Journal of Bacteriology, March 2004, p . 1430-1437, Vol . 186, No . 5

TeiR, a LuxR-Type Transcription Factor Required for Testosterone Degradation in Comamonas testosteroni

José Luis Pruneda-Paz, Mauricio Linares, Julio E . Cabrera, and Susana Genti-Raimondi^*

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, Argentina

Received 27 August 2003/ Accepted 17 November 2003

 
We have identified a new steroid-inducible gene [designatedteiR [testosterone-inducible regulator]] in Comamonas testosteroni that is required for testosterone degradation . Nucleotide sequence analysis of teiR predicts a 391-amino-acid protein which shows homology between residues 327 and 380 [C-terminal domain] tothe LuxR helix-turn-helix DNA binding domain and between residues192 and 227 to the PAS sensor domain . This domain distributionresembles that described for TraR, a specific transcriptionalregulator involved in quorum sensing in Agrobacterium tumefaciens.Analysis of the gene expression indicated that teiR is tightlycontrolled at the transcriptional level by the presence of testosteronein the culture medium . A teiR-disrupted mutant strain was completely unable to use testosterone as the sole carbon and energy source.In addition, the expression of several steroid-inducible geneswas abolished in this mutant . Northern blot assays revealedthat teiR is required for full expression of sip48-ß-HSDgene mRNA [encoding a steroid-inducible protein of 48 kDa and 3ß-17ß-hydroxysteroid dehydrogenase] andalso of other steroid degradation genes, including those encoding3-hydroxysteroid dehydrogenase, ⁵-3-ketoisomerase, 3-oxo-steroid ¹-dehydrogenase, and 3-oxo-steroid ⁴-[5]-dehydrogenase enzymes.Moreover, when teiR was provided to the teiR-disrupted strainin trans, the transcription level of these genes was restored.These results indicate that TeiR positively regulates the transcriptionof genes involved in the initial enzymatic steps of steroiddegradation in C . testosteroni.

 
Steroids, phenylalkanoic acids, resin acids, and different polycyclic aromatic hydrocarbons represent a group of molecules that are widespread in the environment as breakdown products of ligninor other plant-derived molecules [7, 22, 31] . These compounds[known collectively as endocrine disruptors] interfere withthe normal endocrine system physiology of vertebrates, particularlyin the mechanisms governing reproductive development and function[10], and constitute an important group of bioactive environmentalpollutants.

Comamonas testosteroni is a gram-negative bacterium able to use steroids [as well as many other aromatic compounds] as asole carbon source; it is an attractive model for the studyof the mechanisms involved in the mineralization of these bioactive compounds for their removal from the environment [3, 5, 6, 11, 19, 26, 30, 37, 38] . C . testosteroni metabolizes certain steroidsthrough a complex metabolic pathway involving many steps catalyzedby steroid-inducible enzymes [11, 26, 38, 42] . Interestingly,recent works revealed that testosterone simultaneously inducedboth steroid- and PAH-metabolizing enzymes in this bacterium [29, 36] . For this reason, the study of the mechanisms regulatingthe steroid-inducible gene transcription is concerned with understandingboth catabolic pathways.

While the genes encoding some of the enzymes catalyzing the oxidoreduction at different positions of the steroid nucleusand the ring opening of the steroid molecule have been identified[1, 2, 8, 9, 12, 16, 18, 20, 21, 25, 29, 32, 36, 44, 45], onlylimited information is available about the mechanisms governingsteroid-inducible gene expression . The following items havebeen reported to date . Horinouchi et al . [20] suggest that anintermediate compound produced in the course of testosteronedegradation induces expression of tesB, encoding a meta-cleavagesteroid-inducible enzyme . The induction of 3-hydroxysteroid dehydrogenase-carbonyl reductase [-HSD], in contrast, appearsto represent a derepression in which the steroidal inducer preventsthe binding of two repressor proteins [RepA and RepB] to the -HSD gene promoter and mRNA, respectively [44, 45].

