, H. & Hirose, M. (1994). Multiple effects of haemin binding on protease susceptibility of bovine serum albumin and a novel isolation procedure for its large fragment. Biochem J 304, 8186.[Medline]
Smalley, J. W., Charalabous, P., Birss, A. J. & Hart, C. A. (2001). Detection of heme-binding proteins in epidemic strains of Burkholderia cepacia. Clin Diagn Lab Immunol 8, 509514.
Stojiljkovic, I., Evavold, B. D. & Kumar, V. (2001). Antimicrobial properties of porphyrins. Expert Opin Investig Drugs 10, 309320.[Medline]
Strohl, W. R. (1992). Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res 20, 961974.[Abstract]
Taketani, S., Adachi, Y., Kohno, H., Ikehara, S., Tokunaga, R. & Ishii, T. (1998). Molecular characterization of a newly identified heme-binding protein induced during differentiation of urine erythroleukemia cells. J Biol Chem 273, 3138831394.
Thony-Meyer, L. (1997). Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev 61, 337376.[Abstract]
Vara, J., Lewandowska-Skarbek, M., Wang, Y.-G., Donadio, S. & Hutchinson, C. R. (1989). Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J Bacteriol 171, 58725881.[Medline]
Villarejo, M. R., Zamenhof, P. J. & Zabin, I. (1972). -Galactosidase. In vivo
-complementation. J Biol Chem 247, 22122216.
Woloszczuk, W., Sprinson, D. B. & Ruis, H. (1980). The relation of heme to catalase apoprotein synthesis in yeast. J Biol Chem 255, 26242627.
Wood, D. W., Setubal, J. C., Kaul, R. & 48 other authors (2001). The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294, 23172323.
Yrjala, K., Paulin, L. & Romantschuk, M. (1997). Novel organization of catechol meta-pathway genes in Sphingomonas sp. HV3 pSKY4 plasmid. FEMS Microbiol Lett 154, 403408.[CrossRef][Medline]
Zou, P. & Schrempf, H. (2000). The heme-independent manganese-peroxidase activity depends on the presence of the C-terminal domain within the Streptomyces reticuli catalase-peroxidase CpeB. Eur J Biochem 267, 28402849.
Zou, P., Borovok, I., Ortiz de Orué Lucana, D., Müller, D. & Schrempf, H. (1999). The mycelium-associated Streptomyces reticuli catalase-peroxidase, its gene and regulation by FurS. Microbiology 145, 549559.[Medline]
Received 10 February 2004;
revised 22 April 2004;
accepted 20 May 2004.
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