Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni
2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês
10.1074/jbc.m710166200
ISSN1083-351X
AutoresAndré Göhler, Guangming Xiong, Simone Paulsen, Gabriele Trentmann, Edmund Maser,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3α-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3α-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity. The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3α-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3α-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity. Comamonas testosteroni (formerly termed Pseudomonas testosteroni (1Tamaoka J. Ha D.M. Komagata K. Int. J. Syst. Bacteriol. 1987; 37: 52-59Crossref Scopus (158) Google Scholar)) is a Gram-negative bacterium that is able to utilize a variety of steroids and aromatic compounds as the sole carbon and energy source (2Marcus P.I. Talalay P. J. Biol. Chem. 1956; 218: 661-674Abstract Full Text PDF PubMed Google Scholar, 3Ahmad D. Masse R. Sylvestre M. Gene (Amst.). 1990; 86: 53-61Crossref PubMed Scopus (71) Google Scholar, 4Morgan C.A. Wyndham R.C. Can. J. Microbiol. 1996; 42: 423-430Crossref PubMed Scopus (22) Google Scholar). Steroids are widespread in the environment occurring as cholesterol, sex, and adrenal cortical hormones of mammals, molting hormones in insects, or phytosterols in plants (5Kieslich K. J. Basic Microbiol. 1985; 25: 461-474Crossref PubMed Scopus (112) Google Scholar, 6Lafont R. Mathieu M. Ecotoxicology. 2007; 16: 109-130Crossref PubMed Scopus (188) Google Scholar, 7Koolman J. Prog. Clin. Biol. Res. 1990; 342: 704-709PubMed Google Scholar, 8Bishop G.J. Yokota T. Plant Cell Physiol. 2001; 42: 114-120Crossref PubMed Scopus (207) Google Scholar). Accordingly, C. testosteroni inhabits a wide variety of environments, including soil and water as well as animal and plant tissues (9Barbaro D.J. Mackowiak P.A. Barth S.S. Southern Jr., P.M. Rev. Infect. Dis. 1987; 9: 124-129Crossref PubMed Scopus (37) Google Scholar, 10Busse H.J. el Banna T. Oyaizu H. Auling G. Int. J. Syst. Bacteriol. 1992; 42: 19-26Crossref PubMed Scopus (42) Google Scholar, 11Arda B. Aydemir S. Yamazhan T. Hassan A. Tunger A. Serter D. APMIS. 2003; 111: 474-476Crossref PubMed Scopus (29) Google Scholar). The complex degradation pathway for steroids in C. testosteroni has been studied earlier by simultaneous identification of the participating genes and by isolation of the main intermediate compounds that have accumulated in gene-disrupted mutants (12Horinouchi M. Hayashi T. Yamamoto T. Kudo T. Appl. Environ. 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The steroid catabolic pathway is initiated by oxidizing the hydroxyl group at the C3 position, thereby forming a ketone group. This oxidation is followed by isomerization, dehydrogenation, and hydroxylation of the steroid, which leads to the opening of the B-ring. Aromatization and further oxidation reactions result in meta cleavage of the A-ring, finally resulting in common intermediates that enter conventional central metabolism pathways (13Horinouchi M. Hayashi T. Koshino H. Kurita T. Kudo T. Appl. Environ. Microbiol. 2005; 71: 5275-5281Crossref PubMed Scopus (41) Google Scholar). The ring cleavage procedure and the enzymes involved therein are similar to those of a common bacterial aromatic compound degradation pathway (20Skowasch D. Möbus E. Maser E. Biochem. Biophys. Res. Commun. 2002; 294: 560-566Crossref PubMed Scopus (17) Google Scholar, 21Möbus E. Jahn M. Schmid R. Jahn D. Maser E. J. Bacteriol. 