For this study we report the identification and characterization of the teiR gene, which is essential for testosterone degradation in C . testosteroni . teiR encodes a LuxR-type transcription factor required for the expression of several steroid-inducible genes, suggesting that a quorum-sensing mechanism is involved in its regulation.

 
Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are listedin Table 1 . Escherichia coli was grown at 37°C in Luria-Bertani [LB] medium [34] . C . testosteroni was grown at 30°C in LBmedium or in M9 minimal medium [34] plus acetate [0.2% [wt/vol]]or testosterone [0.25 mg ml^-1] or both, as indicated in thetext . Overnight cultures were diluted 1/100 in fresh mediumand incubated for 2 h in LB medium or for 12 h in M9 medium,and cells were washed, diluted 1/50 in fresh medium, and incubatedas indicated for each experiment.

 
Growth of C . testosteroni was monitored by measuring optical density at 600 nm [OD₆₀₀] . Alternatively, growth in M9 medium plus testosterone was monitored by counting colonies that appeared on LB plates [on which appropriately diluted cultures have been spread] after incubation at 30°C . When needed, antibioticswere added at the following concentrations [in micrograms permilliliter]: ampicillin [Ap], 100; chloramphenicol [Cm], 20;gentamicin [Gm], 10; kanamycin [Km], 20; spectinomycin [Sp],600; and tetracycline [Tc], 10.

DNA manipulations and sequence determinations. Standard protocols or manufacturers' instructions were followedfor DNA isolation and recombinant DNA procedures [34] . DNA sequencing was performed on double-stranded templates derived [using the dideoxy chain termination method] [35] from pBIISKMut50 and pBB20H . For TaqDNA polymerase-initiated cycle sequencing reactions with fluorescently labeled dideoxynucleotide terminators [Applied Biosystems Inc.], standard protocols of the manufacturer were used . The sequencing reactions were analyzed using a model 377 automated DNA sequencer [Applied Biosystems Inc.] . Blast softwarewas used to screen DNA and protein databases for similar proteins[4] . Multiple sequence alignments were made with ClustalW software [version 1.7] [40].

Construction of plasmids and allele replacement. Plasmid pBIISKMut50 was constructed by the ligation of NotIDNA fragments from C . testosteroni UT2.5Mut50 into pBIISK . After transformation into E . coli DH5, Tc-resistant [Tc^r] strainswere isolated.

For the construction of pBB20H, a pVK102 C . testosteroni cosmid library was screened using a 0.5-kb PstI/NotI fragment from pBIISKMut50 as a probe [see Southern blot analysis results]and a cosmid containing a teiR gene was isolated [pVK102teiR]. Finally, a 20-kb HindIII fragment from pVK102teiR [containingteiR] was subcloned into the HindIII site of pBBR1MCS2.

Plasmid pGteiR was constructed by PCR amplification of teiR coding sequence with the primers teiR-Fw [5'-ggaagcttgctagcATGTGCCCATATTTCGACAC-3']and teiR-Rv [5'-cccggggctagcaagcttTCACTTGTTCCCCAGCCA-3'] . Theamplification product was ligated into the pGEMT easy vector.

Plasmid pBteiR-3'UR was constructed by insertion of a 2-kb SmaIfragment [obtained from pHP45::] [33] into the NdeI site locatedin the teiR 3' untranslated region of pBIISKMut50 . The recombinant plasmid was transferred [using the mobilizing plasmid pRK2013]into C . testosteroni UT2.5 by triparental mating . Donor, helper, and recipient cells were grown overnight in LB medium . Cell suspensions [0.2 ml each] were mixed, filtered on a 0.4-µm-pore-size nitrocellulose membrane filter, and incubated at 30°C onan LB agar plate for 24 h . Cells were suspended in sterile 1%[wt/vol] NaCl and plated onto LB agar containing Gm [10 µgml^-1] and Sp [600 µg ml^-1] to select transconjugants.Southern hybridization was performed to confirm the genomicstructure of the mutant strain [C . testosteroni UT2.5teiR-3'UR].

