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Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, Emil-Figge-Strasse 66, D-44221 Dortmund, Germany
Correspondence
Cornelius G. Friedrich
cornelius.friedrich{at}udo.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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S is unable to express the soxSR genes, of which soxR encodes a transcriptional regulator. Strain GBS cultivated mixotrophically showed about 22 % of the specific thiosulfate-dependent O2 uptake rate of the wild-type, and when cultivated heterotrophically it produced 35 % activity. However, under both mixotrophic and heterotrophic conditions, strain GBS formed Sox proteins essential for sulfur oxidation in vitro at the same high level as the wild-type produced them during mixotrophic growth. Genetic complementation of strain GBS with soxS restored the activity upon mixotrophic and heterotrophic growth. Chemical complementation by reductants such as L-cysteine, DTT and tris(2-carboxyethyl)phosphine also restored the activity of strain GBS in the presence of chloramphenicol, which is an inhibitor of de novo protein synthesis. The data demonstrate that SoxS plays a key role in activation of the Sox enzyme system, and this suggests that SoxS is part of a novel type of redox control in P. pantotrophus.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Robertson & Kuenen, 1983). The gene region of P. pantotrophus encoding chemotrophic sulfur oxidation comprises 15 genes, namely soxSR and soxVWXYZABCDEFGH. Seven genes encode the proteins SoxYZ, SoxB, SoxCD and SoxXA. Together, these proteins catalyse hydrogen sulfide-, sulfur-, thiosulfate- and sulfite-dependent cytochrome c reduction in vitro (Friedrich et al., 2001, 2005; Rother et al., 2001).
The soxSR genes are transcribed divergently to the other sox genes of the cluster. soxR encodes the repressor SoxR, which is a member of the ArsR family that binds to two intergenic regions (soxS–soxV and soxW–soxX) of the sox locus (Rother et al., 2005), and soxS encodes the periplasmic thioredoxin SoxS (Rother et al., 2001).
The soxS gene is disrupted by the -kanamycin-resistance-encoding interposon Tn5 in the homogenote mutant P. pantotrophus GBS, and this strain is also unable to express soxR. Strain GBS has about 22 % of the specific thiosulfate-dependent O2 uptake rate of the wild-type level under mixotrophic growth conditions with succinate plus thiosulfate. Complementation of strain GBS with soxR restores repression of the specific thiosulfate-oxidizing activity under heterotrophic conditions, suggesting that SoxR is a repressor protein (Rother et al., 2005). The position of the soxS gene upstream of soxR, and the low thiosulfate-oxidizing activity of GBS, seemed to suggest a function of the putative thioredoxin SoxS in the regulation of expression of chemotrophic sulfur oxidation.
In this study, we report the isolation of the periplasmic thioredoxin SoxS, and that it is encoded by the soxS gene. We demonstrate that a mutant unable to express soxS forms high levels of Sox proteins that are not active, and that SoxS is involved in activation of the thiosulfate-oxidizing enzyme system, and we exclude the involvement of SoxS in the expression of the sox genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The DNA sequence of the sox gene region is accessible under GenBank/EMBL accession number X79242.
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) at 37 °C. P. pantotrophus strains were cultivated under mixotrophic growth conditions in mineral medium, initial pH 7.2, with 20 mM thiosulfate plus 20 mM disodium succinate (Bardischewsky & Friedrich, 2001), unless otherwise stated. Heterotrophic growth was performed in mineral medium at an initial pH of 6.8, with 20 mM succinate at 30 °C. L-Cysteine, DTT and tris(2-carboxyethyl)phosphine (TCEP) were added as specified in the text. Antibiotics were added where appropriate (for E. coli: 100 µg ampicillin ml–1, 50 µg chloramphenicol ml–1, and 50 µg kanamycin ml–1; for P. pantotrophus: 300 µg kanamycin ml–1, 10 µg chloramphenicol ml–1, and 40 µg rifampicin ml–1).
DNA techniques.