1997; 179: 5951-5955Crossref PubMed Google Scholar). It has been estimated that the complete degradation of the steroid nucleus to CO2 and H2O requires more than 20 enzymatic reactions. Interestingly, the catabolic enzymes for steroid degradation are usually not constitutively expressed but, rather, are induced by their respective substrates (20Skowasch D. Möbus E. Maser E. Biochem. Biophys. Res. Commun. 2002; 294: 560-566Crossref PubMed Scopus (17) Google Scholar, 21Möbus E. Jahn M. Schmid R. Jahn D. Maser E. J. Bacteriol. 1997; 179: 5951-5955Crossref PubMed Google Scholar, 22Florin C. Köhler T. Grandguillot M. Plesiat P. J. Bacteriol. 1996; 178: 3322-3330Crossref PubMed Google Scholar, 23Möbus E. Maser E. J. Biol. Chem. 1998; 273: 30888-30896Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 24Oppermann U.C.T. Belai I. Maser E. J. Steroid Biochem. Mol. Biol. 1996; 58: 217-223Crossref PubMed Scopus (39) Google Scholar, 25Oppermann U.C.T. Maser E. Eur. J. Biochem. 1996; 241: 744-749Crossref PubMed Scopus (64) Google Scholar). However, the organization and regulation of their corresponding genes is largely unknown, although two steroid degradation gene clusters in C. testosteroni TA441 were identified (12Horinouchi M. Hayashi T. Yamamoto T. Kudo T. Appl. Environ. Microbiol. 2003; 69: 4421-4430Crossref PubMed Scopus (94) Google Scholar, 14Horinouchi M. Kurita T. Yamamoto T. Hatori E. Hayashi T. Kudo T. Biochem. Biophys. Res. Commun. 2004; 324: 597-604Crossref PubMed Scopus (59) Google Scholar, 15Horinouchi M. Hayashi T. Kudo T. J. Steroid Biochem. Mol. Biol. 2004; 92: 143-154Crossref PubMed Scopus (35) Google Scholar, 26Horinouchi M. Hayashi T. Koshino H. Yamamoto T. Kudo T. Appl. Environ. Microbiol. 2003; 69: 2139-2152Crossref PubMed Scopus (38) Google Scholar). In previous investigations we identified 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) 2The abbreviations used are: 3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase; ELISA, enzyme-linked immunosorbent assay; RepA, repressor A; RepB, repressor B; TeiR, testosterone-inducible regulator; GFP, green fluorescence protein; SIN, standard I nutrition. 2The abbreviations used are: 3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase; ELISA, enzyme-linked immunosorbent assay; RepA, repressor A; RepB, repressor B; TeiR, testosterone-inducible regulator; GFP, green fluorescence protein; SIN, standard I nutrition. as an important enzyme in steroid degradation of C. testosteroni (21Möbus E. Jahn M. Schmid R. Jahn D. Maser E. J. Bacteriol. 1997; 179: 5951-5955Crossref PubMed Google Scholar, 23Möbus E. Maser E. J. Biol. Chem. 1998; 273: 30888-30896Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 25Oppermann U.C.T. Maser E. Eur. J. Biochem. 1996; 241: 744-749Crossref PubMed Scopus (64) Google Scholar). By catalyzing the interconversion of hydroxyl and oxo groups at position 3 of the steroid ring structure, 3α-HSD/CR initiates steroid ring opening and is, therefore, of importance for complete steroid mineralization. Moreover, this enzyme is capable of catalyzing the carbonyl reduction of nonsteroidal xenobiotic carbonyl compounds (25Oppermann U.C.T. Maser E. Eur. J. Biochem. 1996; 241: 744-749Crossref PubMed Scopus (64) Google Scholar, 27Oppermann C.T. Netter K.J. Maser E. Adv. Exp. Med. Biol. 1993; 328: 379-390Crossref PubMed Scopus (29) Google Scholar). It has been demonstrated that this substrate pluripotency not only enhances the metabolic capacity of insecticide degradation but also increases the resistance of C. testosteroni towards the steroid antibiotic fusidic acid (24Oppermann U.C.T. Belai I. Maser E. J. Steroid Biochem. Mol. Biol. 1996; 58: 217-223Crossref PubMed Scopus (39) Google Scholar). Because the expression of the encoding