TeiR, a LuxR-type transcription factor required for testosterone degradation in Comamonas testosteroni

Abstract

We have identified a new steroid-inducible gene (designated teiR [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) to the LuxR helix-turn-helix DNA binding domain and between residues 192 and 227 to the PAS sensor domain. This domain distribution resembles that described for TraR, a specific transcriptional regulator involved in quorum sensing in Agrobacterium tumefaciens. Analysis of the gene expression indicated that teiR is tightly controlled at the transcriptional level by the presence of testosterone in 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 genes was abolished in this mutant. Northern blot assays revealed that teiR is required for full expression of sip48-beta-HSD gene mRNA (encoding a steroid-inducible protein of 48 kDa and 3beta-17beta-hydroxysteroid dehydrogenase) and also of other steroid degradation genes, including those encoding 3alpha-hydroxysteroid dehydrogenase, Delta(5)-3-ketoisomerase, 3-oxo-steroid Delta(1)-dehydrogenase, and 3-oxo-steroid Delta(4)-(5alpha)-dehydrogenase enzymes. Moreover, when teiR was provided to the teiR-disrupted strain in trans, the transcription level of these genes was restored. These results indicate that TeiR positively regulates the transcription of genes involved in the initial enzymatic steps of steroid degradation in C. testosteroni.

FIG. 1.

Restriction map of the 3.2-kb HindIII fragment cloned into pSL9 plasmid. The regions carrying stdC and sip48 and the β-HSD gene are indicated by boxes. H, HindIII; Hc, HincII; P, PstI; Ev, EcoRV. A schematic representation of pUT2.5 carrying the transcriptional fusion of the β-HSD gene upstream region (thick line) to a promoterless lacZ gene (open box) is shown. The black box and arrow indicate the sip48-β-HSD gene steroid-inducible promoter.

FIG. 2.

Activity of sip48-β-HSD gene promoter in C. testosteroni UT2.5 and UT2.5Mut50 strains. The levels of β-galactosidase activity of a sip48-β-HSD gene promoter-lacZ transcriptional fusion in C. testosteroni UT2.5 and UT2.5Mut50 strains growing in LB medium and M9 minimal medium supplemented with acetate (M9) in the presence (black bars) and the absence (white bars) of testosterone are shown. β-Galactosidase activities were measured with permeabilized cells as described in Materials and Methods. Each value is the average of the results from three independent experiments (error bars indicate standard deviations).

FIG. 3.

(A) Alignment of the helix-turn-helix DNA binding domains of three transcriptional regulators (CsgD [COG2771], LuxR [smart00421], and GerE [pfam00196]) and amino acids 327 to 380 of the TeiR protein (accession number AY363220). (B) Alignment of the PAS sensor domain (smart00091) and amino acids 192 to 227 of the TeiR protein. For residues that were identical in all the aligned proteins, the characters representing the residues appear on a dark-gray background. For residues that were identical in 60% of the aligned proteins, the characters representing the residues appear on a light-gray background.

FIG. 4.

Expression of teiR in C. testosteroni UT2.5 and UT2.5Mut50 strains in response to the presence of testosterone. Total RNA samples (20 μg per lane) were prepared from bacteria grown on LB medium in the absence (LB) or presence (LB+T) of testosterone during 9, 12, and 24 h of culture growth. (A) The membrane was hybridized with a DNA fragment complementary to the teiR gene. (B) The samples were analyzed by electrophoresis on formaldehyde-agarose gels, transferred to a nylon membrane, and stained with methylene blue.

FIG. 5.

Expression of steroid-inducible genes in C. testosteroni UT2.5 and UT2.5Mut50 strains. Total RNA samples (20 μg per lane) were prepared from bacteria grown on LB medium in the presence or absence of testosterone during 6, 9, 12, and 24 h of culture growth. (A and B) The membrane was hybridized with a DNA fragment complementary to α-HSD and Δ⁵-KSI (αhsd/ksi) (A) and to Δ¹-DH and Δ⁴-DH (Δ¹dh/Δ⁴-dh) (B) steroid-inducible genes. (C) The samples were analyzed by electrophoresis on formaldehyde-agarose gels, transferred to a nylon membrane, and stained with methylene blue.

FIG. 6.

Growth of C. testosteroni UT2.5 (white shading) and UT2.5Mut50 (grey shading) strains in LB medium (circle), M9 medium plus acetate (square), and M9 medium plus testosterone (triangle). Growth of bacteria in LB medium or M9 plus acetate was monitored by measuring OD₆₀₀. Growth of bacteria in M9 medium plus testosterone was monitored by counting colonies (in CFU per milliliter) that appeared on LB plates (on which appropriately diluted cultures have been spread) after incubation at 30°C.

FIG. 7.

Growth and sip48-β-HSD gene promoter activity in C. testosteroni UT2.5, UT2.5Mut50, and UT2.5teiR-3′URΩ strains and in the UT2.5Mut50 strain complemented with teiR. (A) Growth of C. testosteroni UT2.5 (black circle), UT2.5teiR-3′URΩ (black square), and UT2.5Mut50 (black triangle) strains and of the UT2.5Mut50 strain complemented with pBBteiR (white triangle). Growth of bacteria in LB medium or M9 plus acetate was monitored by measuring OD₆₀₀. Growth of bacteria in M9 minimal medium plus testosterone was monitored by counting colonies (in CFU per milliliter) that appeared on LB plates (on which appropriately diluted cultures have been spread) after incubation at 30°C. (B) β-Galactosidase activity levels of sip48-β-HSD gene promoter-lacZ transcriptional fusion in C. testosteroni strains UT2.5, UT2.5Mut50, UT2.5teiR-3′URΩ, and UT2.5Mut50 complemented with pBBteiR (UT2.5Mut50 + pBBteiR) growing in LB medium and in M9 minimal medium supplemented with acetate (M9) in the presence (black bars) and the absence (white bars) of testosterone. β-Galactosidase activities were measured with permeabilized cells as described in Materials and Methods. Each value is the average of the results from three independent experiments (error bars indicate standard deviations).