Standard DNA techniques were applied (Sambrook et al., 1989). Plasmids were prepared in small scale from E. coli, as described by Kieser (1984). For DNA sequencing, plasmid DNA was prepared with the Qiagen Plasmid Midi Kit (Qiagen). Starting from the known sequence, 2674 bp of pOG7 were further sequenced downstream of soxR. DNA sequencing was done by Invitec. Restriction enzymes, T4 DNA ligase, alkaline phosphatase and Klenow polymerase were obtained from Fermentas, Promega or Roche, and used as recommended by the manufacturer. Agarose gel electrophoresis was done in TAE (Sambrook et al., 1989). DNA fragments were eluted from agarose gels using the QIAquick gel extraction kit (Qiagen).
Construction of pRIsoxR, pRIsoxS and pRIsoxSR.
The soxS gene was disrupted by inserting an -kanamycin interposon by gene replacement, yielding the homogenote mutant P. pantotrophus GBS, as described by Rother et al. (2005). Strain GBS was complemented with soxR by plasmid pRIsoxR (Rother et al., 2005). Plasmid pRIsoxS was constructed for complementation of strain GBS with soxS. Plasmid pOG7 (Table 1
) was cut with BamHI and KpnI, and the generated 608 bp fragment containing soxS was cloned into pRI1, resulting in the plasmid pRIsoxS. For complementation with SoxR and SoxS, pOG7 was cut with KpnI and SalI. The generated 3192 bp fragment containing soxSR was cloned into pRI1, resulting in the plasmid pRIsoxSR. E. coli strains were transformed as described by Chung et al. (1989). E. coli S17-1 was used to mobilize the plasmids pRIsoxR, pRIsoxS and pRIsoxSR into P. pantotrophus GB17 and GBS (Simon et al., 1983).
Preparation of cell fractions.
Periplasmic proteins were extracted from the cells by osmotic shock according to the QIAexpressionist (2nd edn, Protocol 4; Qiagen). Tris/HCl was added to the extract to give a final concentration of 25 mM.
Cell-free extracts were prepared by passing cell suspensions (0.1 g dry cell weight ml–1) twice through a French pressure cell at 150 MPa, and subjecting the extracts to differential centrifugation. The 200 000 g supernatant was used for ammonium sulfate fractionation. Proteins precipitating between 44 and 65 % ammonium sulfate saturation were dialysed against 25 mM sodium/potassium phosphate buffer, pH 6.5, and designated as the A65 fraction (Friedrich et al., 2000).
Purification of the thioredoxin SoxS.
SoxS was identified by antibodies raised against the immunogenic oligopeptides OP-S with the amino acid sequences LQMRDPLPPGLELAR and DVESGRLEGYPGED, as deduced from the soxS nucleotide sequence. SoxS was purified from the A65 fraction of cells, which were cultivated mixotrophically with succinate plus thiosulfate, using similar methods to those described for the Sox proteins that constitute the Sox enzyme system (Rother et al., 2001). The protein was subjected to chromatography on Q-Sepharose Fast Flow, and eluted in the 0.10 M sodium chloride fraction of the respective step gradient. The SoxS-containing fractions were identified by Western blot analysis (Towbin et al., 1979), and then combined, concentrated by ultrafiltration and subjected to gel-filtration chromatography on Sephacryl S200 HR. For further purification, SoxS-containing fractions were subjected to chromatography on Phenyl Sopharose High Performance, which is a hydrophobic interaction matrix. This step yielded a partially purified preparation of SoxS that was analysed by immunoblotting.
Analytical procedures.
Denatured proteins were separated by SDS-PAGE according to Laemmli (1970). Proteins were stained with Coomassie blue, as described by Weber et al. (1972). Protein from cell-free extracts was determined by the method of Bradford (1976).
Immunoblots (Western blots) (Towbin et al., 1979) were performed according to the ‘semi-dry’ procedure, using the multiphor electrophoresis system (Pharmacia) and antibodies raised in rabbits, at the facilities of Eurogentec. SoxYZ antigens were detected with polyclonal antibodies raised against homogeneous SoxYZ. SoxCD antigens were detected with polyclonal antibodies raised against homogeneous SoxCD. Antibodies raised against the cytochrome complex SoxXA of P. pantotrophus were used to detect SoxXA. SoxB antigens were detected with antibodies against SoxB antigens expressed in E. coli (Friedrich et al., 2000).