gene, hsdA, is inducible by steroids such as testosterone and progesterone, elucidation of the hsdA-inducing mechanism may shed light on the regulation and genetics of the entire steroid degradation pathway in C. testosteroni. Recently, we identified two genes involved in hsdA regulation and reported a two repressor model to control hsdA gene expression. Repressor A (RepA) was found to bind to two operator sequences upstream of hsdA, thereby preventing hsdA transcription (28Xiong G. Maser E. J. Biol. Chem. 2001; 276: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Repressor B (RepB) turned out to bind to the mRNA of 3α-HSD/CR, thereby interfering with hsdA translation (29Xiong G. Martin H.J. Maser E. J. Biol. Chem. 2003; 278: 47400-47407Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). By transposon mutagenesis, teiR (testosterone-inducible regulator) was identified, a gene that was hypothesized to encode a positive regulator of steroid degradation in C. testosteroni ATCC11996 in the presence of testosterone (30Pruneda-Paz J.L. Linares M. Cabrera J.E. Genti-Raimondi S. J. Bacteriol. 2004; 186: 1430-1437Crossref PubMed Scopus (28) Google Scholar). A similar positive regulator (TesR) was cloned and postulated to regulate a steroid degradation gene clusters in C. testosteroni TA441 (14Horinouchi M. Kurita T. Yamamoto T. Hatori E. Hayashi T. Kudo T. Biochem. Biophys. Res. Commun. 2004; 324: 597-604Crossref PubMed Scopus (59) Google Scholar). However, the mechanism and mode of action by which these positive regulatory elements control the steroid degradation gene cluster remained obscure. On the other hand, detailed knowledge on hsdA regulation is of great importance and might give a general view on steroid dependent gene regulation in bacteria. In the present investigation we could unravel the role of TeiR in steroid-dependent gene regulation in C. testosteroni and demonstrate that TeiR is a kinase that drives steroid sensing and metabolism in C. testosteroni. We cloned the teiR gene, produced recombinant TeiR protein as well as respective antibodies, and by constructing a TeiR-GFP fusion protein, proved that TeiR is a membrane protein with almost exclusive polar localization. Studies with wild type and teiR knock-out mutants revealed that TeiR induces hsdA expression and testosterone catabolism in C. testosteroni. These knock-out studies also showed that, in addition to testosterone, a variety of other steroids can bind to TeiR. Deletion mutants of the teiR gene disclosed that the protein consists of three main domains, an N-terminal membrane attachment site, a central hydrophobic steroid binding sequence, and a C-terminal part mediating its regulatory function. More detailed analysis showed that TeiR is required for twitching and swimming of C. testosteroni in the medium to the steroid source and that the mode of intracellular signal transduction occurs via its kinase activity. Bacterial Strains and Plasmids—Host strains Escherichia coli HB101 (Promega) and C. testosteroni ATCC 11996 (Deutsche Sammlung von Mikroorganismen) were used for cloning and gene expression. Subcloning of fragments was carried out in plasmids pBBR1MCS-2 (containing the kanamycin resistance gene; a gift from Peterson and co-workers (31Kovach M.E. Elzer P.H. Hill D.S. Robertson G.T. Farris M.A. Roop II, R.M. Peterson K.M. Gene (Amst.). 