ß-Galactosidase assays. The standard procedures described by Miller [27] were used forquantitative measurements of ß-galactosidase activity.Samples were collected after 12 h [LB medium] or 17 h [M9 medium]of incubation . The values given throughout this paper representthe averages of the results of three independent experiments,each of which was conducted with duplicate samples.

Southern blot analysis. Genomic DNAs were prepared essentially as described by Sambrooket al . [34] . Southern blot analysis was performed as describedpreviously [8] . DNA fragments were transferred from agarosegels or from bacterial colonies to nylon membranes after alkalidenaturation . A 650-bp EcoRV-HindIII fragment from pSL9 [complementaryto the 3' end of the gene encoding 3ß-17ß-hydroxysteroiddehydrogenase [ß-HSD]] and a 540-bp PstI-NotI fragmentfrom pBIISKMut50 [complementary to the teiR 3' untranslatedregion] were labeled with [³²P]dATP [3,000 Ci mmol^-1] by a randompriming method [14] and used as probes as indicated in the text.

Testosterone degradation. Testosterone degradation was performed as described previously[18] . Briefly, bacterial cells [grown in LB medium plus testosteroneduring 12 h of culture] were harvested by centrifugation at4°C . Aliquots of culture supernatants were extracted threetimes with 5 vol of ethyl ether and submitted [using benzene-ethanol[95:5 {vol/vol}] as a solvent system] to thin-layer chromatographyon silica gel GF254 plates . The pattern of testosterone degradationwas visualized using 254-nm UV light . Testosterone, 4-androstene-3,17-dione,and 1,4-androstadiene-3,17-dione were used as standards.

RNA isolation and Northern blot analysis. C . testosteroni was grown in LB medium or M9 medium plus acetateduring the indicated periods of culture growth in the absenceor presence of testosterone . Total RNA was extracted as describedpreviously [8] . RNA samples [20 µg per lane] were electrophoresedon a 1.2% [wt/vol] agarose gel containing 18% [vol/vol] formaldehydeand transferred to nitrocellulose membranes [8] . Equal levelsof loading and transfer were assessed by methylene blue stainingof membranes . Prehybridization and hybridization reactions were performed as described previously [8] . A 600-bp HincII-PstI restriction fragment from pSL9 [complementary to the sip48 gene], a 650-bp EcoV-HindIII restriction fragment from pSL9 [complementaryto the ß-HSD gene], a 1,400-bp PstI restriction fragmentfrom pAK1370 [complementary to the -HSD and ⁵-KSI genes], a2,200-bp KpnI restriction fragment from pTEK21 [complementaryto the 3-oxo-steroid ¹-dehydrogenase [¹-DH] and 3-oxo-steroid ⁴-[5]-dehydrogenase [⁴-DH] genes], and a 1,200-bp EcoRI restrictionfragment from pGteiR [complementary to teiR] were labeled with[³²P]dATP [3,000 Ci mmol^-1] by a random priming method [14] and used as probes as indicated in the text.

Genetic complementation of teiR mutant. The complete coding sequence of teiR was obtained as a 1.2-kbEcoRI fragment from pGteiR and then subcloned into the EcoRI site of pBBR1MCS2 to generate pBBteiR . Plasmids pBBR1MCS2 [negative control] and pBBteiR were mobilized [using the mobilizing plasmid pRK2013] from E . coli to C . testosteroni UT2.5Mut50 by triparentalmating . Donor, helper, and recipient cells were grown overnightin LB medium . Cell suspensions [0.2 ml each] were mixed, filteredon a 0.4-µm-pore-size nitrocellulose membrane filter, and incubated on an LB agar plate for 8 h at 30°C . Cellswere suspended in sterile 1% NaCl, and transformants were selectedon LB agar plates containing Gm [10 µg ml^-1] and Km [500µg ml^-1].

Nucleotide sequence accession number. The nucleotide sequences reported in this paper have been depositedin the GenBank database under accession number AY363220 . The3.1-kb HindIII fragment of pSL9 bearing stdC, sip48, and theß-HSD gene has the following accession number: U41265.