The N-terminal amino acid sequence was determined from purified SoxS, which was separated by SDS-PAGE, and transferred to ProBlot membranes. The major protein band of 11 kDa was cut out of the membrane, and subjected to automated Edman degradation using the protein sequencer system model 494A/190A (Applied Biosystems), as detailed previously (Fischer et al., 1996). The signal peptide cleavage site was predicted by the PSORT program package (Nakai & Kaneshisa, 1991).
The PHYLIP package (Felsenstein, 1989) was used to calculate protein distance matrices, and to construct phylogenetic trees. For settings, standard options were used. Trees were drawn using TREEVIEW, version 1.6.6. (Page, 1996).
Enzyme assays.
The thiosulfate oxidation rate of whole cells was determined with an oxygen electrode (Rank Brothers). The assay (3 ml) contained 50 µl cell suspension (OD436 30, equivalent to 150 µg protein) and 100 µmol sodium/potassium phosphate buffer (pH 8.0). Reactions were started with 30 µl 0.20 M sodium thiosulfate. The specific thiosulfate-dependent O2 uptake rate was measured as µmol O2 consumed min–1 (mg protein)–1.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). As expected, no SoxS antigens were detected from the A65 fraction of extracts of the homogenate mutant strain GBS (Fig. 1b) harbouring a soxS : : -disruption (Rother et al., 2005). To complement strain GBS with soxS or soxSR, the two plasmids pRIsoxS and pRIsoxSR (Table 1) were constructed, and conjugated into strain GBS. In both strains GBS(pRIsoxS) and GBS(pRIsoxSR), SoxS antigens were present after mixotrophic growth (Fig. 1b). In periplasmic extracts, the signal of SoxS antigens was stronger, as observed from the A65 fraction of extracts of the wild-type GB17 that indicated the selectivity of the procedure. The faint signal of SoxS in the A65 fraction of strain GB17 indicated the low concentration of SoxS in vivo, and the stronger signals observed from the respective extracts of the complemented strains of GBS showed the higher rate of expression of the soxS gene from the plasmids pRIsoxS and pRIsoxSR (Fig. 1b).
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).
Sequence analysis of SoxS
The low molecular mass (11 077 Da) and the CXXC motif were diagnostic for thioredoxins. Paracoccus denitrificans 1222 is the closest relative of P. pantotrophus GB17 (Rainey et al., 1999), which was previously classified as P. denitrificans GB17 (Ludwig et al., 1993). From the genome sequence of Paracoccus denitrificans 1222 (accession no. AAIT00000000), eight proteins with characteristics of thioredoxins were identified. Of these, six were predicted to be located in the periplasm, and two in the cytoplasm (Fig. 2), all being rather diverse, and suggesting different functions and partner proteins. The amino acid sequences of thioredoxins predicted from the genomes of other bacteria were aligned together with those for which redox function has been shown. The alignment revealed that SoxS-homologous thioredoxins clustered in a distinct group differentiated from the other thioredoxins (data not shown). The BLAST search demonstrated that proteins highly homologous to SoxS were exclusively present in sulfur-oxidizing chemotrophic and phototrophic Alphaproteobacteria, which harbour sox gene clusters (data not shown). These data revealed that thioredoxins of the SoxS-type were distinct from other periplasmic thioredoxins, and from those located in the cytoplasm or attached to the cytoplasmic membrane. This analysis suggests a specific function for SoxS thioredoxins in chemotrophic sulfur metabolism.
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). To analyse whether SoxS was involved in the regulation of the transcription of the sox genes, immunoblot analysis was performed to detect the Sox-relevant proteins in cell-free extracts of strains GB17, GBS, GBS(pRIsoxR), GBS(pRIsoxS) and GBS(pRIsoxSR). The proteins SoxXA, SoxYZ, SoxB and SoxCD were present at high concentrations in cell-free extracts of the wild-type GB17 grown mixotrophically with thiosulfate, while only traces of the proteins were detected in wild-type cells cultivated heterotrophically (Fig. 3). Mixotrophically, as well as heterotrophically, cultivated cells of strain GBS and GBS(pRIsoxS) formed the Sox proteins at the same level as the wild-type when it was cultivated under mixotrophic conditions. However, expression of soxR in trans by plasmids pRIsoxR and pRIsoxSR restored the wild-type phenotype in the complemented strains of GBS (Fig. 3). Therefore SoxR, but not SoxS, was involved in repression of the transcription of the sox genes under heterotrophic growth conditions.
