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Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor.

Brett PJ, Burtnick MN, Fenno JC, Gherardini FC - Mol. Microbiol. (2008)

Bottom Line: Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR(Td)) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members.In addition, we demonstrate that although TroR(Td) possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR(Td).Based upon these findings, we propose that TroR(Td) represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA.

ABSTRACT
Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes (troABCDR) encoding for the components of an ATP-binding cassette cation-transport system (troABCD) and a DtxR-like transcriptional regulator (troR). In addition, a sigma(70)-like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroR(Tp)) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR(Td)) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroR(Td) is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola. In addition, we demonstrate that although TroR(Td) possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR(Td). Based upon these findings, we propose that TroR(Td) represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.

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TroRTdΔ157−222 repressor responds to Mn2+ and Fe2+ in E. coli. (A) Strain DEN113A harbouring pTDE100 (T. denticola troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) or (B) strain PAL113A harbouring pPAL100 (T. pallidum troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) were grown aerobically overnight in M9CG assay media supplemented with various divalent cations (5 μM) or in unsupplemented M9CG media (control). β-Galactosidase activity of the overnight cultures was determined as described by Miller (1972). Results represent the means and standard deviations of three independent experiments.
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fig07: TroRTdΔ157−222 repressor responds to Mn2+ and Fe2+ in E. coli. (A) Strain DEN113A harbouring pTDE100 (T. denticola troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) or (B) strain PAL113A harbouring pPAL100 (T. pallidum troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) were grown aerobically overnight in M9CG assay media supplemented with various divalent cations (5 μM) or in unsupplemented M9CG media (control). β-Galactosidase activity of the overnight cultures was determined as described by Miller (1972). Results represent the means and standard deviations of three independent experiments.

Mentions: Unlike other members of the DtxR family of metalloregulators, both TroRTp and B. subtilis MntR are manganese-responsive and lack C-terminal SH3-like domains. In order to assess the importance of the TroRTd C-terminal domain with respect to metal specificity, β-galactosidase activity from strains DEN113A and PAL113A was measured in response to metal ion supplementation. In the presence of TroRTdΔ157−222 expression, β-galactosidase activity from both DEN113A and PAL113A decreased in response to Mn2+ and Fe2+ and also decreased slightly in response to Co2+ (Fig. 7A and B). β-Galactosidase activity remained unchanged in the presence of Cd2+, Cu2+, Ni2+ and Zn2+ (Fig. 7A and B). These results indicated that similar to full-length TroRTd, TroRTdΔ157−222 maintained repressor activity in the presence of Mn2+, Fe2+ and Co2+. Based on these results, it appears that the C-terminal domain of TroRTd is not important for metal specificity and the role of this region of the protein in metal binding, if any, remains unclear.


Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor.

Brett PJ, Burtnick MN, Fenno JC, Gherardini FC - Mol. Microbiol. (2008)

TroRTdΔ157−222 repressor responds to Mn2+ and Fe2+ in E. coli. (A) Strain DEN113A harbouring pTDE100 (T. denticola troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) or (B) strain PAL113A harbouring pPAL100 (T. pallidum troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) were grown aerobically overnight in M9CG assay media supplemented with various divalent cations (5 μM) or in unsupplemented M9CG media (control). β-Galactosidase activity of the overnight cultures was determined as described by Miller (1972). Results represent the means and standard deviations of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2628430&req=5

fig07: TroRTdΔ157−222 repressor responds to Mn2+ and Fe2+ in E. coli. (A) Strain DEN113A harbouring pTDE100 (T. denticola troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) or (B) strain PAL113A harbouring pPAL100 (T. pallidum troP/O-lacZ reporter) and pPJB113A (expresses TroRTdΔ157−222) were grown aerobically overnight in M9CG assay media supplemented with various divalent cations (5 μM) or in unsupplemented M9CG media (control). β-Galactosidase activity of the overnight cultures was determined as described by Miller (1972). Results represent the means and standard deviations of three independent experiments.
Mentions: Unlike other members of the DtxR family of metalloregulators, both TroRTp and B. subtilis MntR are manganese-responsive and lack C-terminal SH3-like domains. In order to assess the importance of the TroRTd C-terminal domain with respect to metal specificity, β-galactosidase activity from strains DEN113A and PAL113A was measured in response to metal ion supplementation. In the presence of TroRTdΔ157−222 expression, β-galactosidase activity from both DEN113A and PAL113A decreased in response to Mn2+ and Fe2+ and also decreased slightly in response to Co2+ (Fig. 7A and B). β-Galactosidase activity remained unchanged in the presence of Cd2+, Cu2+, Ni2+ and Zn2+ (Fig. 7A and B). These results indicated that similar to full-length TroRTd, TroRTdΔ157−222 maintained repressor activity in the presence of Mn2+, Fe2+ and Co2+. Based on these results, it appears that the C-terminal domain of TroRTd is not important for metal specificity and the role of this region of the protein in metal binding, if any, remains unclear.

Bottom Line: Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR(Td)) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members.In addition, we demonstrate that although TroR(Td) possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR(Td).Based upon these findings, we propose that TroR(Td) represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA.

ABSTRACT
Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes (troABCDR) encoding for the components of an ATP-binding cassette cation-transport system (troABCD) and a DtxR-like transcriptional regulator (troR). In addition, a sigma(70)-like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroR(Tp)) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR(Td)) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroR(Td) is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola. In addition, we demonstrate that although TroR(Td) possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR(Td). Based upon these findings, we propose that TroR(Td) represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.

Show MeSH
Related in: MedlinePlus