 
Identification of a gene required for sip48-ß-HSDgene steroid-inducible expression. Previously, we characterizedtwo steroid-inducible genes encoding a protein of unknown function [Sip48] and ß-HSD [which are transcribed as a polycistronicmessage] . We localized the promoter activity responsible forsip48-ß-HSD gene steroid-inducible transcription inthe sip48 5' untranslated region [unpublished data] . A lacZtranscriptional fusion containing the promoter region insertedinto the chromosome of C . testosteroni [C . testosteroni UT2.5][Fig. 1] allowed us to measure promoter activity by quantifying the ß-galactosidase activity produced by this strainin different experimental conditions . High levels of ß-galactosidase activity [3,000 to 4,000 Miller units] were found when C . testosteroni UT2.5 was grown in presence of testosterone irrespective of whether LB medium or M9 medium was used [Fig . 2] . In addition, this strain showed the same growth rate as wild-type C . testosteroni cells in LB medium and M9 minimal medium supplemented with acetate or testosterone [data not shown].

 
To characterize genes involved in the regulation of sip48-ß-HSD gene steroid-inducible expression, a mini-Tn5 insertional mutagenesisprocedure was carried out by the transference of a Tc minitransposonelement [pUTminiTn5] into C . testosteroni UT2.5 . A number ofmutant strains that exhibited resistance to Tc were isolated.Screening of the resulting transconjugants revealed the presenceof white-colored colonies grown on induction medium containingX-Gal [5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside] and testosterone . One of these colonies [designated UT2.5Mut50] was isolated and further characterized . The effect of the minitransposon insertion on sip48-ß-HSD gene promoter activity inthis mutant bacterium was confirmed by comparing the ß-galactosidase activity produced by this strain to that produced by C . testosteroni UT2.5 when the strains were grown in LB medium and M9 minimal medium supplemented with acetate in the presence and the absenceof testosterone [Fig . 2] . The results clearly demonstrate that the testosterone-inducible expression of the reporter gene controlled by the sip48-ß-HSD gene promoter is absent from the C . testosteroni UT2.5Mut50 mutant strain . To assess whetherthe ß-galactosidase levels determined for the UT2.5Mut50strain reflected the transcriptional state of the correspondingsip48-ß-HSD gene transcript, their mRNA levels wereanalyzed by Northern blot analysis using sip48 and ß-HSDgene probes as described in Materials and Methods . Strong signalswere revealed in the lanes corresponding to mRNAs from C . testosteroniwild-type and UT2.5 strains growing in the presence of testosterone.In contrast, no sip48-ß-HSD gene transcript was observedwith mRNAs from C . testosteroni UT2.5Mut50 grown under the sameexperimental conditions [data not shown], thus confirming theresults obtained with the ß-galactosidase reporterfusion.

Characterization of the identified gene. Chromosomal DNA of the C . testosteroni UT2.5Mut50 strain wasisolated and digested with a NotI enzyme, which does not cutwithin the minitransposon . The digested mixture was ligatedinto the NotI site of pBIISK plasmid, and transformants ableto grow on Tc were selected . The resulting plasmid [pBIISKMut50]was found to contain a 2.5-kb insert [including the 1.8-kb Tc^r fragment] . Complete analysis of the 0.7-kb fragment sequencelocated downstream of the Tc^r gene revealed an incomplete openreading frame [3' end] . To obtain the 5' coding sequence ofthe interrupted gene, a DNA pVK102 C . testosteroni cosmid librarywas screened with a probe complementary to the 3' end of thedisrupted gene . A positive-testing clone was isolated and characterizedby Southern blot analysis . A 20-kb HindIII fragment was subclonedinto pBBR1MCS2 [pBB20H] and partially sequenced, and the completesequence of the disrupted open reading frame was obtained . Thededuced amino acid sequence of this gene predicts a 391-amino-acidprotein with a molecular mass of 43 kDa . Computer analysis showedthat this amino acid sequence shows high-level similarity [93.1%]between residues 327 and 380 [C-terminal domain] to that ofthe LuxR helix-turn-helix DNA binding domain [smart00421] [Fig.3A] . In addition, the sequence between residues 192 and 227shows mild similarity [53.7%] to that of the PAS sensor domain[smart00091] [Fig . 3B] . This novel gene [called teiR [testosterone-inducible regulator]] encodes a protein with 28% identity [44% similarity]over an aligned length of 384 amino acid residues with a putativeprotein of Novosphingobium aromaticivorans [accession number ZP 00092325.1] . Other related proteins are putative transcription regulator proteins of Mesorhizobium loti [accession number NP 103400.1],Bradyrhizobium japonicum [accession number NP 770508.1],Ralstonia solanacearum [accession number NP 523029.1], and aDNA-binding protein of Vibrio vulnificus [accession number NP 761512.1].A 60-amino-acid region in the C-terminal domain [wherea potential helix-turn-helix DNA binding motif is located alongwith several highly conserved amino acids such as those found in CsgD [COG2771], GerE [pfam00196], and LuxR [smart00421] regulatory proteins] is typically present in these proteins.