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S was about 22 % of that of the wild-type. Moreover, trans-complementation of strain GBS with soxR had no effect on the activity of whole cells (Rother et al., 2005).
To determine if SoxS affected the activity of the Sox enzyme system in vivo, the specific thiosulfate-oxidizing activity was determined from mixotrophically cultivated cells of the wild-type GB17, and strains GBS, GBS(pRIsoxR), GBS(pRIsoxS) and GBS(pRIsoxSR).
As expected, under mixotrophic growth conditions, the wild-type formed a high specific thiosulfate-oxidizing activity of 0.57 µmol O2 min–1 (mg protein)–1. Strain GBS exhibited a low specific thiosulfate-oxidizing activity of 0.13 µmol O2 min–1 (mg protein)–1 (Table 2), while the Sox proteins were present at the same concentration as in the wild-type (Fig. 3).
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S with the soxS gene resulted in a full restoration of the specific thiosulfate-oxidizing activity, and complementation to soxSR increased the specific thiosulfate-oxidizing activity of whole cells to about 48 %, as compared with the wild-type. However, expression of soxR alone had no effect on the specific thiosulfate-dependent O2 uptake rate (data not shown). This result strongly suggests that SoxS plays a key role in activation of one or more proteins of the Sox enzyme system of P. pantotrophus.
Reductants activate the Sox enzyme system in vivo
The membrane protein SoxV is essential for the function of the Sox enzyme system in P. pantotrophus and the phototroph Rhodovulum sulfidophilum (Bardischewsky et al., 2006; Appia-Ayme & Berks, 2002). However, the function of SoxV can be chemically compensated by addition of the reductant DTT, resulting in the transient restoration of the specific thiosulfate-oxidizing activity under aerobic conditions (Bardischewsky et al., 2006). SoxV of P. pantotrophus is a paralogue of the DsbD/CcdA family, which also transports electrons from the cytoplasm to the periplasm. However, DsbD/CcdA proteins exclusively function in reduction of apocytochromes to enable addition of the haem moiety. Also, the phenotypes of some dsbAB- and ccdA/dsbD-defective strains can be restored by addition of thiol compounds to the medium (Bardwell et al., 1993; Sambongi & Ferguson, 1994). Therefore, the effect of reductants on the specific thiosulfate-oxidizing activity of strain GBS when cultivated mixotrophically with succinate plus thiosulfate was examined.
Addition of 1 mM L-cysteine to the medium led to a 1.6-fold increase in specific thiosulfate-oxidizing activity of strain GBS, but no increase was seen for the wild-type. The activity increase depended on the concentration of L-cysteine, and 5 mM L-cysteine increased the activity threefold (Table 2). The increase occurred within 30 min, and was maintained at the high level for at least 3 h (data not shown). Also, addition of DTT (1 mM) and the non-sulfur reductant TCEP (1 mM) increased the specific thiosulfate-oxidizing activity of whole cells. Inclusion of chloramphenicol, an inhibitor of translation, or rifampicin, an inhibitor of transcription, prior to the addition of DTT, did not affect the increase in activity (Table 2). From these studies, and the rapid effect of the various reductants, it was concluded that one or more Sox proteins present in strain GBS were activated by reduction, while de novo protein synthesis was not involved in the observed increase in specific thiosulfate-oxidizing activity.
The Sox enzyme system is active in extracts of strain GBS
Immunoblot analysis of strain GBS revealed a high concentration of the Sox proteins (Fig. 3). However, strain GBS exhibited a low in vivo specific thiosulfate-dependent O2-uptake rate (Table 2). For high activity, strain GBS required either genetic complementation to produce SoxS, or chemical complementation by reductants (Table 2). The in vitro specific thiosulfate-dependent cytochrome c reduction rate, as examined from the ammonium sulfate fraction of the cell-free extract of strain GBS, was 2.2 mU (mg protein)–1, and thus similar to that of the wild-type [1.9 mU (mg protein)–1]. From these data, SoxS appears to be required for the activation of one or more proteins of the specific thiosulfate-oxidizing enzyme system in vivo. The data do not suggest that SoxS is involved in the oxidation mechanism of thiosulfate, since the reconstituted Sox enzyme system functioned well without SoxS. This was evident from the in vitro studies of strain GBS specified above, and from biochemical studies (Rother et al., 2001). The membrane-bound protein SoxV is specifically required for sulfur oxidation in vivo, while the periplasmic thioredoxin SoxW is not. Therefore, one partner of SoxS might be SoxV, while the other partner is very likely to be at least one of the Sox proteins that constitute the Sox enzyme system. Future studies using site-directed mutagenesis will aim to identify the redox partners of SoxS.