1995; 166: 175-176Crossref PubMed Scopus (2650) Google Scholar)) and pUC18 (containing the ampicillin resistance gene and obtained from Invitrogen). Plasmid pK18 containing the kanamycin resistance gene was a gift from Ciba Pharmaceuticals, Inc., Department of Biotechnology (Basel, Switzerland). The plasmid copy numbers determined were 80 copies of pK18 and pUC18 and 5 copies of pBBR1MCS-2 per cell in E. coli. For overexpression and purification of TeiR, E. coli strain BL21(DE3)pLysS together with plasmid pET15b from Novagen was used. The tac promoter (274 bp) was obtained by BamHI digestion from plasmid pHA10, which was a gift from H. Arai (32Arai H. Igarashi Y. Kodama T. Agric. Biol. Chem. 1991; 55: 2431-2432Crossref PubMed Scopus (26) Google Scholar). With plasmid pcDNA3.1/CT-GFP-TOPO (Invitrogen), a TeiR-GFP fusion protein was prepared, and plasmid pCR2.1-TOPO (Invitrogen) served for PCR cloning of teiR fragments and sequencing. Growth Media and Growth Conditions—Bacterial cells were grown in a shaker (180 rpm) in Standard I Nutrient broth medium (Merck) or LB medium at 37 °C (E. coli) or 27 °C (C. testosteroni). Growth media contained 60 μg/ml ampicillin and 30 μg/ml kanamycin. Restriction Enzymes and Other Reagents—Restriction enzymes, T4 ligase, S1 nuclease, and shrimp alkaline phosphatase were obtained from Roche Applied Science, New England Biolabs, MBI, Promega, and Amersham Biosciences, and used according to the manufacturers' instructions. Sodium lauroyl sarcosinate was from Fluka. The steroid compounds were supplied by Sigma. Ampicillin and kanamycin were from Appli-Chem and Calbiochem, respectively. [γ-32P]ATP and [γ-32P]GTP was obtained from Amersham Biosciences. DNA Manipulations—Recombinant DNA work was carried out following standard techniques according to Sambrook and Russel (33Sambrook J. Russel D.W. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York2001Google Scholar). The fragments cloned in this work are shown in Fig. 1. All of the primers were prepared by MWG (Ebersberg, Germany). Before further cloning, fragments prepared by PCR were cloned into pCR2.1-TOPO and then checked for correct sequence by MWG. Transformation of Bacteria—All constructs were verified by restriction enzyme analysis of the resulting band patterns. Plasmids were purified with the Tip-100 kit from Qiagen. Ligated constructs were transferred into competent E. coli or C. testosteroni cells prepared by the calcium chloride or electroporation method. Double plasmid cotransformations were performed by exploiting the kanamycin resistance gene of pBBR1MCS-2 or pK18 and the ampicillin resistance gene of pUC18. In these cases, both antibiotics were added to the culture medium. Plasmid isolation and agarose gel electrophoresis were performed to prove successful double transformations. Cloning of the teiR Gene from C. testosteroni and Subcloning of teiR Gene Fragments—The teiR gene was cloned from C. testosteroni (ATCC 11996) chromosomal DNA by using the following pair of primers: forward primer 5′-CGAGCTCCATCGCTTGCGTG-3′ and reverse primer 5′-GCGGCCGCTCTATGCCCG-3′ (30Pruneda-Paz J.L. Linares M. Cabrera J.E. Genti-Raimondi S. J. Bacteriol. 2004; 186: 1430-1437Crossref PubMed Scopus (28) Google Scholar). The full teiR gene was then cloned into pCR2.1-TOPO to yield plasmid pTOPO4, which after sequence confirmation (MWG) was used as template for further PCR reactions (Fig. 1A). To generate pKteiR10, forward primer 5′-GGAATTCCATATGTGCCCATATTTCGAC-3′ and reverse primer 5′-GGAATTCTCGAATCACTTGTTCCCCAGC-3′ were used in a PCR reaction with plasmid pTOPO4 as template, and the resulting fragment was digested with EcoRI and introduced into pK18 downstream from the lacZ promoter. After sequence confirmation, the same fragment was digested by NdeI and XhoI and introduced into pET-15b downstream from the N-terminal His tag coding sequence to yield pET-TeiR1. To obtain the TeiR-GFP fusion plasmid pBBtac-teir-GFP1, forward primer 