 
teiR gene expression was investigated in C . testosteroni UT2.5 and UT2.5Mut50 strains growing on LB medium with or without testosterone . Total RNA was extracted at different culture times under each set of experimental conditions, and Northern blotassays were performed . The teiR probe recognized strong signals corresponding to a 1,150-nucleotide transcript when C . testosteroni UT2.5 bacteria were grown in the presence of testosterone, indicating that this is a steroid-inducible gene . In contrast, no teiR expression was found when the UT2.5Mut50 RNA was probed, thus indicating the absence of teiR expression in the UT2.5Mut50 strain [Fig . 4].

 
Expression analysis of other steroid-inducible genes in C . testosteroni UT2.5Mut50. To establish whether the teiR gene is required for the transcriptionof other steroid-inducible genes, Northern blot assays wereperformed . Total RNAs from C . testosteroni UT 2.5 and UT2.5Mut50strains grown on LB medium with or without testosterone wereisolated . DNA fragment probes complementary to the sequencesof the steroid-inducible genes encoding -HSD-KSI and ¹-DH-⁴-DH recognized the corresponding strong signals in the RNA samples obtained from C . testosteroni UT 2.5 [the control strain] grown in the presence of testosterone . In contrast, no steroid-inducible gene expression was observed in C . testosteroni UT2.5Mut50 strain [Fig . 5] . These results demonstrate that in addition to sip48-ß-HSDgene transcription, the expression of other testosterone-induciblegenes such as the -HSD-KSI and ¹-DH-⁴-DH genes is impaired byteiR gene disruption.

 
Altered phenotypes in C . testosteroni UT2.5Mut50. Having obtained evidence that teiR disruption abolishes notonly sip48-ß-HSD gene testosterone-inducible expressionbut also the transcription of other steroid-inducible genes,we analyzed the growth of C . testosteroni UT2.5Mut50 in M9 minimalmedium supplemented with testosterone as the sole carbon andenergy source . The results [shown in Fig . 6] indicate that the mutant strain is completely unable to use testosterone as thesole carbon source . Moreover, measurement of testosterone degradationin C . testosteroni UT2.5Mut50 indicates that this strain cannot transform testosterone into androstenedione [data not shown]. Nevertheless, the mutant and wild-type strains showed identical duplication times [70 min] when they were grown in LB mediumor M9 minimal medium supplemented with acetate [Fig . 6] . These results demonstrate that teiR disruption abolishes testosterone metabolism and [as a consequence] C . testosteroni growth in medium containing this steroid as the sole carbon source.