Edited by: H. L. Drake
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bardischewsky, F. & Friedrich, C. G. (2001). The shxVW locus is essential for oxidation of inorganic sulfur and molecular hydrogen by Paracoccus pantotrophus GB17: a novel function in lithotrophy. FEMS Microbiol Lett 202, 215–220.[CrossRef][Medline]
Bardischewsky, F., Fischer, J., Höller, B. & Friedrich, C. G. (2006). SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation. Microbiology 152, 465–472.
Bardwell, J. C., Lee, J.-O., Jander, G., Martin, N., Belin, D. & Beckwith, J. (1993). A pathway for disulphide bond formation in vivo. Proc Natl Acad Sci U S A 90, 1038–1042.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]
Chung, C. T., Niemela, S. L. & Miller, R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86, 2172–2175.
Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Fischer, J., Quentmeier, A., Kostka, S., Kraft, R. & Friedrich, C. G. (1996). Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans. Arch Microbiol 165, 289–296.[CrossRef][Medline]
Friedrich, C. G., Quentmeier, A., Bardischewsky, F., Rother, D., Kraft, R., Kostka, S. & Prinz, H. (2000). Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17. J Bacteriol 182, 4677–4687.
Friedrich, C. G., Rother, D., Bardischewsky, F., Quentmeier, A. & Fischer, J. (2001). Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl Environ Microbiol 67, 2873–2882.
Friedrich, C. G., Bardischewsky, F., Rother, D., Quentmeier, A. & Fischer, J. (2005). Prokaryotic sulfur oxidation. Curr Opin Microbiol 8, 253–259.[CrossRef][Medline]
Kieser, T. (1984). Factors affecting the isolation of ccc DNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 19–36.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Ludwig, W., Mittenhuber, G. & Friedrich, C. G. (1993). Transfer of Thiosphaera pantotropha to Paracoccus denitrificans. Int J Syst Bacteriol 43, 363–367.[CrossRef][Medline]
Nakai, K. & Kaneshisa, M. (1991). Expert system for predicting protein localization sites in Gram-negative bacteria. Proteins 11, 95–110.[CrossRef][Medline]
Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Applic Biosci 12, 357–358.
Pfitzner, U., Odenwald, A., Ostermann, T., Weingard, L., Ludwig, B. & Richter, O. M. (1998). Cytochrome c oxidase (heme aa3) from Paracoccus denitrificans: analysis of mutations in putative proton channels of subunit I. Bioenerg Biomembr 30, 89–97.[CrossRef][Medline]
Rainey, F. A., Kelly, D. P., Stackebrandt, E., Burghardt, J., Hiraishi, A., Katayama, Y. & Wood, A. P. (1999). A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb. nov. Int J Syst Bacteriol 49, 645–651.[CrossRef][Medline]
Robertson, L. A. & Kuenen, J. G. (1983). Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultative autotrophic sulphur bacterium. J Gen Microbiol 129, 2847–2855.
Rother, D., Henrich, H.-J., Quentmeier, A., Bardischewsky, F. & Friedrich, C. G. (2001). Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur oxidizing system in Paracoccus pantotrophus GB17. J Bacteriol 183, 4499–4508.
Rother, D., Orawski, G., Bardischewsky, F. & Friedrich, C. G. (2005). SoxRS-mediated regulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus. Microbiology 151, 1707–1716.
Sambongi, Y. & Ferguson, S. J. (1994). Specific thiol compounds complement deficiency in c-type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane bound disulphide isomerase-like protein. FEBS Lett 353, 235–238.[CrossRef][Medline]
Sambrook, J., Maniatis, T. & Fritsch, E. F. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 784–790.
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 6762–6766.
Weber, K., Pringle, J. R. & Osborn, M. (1972). Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol 26, 3–27.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]
Received 8 November 2006;
revised 18 December 2007;
accepted 22 December 2006.
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