5′-CTAGCTAGCATGTGCCCATATTTCGAC-3′ and reverse primer 5′-TAGCTAGCCTTGTTCCCCAGCCAGGC-3′ were used in a PCR reaction with pTOPO4 as template. The resulting fragment was digested with NheI and cloned into pcDNA3.1/CT-GFP-TOPO to yield pcGFP-teiR (not shown). After digestion with XbaI, pcGFP-teiR was introduced into pBBR1MCS-2 under the control of the tac promotor. Plasmids pKteiRA, pKteiRB, and pKteiRC, which harbor different fragments of the TeiR gene, were obtained by partial digestion with PstI and identification by restriction fragment analysis. Plasmid pBBtacIII was generated by producing a PCR fragment with forward primer 5′-TCCCCCCGGGATGACCGAAGGCATG-3′ and reverse primer 5′-CCCAAGCTTTCACTTGTTCCCCA-3′ and plasmid pTOPO4 as template. The resulting fragment was digested with XmaI and HindIII and ligated into pBBR1MCS-2. Plasmid pBBtacIV was obtained by two-step PCR. In the first PCR forward primer 5′-TCCCCCCGGGATGTGCCCATATTTCGAC-3′ and reverse primer 5′-ATCTGTCGGTGAAC CGTCTGTCGATGAC-3′ were used with template plasmid pTOPO4 to generate a fragment that was used as forward primer for the second PCR. The latter was used in combination with reverse primer 5′-CCCAAGCTTTCACTTGTTCCCCA-3′ to yield a fragment that was digested with XmaI and HindIII and ligated into pBBR1MCS-2. N-terminal deleted constructs were produced by PCR, digested with XmaI and HindIII, and cloned into vectors pCR2.1-TOPO or pK18. Primers for yielding pTOPON5teiR (not shown) or pKN5teiR were forward primer 5′-TCCCCCCGGGATGCCTACCGACCGTATCG-3′ and reverse primer 5′-CCCAAGCTTTCACTTGTTCCCCA-3′, and primers for yielding pTOPON8teiR (not shown) or pKN8teiR were forward primer 5′-TCCCCCCGGGATGATGCTGCCGCACATC-3′ and reverse primer 5′-CCCAAGCTTTCACTTGTTCCCCA-3′. Plasmids pKteiR627 and pKteiR567, bearing C-terminal-deleted fragments of teiR, were constructed by standard PCR and cloned into pK18 via XmaI and HindIII restriction sites. Primers for pKteiR627 were forward primer 5′-TCCCCCCGGGATGTGCCCATATTTCGAC-3′ and reverse primer 5′-CCCAAGCTTTCAATCGAGGATGACCAC-3′, and primers for pKteiR567 were forward primer 5′-TCCCCCCGGGATGTGCCCATATTTCGAC-3′ and reverse primer 5′-CATTGCCGTTCTGAAGCTTCTGACGATCCTGG-3′. An additional fragment between teiR sequence 627 bp and 1176 bp (including a HindIII restriction site) was received by PCR (forward primer 5′-GGATCGTCAGAAGCTTCAGAACGGCAATGTG-3′; reverse primer 5′-CCCAAGCTTTCACTTGTTCCCCA-3′) and cloned into pKteiR567 to yield pKteiRdY. Generation of teiR Gene Knockout Mutants of C. testosteroni—Two different teiR knock-out mutants of C. testosteroni were prepared by homologous integration (Fig. 1B). For knock-out mutant teiRkoI, plasmid pTOPO-teiR14 (not shown) was generated by PCR using forward primer 5′-CGGAATTCAGGCCGACCGCCGCG-3′ and reverse primer 5′-CGGAATTCACGGCGACCGCTGGGG-3′, both containing an additional cytosine (underlined), against pTOPO4. The resulting fragment harboring a frameshift mutation within the teiR gene was digested with EcoRI and cloned into pK18 to yield pKPCR. For knockout mutant teiRkoII, a PstI fragment (384–750 bp) was isolated after PstI digestion of pTOPO4 and cloned into pK18 to yield pKteiR5. Because of its sensitivity to kanamycin, C. testosteroni can only grow after homologous integration of the kanamycin resistance gene from pK18, which on the other hand, cannot replicate as a plasmid in C. testosteroni. Accordingly, 10 μg of the pK18 descendent pKPCR or pKteiR5 plasmids, which also contain teiR sequences homologous to C. testosteroni chromosomal DNA, were transformed into C. testosteroni by electroporation (1.8 kV, 1-mm cuvette, Bio-Rad). The cells were cultured in 0.4 ml of SIN medium at 27 °C for 3 h (110 rpm). The culture was spread on 30 μg/ml kanamycin SIN agar plates and cultured in a 27 °C incubator overnight. Only cells with pKPCR or pKteiR5 integrated into the chromosomal DNA could grow in the kanamycin-containing medium. Total DNA from the colonies was isolated and checked for knock-out mutations by Southern blot hybridization. Subcloning of the 3α-HSD/CR Gene—As described previously, a 5.257-kilobase EcoRI fragment of C. testosteroni chromosomal DNA was cloned into pUC18 (ampicillin-resistant) to yield p6 (29Xiong G. Martin H.J. Maser E. J. Biol. Chem. 2003; 278: 47400-47407Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Plasmid p6 contains the 3α-HSD/CR gene, hsdA, together with its regulatory region and including the two repressor genes repA and repB (28Xiong G. Maser E. J. Biol. Chem. 2001; 276: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 29Xiong G. Martin H.J. Maser E. J. Biol. Chem. 2003; 278: 47400-47407Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The expression of hsdA served as a detection system for TeiR-dependent steroid regulation in cotransformation experiments with plasmid p6. Preparation and Purification of Recombinant TeiR Protein—Overexpression of TeiR was performed in E. coli strain BL21(DE3)pLysS and plasmid pET15b (Novagen), and the recombinant protein was purified by its His-tag sequence. Cells transformed with plasmid pET-TeiR1 (Fig. 1A) were grown at 37 °C in a shaker (180 rpm), and maintenance of plasmids was ensured by adding 60 μg/ml ampicillin to the culture medium. 100 μl of a culture grown overnight was used to inoculate 3 ml of fresh medium. At an A595 of 0.6, expression was induced by the addition of isopropyl-β-d-thiogalactoside to a final concentration of 1 mm. After 4 h cells were sedimented by centrifugation. The cell pellet was either stored at -80 °C for further usage or directly suspended in 200 μl of buffer B (100 mm sodium phosphate, 10 mm Tris-HCl, 8 m urea, pH 8.0) (Qiagen). Cells were lysed by freezing (-20 °C, 30 min) and thawing (room temperature, 30 min) 3 times, and the resulting mixture was centrifuged (20 min, 13,000 rpm, 4 °C). The supernatant was applied to a mini nickel-agarose affinity column (Qiagen). After washing 2 times with 600 μl of buffer C (100 mm sodium phosphate, 10 mm Tris-HCl, 8 m urea, pH 6.3) (Qiagen) and washing one time with washing buffer (50 mm sodium phosphate, 300 mm sodium chloride, 20 mm imidazole, 1% sodium lauroyl sarcosinate, pH 8.0), TeiR was eluted from the column by applying 100 μl of elution buffer 4 times (50 mm sodium phosphate, 300 mm sodium chloride, 250 mm imidazole, 1% sodium lauroyl sarcosinate, pH 8.0). Samples containing pure and soluble TeiR protein, as assessed by SDS-polyacrylamide gel electrophoresis (Fig. 2), were used to prepare antibodies, to determine steroid binding specificity, and to prove TeiR kinase activities. Protein Determination—Protein concentration was determined by the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Protein analysis by SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Western Blot Analysis—Electroblotting was performed in a semidry blotting system. After separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred to a nitrocellulose membrane, and antigen-antibody complexes were visualized by chemiluminescence (ECL PLUS-detection system, Amersham Biosciences). For GFP detection, membranes were incubated with primary antisera against GFP (raised in mice) in a dilution of 1:2000 for1 h at room temperature. The secondary antibody (peroxidase conjugated gout anti-mouse immunoglobulin) was used in a 1:10,000 dilution for 1 h at room temperature. After 