 
Insertional transcription inactivation downstream of the teiR coding sequence. To investigate whether the phenotype observed in the C . testosteroniUT2.5Mut50 strain was due to a polar mutation caused by teiRdisruption, we inserted a Sp cassette after the coding sequenceof this gene . A new C . testosteroni UT2.5 mutant strain wasconstructed by insertion of the Sp interposon into the NdeIrestriction site located 50 bp downstream of a teiR stop codon.Southern blot analysis of the selected mutant strain [C . testosteroniUT2.5teiR-3'UR] allowed us to confirm that the wild-type sequencewas completely replaced by the interposon-disrupted construct[data not shown] . The UT2.5teiR-3'UR mutant strain grew on LBmedium or M9 minimal medium plus acetate at the same growthrate [70 min] as C . testosteroni UT2.5 . Moreover, the C . testosteroniUT2.5teiR-3'UR strain was able to grow on M9 minimal mediumsupplemented with testosterone as the sole carbon source [Fig.7A], indicating that no genes required for growth on testosteroneare 3' of teiR and cotranscribed with teiR . In addition, the level of sip48-ß-HSD gene promoter activity [measuredas the level of ß-galactosidase activity in C . testosteroni UT2.5teiR-3'UR grown in testosterone-containing medium] wasequal to that of the control strain [UT2.5] [Fig . 7B] . Thus,no genes required for testosterone-inducible gene expressionare located downstream of teiR coding sequence and cotranscribedwith it.

 
Genetic complementation of teiR mutant. To demonstrate that teiR encodes a protein involved in the testosterone-inducible expression of sip48-ß-HSD genes and to confirm thatthis gene is essential for growth on testosterone as the solecarbon source, we performed a complementation assay . The aimof this experiment was to analyze the expression of the reporterlacZ fusion in C . testosteroni UT2.5Mut50 after trans-complementation with teiR and to ascertain whether the complemented strain regains the growing phenotype of the C . testosteroni UT2.5 parental strain . The complementation procedure was performed with pBBteiR plasmid containing the teiR gene . As expected, the testosterone-induciblesip48-ß-HSD gene promoter activity was restored inthe complemented UT2.5Mut50 strain . The ß-galactosidase levels determined when the cells were growing in the presence of testosterone were similar to those of the isogenic UT2.5strain [Fig . 7B] . In addition, the complemented bacterium was able to grow when testosterone was used as the sole carbon source [Fig . 7A] . In both experiments, the behavior of a complementedstrain carrying pBBR1MSC2 without an insert was indistinguishablefrom that of C . testosteroni UT2.5Mut50 [data not shown] . Northernblot assays performed with the complemented strain also demonstratedsteroid-inducible expression of the sip48-ß-HSD gene,the -HSD-KSI gene, and the ¹-DH-⁴-DH genes [data not shown].Altogether, these data confirm that teiR is essential for thesteroid-inducible transcription of different steroid-induciblegenes and for steroid metabolism in C . testosteroni.

 
In this work, we report the identification and characterizationof teiR, a novel steroid-inducible gene that is necessary for testosterone degradation in C . testosteroni strains . The C-terminal domain of TeiR has a high level of similarity to the LuxR DNA binding domain [belonging to the family of LuxR-type transcription regulators] . It has been established by genetic analyses thatLuxR is composed of two functional modules or domains: an amino-terminal domain with an autoinductor binding region and a carboxy-terminal transcription regulatory domain [28] . LuxR-like proteins bind autoinducers that have achieved a critical threshold concentration, after which the LuxR-autoinducer complexes generally activate gene transcription . Two interesting features among LuxR-type transcriptional regulators are the high level of homology inthe DNA binding domain and the variability in the sensor domain[17] . In this regard, the TeiR C-terminal domain shows high-levelhomology to the helix-turn-helix DNA binding motif present inLuxR and other related transcriptional regulators . In additionto the LuxR helix-turn-helix DNA binding domain [residues 327to 380], a PAS sensor domain located between amino acids 192and 227 was observed . This domain distribution resembles thatdescribed for different LuxR-type proteins; it is particularlysimilar to that of TraR, a specific transcriptional regulatorinvolved in quorum sensing in Agrobacterium tumefaciens [whosethree-dimensional structure has been recently described] [41].