4 × TBST (50 mm Tris-HCl, 150 mm NaCl, 0.1% Tween 20, pH 7.5) (Roche Diagnostics) washing, 2 ml of fluorescence reagent was spread on the membranes, covered with plastic membranes, and exposed to x-ray films. Laser-scanning Microscopy—C. testosteroni and E. coli cells from the late log-phase and expressing the GFP-TeiR fusion protein (after pBBtac-teiR-GFP1 transformation) were taken and fixed with 4% formaldehyde in phosphate-buffered saline. The samples were illuminated with an argon laser (488 nm for detection of GFP fluorescence) and recorded using a Leica TCS SP1 confocal laser-scanning microscope. Swimming and Twitching Motility Assay—Swimming and twitching activities of C. testosteroni wild type and teiR knockout mutants were assayed by the agar stab method. For swimming, C. testosteroni wild type and teiRkoI knock-out cells (5 μl) were inoculated onto 0.3% agar plates. Testosterone was stab-spotted (5 μl of 1 mm each) into the agar surface in some distance to the bacterial inoculates. For twitching, bacterial cells (5 μl) were inoculated in a line onto 0.5% agar plates. Testosterone was stab-spotted (5 μl of 1 mm each) into the agar surface in increasing distances to the bacterial inoculates. After 2 days of incubation at 27 °C, the size of the swimming and twitching zones around the bacterial inoculation site at the interface to the testosterone spots was determined. Testosterone Binding and Degradation—To test the [3H]testosterone binding activities, wild type and teiR knock-out mutant cells of C. testosteroni were grown overnight and then diluted to an A595 of 1.0 with water. An aliquot of 400 μl was taken and centrifuged at 13,000 rpm for 20 s and resuspended in 100 μl of water. For preparation of the bacterial membranes, 1 μl of lysozyme (10 mg/ml) was added and incubated at 37 °C for 30 min. The lysed cells were centrifuged at 13,000 rpm for 20 min, and the pellet containing the membranes was resuspended in 100 μl of water. In the testosterone binding assay, 10 μl of intact cells were mixed with 0.4 × TEN buffer (10 × TEN buffer: 1 m NaCl, 0.01 m EDTA, 0.1 m Tris-HCl, pH 8.0) and 0.1 μl of [3H]testosterone (1 mCi/ml) to a final volume of 20 μl. After incubation times of 5 min or 30 min at 27 °C, the incubates were transferred onto 1-cm2 pieces of Whatman No. 3MM paper. The pieces were put on Whatman No. 3MM stripes by adhesion and continuously rinsed by gravity with a solvent containing 0.2 × TEN and 0.3% Tween. After 3 h of washing, the 1-cm2 pieces were dried at 37 °C for 1 h and mixed with 3 ml of scintillation solution, and [3H]testosterone measured as cpm in a liquid scintillation counter (Wallac 1409; machine efficiency = 35%). The same procedure was performed to analyze the testosterone binding capabilities of teiR gene deletion mutants that were expressed in E. coli to identify the steroid binding domain within the TeiR protein. For measuring the testosterone uptake activities, C. testosteroni wild type cells and teiR knock-out mutants were cultured in 1 ml of SIN medium. After an A595 nm of 0.6 was reached, 1 μCi (1 μl) of [3H]testosterone was added to the medium, and the cells allowed to grow for up to 3 h. Cells were harvested, and [3H]testosterone was measured in the remaining culture medium as described above. Protein Extraction—The probes for 3α-HSD/CR ELISA detection were prepared from 3 ml of bacterial cell culture and subsequent centrifugation at 13,000 × g for 10 s. The pellet was washed 3 times with 1 ml of phosphate-buffered saline and resuspended in 200 μl of phosphate-buffered saline with 100 μg/ml lysozyme. To complete
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