It has been suggested that quorum sensing is an integral component of gene regulatory networks in gram-negative bacteria [43]. Particularly, C . testosteroni teiR-disrupted strains are unable to induce the expression of several steroid-inducible genes, such as those encoding a steroid-inducible protein of 48 kDa,ß-HSD, -HSD, ⁵-KSI, ¹-DH, and ⁴-DH enzymes . The absenceof these proteins probably determines the complete impairmentof the C . testosteroni teiR mutant strain with respect to theuse of different steroid compounds as a sole carbon and energysource . The results of the complementation assay and the transcriptionalinterruption downstream of the teiR coding sequence clearlydemonstrate the importance of this gene for the observed phenotypes.Taken together, these results suggest that TeiR is a globalregulator of the steroid catabolic pathway in C . testosteroni.

One or more regulatory proteins often control the expressionof bacterial catabolic pathways for aromatic compounds, andthe effectors of these regulatory proteins are usually eitherthe initial substrates or catabolic intermediates of the pathways[39] . It has been reported in particular that the regulationof -HSD gene expression appears to be a derepression mechanismin which the steroidal inducer [testosterone] prevents the bindingof two repressor proteins [RepA and RepB] to the -HSD gene promoterand mRNA, respectively [45] . The complete lack of activationof -HSD gene steroid-inducible transcription when the teiR-disrupted mutant was grown in the presence of testosterone indicates thatthis steroid is not the true inducer of this pathway, suggestingthat the mechanisms regulating -HSD gene expression might bemore complex than previously reported . In agreement with ourresults, the data obtained by Horinouchi et al . [20] suggestthat expression of tesB [encoding a meta-cleavage steroid-inducibleenzyme] in C . testosteroni TA441 is induced by an intermediatecompound produced in the course of testosterone degradation.

In conclusion, mutation of teiR is sufficient to block the ability of C . testosteroni to use testosterone as a sole carbon source, indicating that TeiR is an integral part of the degradation portion of the steroid catabolic pathway . Furthermore, teiR encoding a LuxR-type transcription factor is required for the expression of several steroid-inducible genes, suggesting thata quorum-sensing mechanism might be involved in its regulation.

 
We are grateful to Victor de Lorenzo for kindly providing pUTminiTn5 [Tc^r], to Athan Kuliopolus for providing the pAK1370 plasmid encoding the C-terminal sequences of the 3-HSD and ⁵--KSI genes,and to Patrick Plessiat for providing the pTEK21 plasmid encoding ¹-DH and the N end of ⁴-DH . We are grateful to Luis Patrito,Alfredo Flury, and Graciela Panzetta-Dutari for discussionsand critical reading of the manuscript.

This work was supported by grants from the Consejo Nacionalde Ciencia y Tecnología [CONICET] and the Secretaríade Ciencia y Tecnología de Universidad Nacional de Córdoba[SECyT] . J.L.P.-P . was supported by a fellowship from the SECyT.

 
* Corresponding author . Mailing address: Universidad Nacional de Córdoba, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina . Phone: 54 351 4334164 . Fax: 54 351 4333048 . E-mail: sgenti@fcq.unc.edu.ar .

Present address: Developmental Genetics Section, Laboratoryof Molecular Biology, National Cancer Institute, National Institutesof Health, Bethesda, MD 20892.

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What Is Biotechnology?, What Is Pcr?, What Is Nitrification?, What Is Water Purification?, What Is Genome?, e, Microbes, o, Microbiology, e, Bacteriology, i, Microorganisms, n, Microorganism, a, Pseudomonas aeruginosa, s, S. cerevisiae, i, Minimum inhibiting concentration, o, Candida albicans, n, Escherichia coli, n, Antibiotic prophylaxis, o, Streptococcal, a, Beta lactamase, c, Escherichia coli, n, Prokaryotes, n, S. cerevisiae, a, Neisseria, c, Acinetobacter, o, Acinetobacter, i, Bacteria, r, Minimal inhibiting concentration, a, Antimicrobial, s, Penicillin, c, Vibriosis, i, Bacillus, n, Penicillin




 

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