Characterization of the Bacteroides CTnDOT Regulatory Protein RteC

Jiyeon Park; Abigail A Salyers

doi:10.1128/JB.01015-10

. 2010 Oct 29;193(1):91–97. doi: 10.1128/JB.01015-10

Characterization of the Bacteroides CTnDOT Regulatory Protein RteC^▿

Jiyeon Park ^1,^*, Abigail A Salyers ¹

PMCID: PMC3019952 PMID: 21037014

Abstract

Excision of the Bacteroides conjugative transposon CTnDOT is stimulated by tetracycline. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the orf2c operon, which contains the excision genes. The protein encoded by this gene, RteC, did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, we sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains and that the C-terminal domain might have a winged helix motif. Based on the Robetta prediction, the human transcriptional factors E2F-4 and DP2 were identified as the most likely structural homologs of RteC. We made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of orf2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of P_E. The upstream region of orf2c contained two inverted-repeat half sites. Mutational analysis of these half sites showed that both half sites are important for activity. Thus, we have identified the DNA binding portion of RteC and the DNA site to which it binds.

Bacteroides spp. are one of the numerically predominant groups of bacteria in the human colon, where they account for about 30% of the microbiota (15). Bacteroides spp. can cause serious opportunistic infections if they escape from the colon due to abdominal surgery or other trauma. Infections caused by Bacteroides spp. are difficult to treat because of increasing antibiotic resistance. Antibiotic resistance genes are being spread among Bacteroides spp. by horizontal gene transfer (16, 18, 19). CTnDOT is a conjugative transposon that was found originally in a Bacteroides strain that was isolated from a patient with a serious Bacteroides infection (16, 22). Subsequent studies showed that conjugative transposons such as CTnDOT-type elements play an important role in transferring antibiotic resistance genes among Bacteroides spp. in the colon (22). The first step in CTnDOT transfer is excision from the chromosome to form a nonreplicating circular intermediate (28). Previously, an operon containing genes important for excision (orf2c, orf2d, and exc) was identified (4). These genes are regulated at the transcriptional level (14). Excision is induced by tetracycline (3, 4, 20, 23). Regulation appeared to be mediated by three regulatory proteins encoded by rteA, rteB, and rteC (17). The tetQ gene is the first gene in the operon that contains rteA and rteB (27). Transcription of tetQ, rteA, and rteB is constitutive, whereas the translation of these genes is increased during exposure to tetracycline (27). Increased protein production is due to a translational attenuation mechanism (26, 27). Both RteA and RteB are clearly regulatory proteins based on amino acid similarity to known two-component regulatory systems, with RteA being a histidine kinase environmental sensor and RteB being a transcriptional activator (24). RteB activates the expression of rteC, which in turn controls the expression of genes essential for excision (14, 24). Previously, it was demonstrated that disruption of rteC abolished excision, suggesting that RteC acts as a positive regulator of excision genes (4). When rteC was placed under the control of the tetQ promoter, so that rteA and rteB were no longer needed, and introduced in trans with both the transcriptional and translational orf2c-uidA fusions, the construct stimulated the expression of the fusions (14). Therefore, RteC itself is sufficient to express the excision gene operon (14, 24). Our hypothesis was that RteC is a DNA binding protein that binds upstream of the promoter of the orf2c operon, P_E. However, RteC did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, we decided to search for structural homologs of RteC to obtain insights into the possible structure and function of RteC.

MATERIALS AND METHODS

Strains and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 1 . The concentrations of antibiotics used were as follows: ampicillin (Ap), 100 μg/ml; chloramphenicol (Cm), 15 μg/ml; kanamycin (Kn), 50 μg/ml; cefoxitin (Cef), 20 μg/ml; erythromycin (Em), 10 μg/ml; gentamicin (Gen), 200 μg/ml; and rifampin (Rif), 10 μg/ml. All the Escherichia coli strains were grown in Luria broth (LB) or on LB agar at 37°C, and the Bacteroides strains were grown in either Trypticase yeast extract-glucose (TYG) medium or supplemented brain heart infusion (BHI) medium at 37°C under anaerobic conditions.

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid	Relevant phenotype^a	Description (reference or source)
E. coli strains
DH5αMCR	RecA	From Gibco BRL
HB101(RP1)	RecA Str^r	HB101 containing IncPα plasmid RP1 (21)
BL21-Codon Plus (DE3) RIL	Cm^r	Cells enable high-level expression of heterologous proteins in E. coli (Stratagene)
B. thetaiotaomicron 5482A strains
BT4001	Rif^r	Spontaneous rifampin-resistant mutant
BT4007	Rif^r Tc^r Em^r	BT4001 containing wild-type CTnDOT
Plasmids
pMJF2	Ap^r (Em^r)	Cloning vector to create a uidA fusion, also an E. coli-Bacteroides shuttle vector (6)
pAFD1	Ap^r (Em^r)	E. coli-Bacteroides shuttle vector containing ermF
pC-COW	Ap^r Tc^r Cm^r (Cm^r)	E. coli-Bacteroides shuttle vector with IS4351-cat and Bacteroides plasmid pB8-51 that is compatible with pAFD1and pMJF2-based vectors (9)
pCR-Blunt	Kn^r	3.5-kb cloning vector for PCR product (Invitrogen)
pET28b	Kn^r	Plasmid for the overexpression of His₆-tagged proteins in E. coli (Novagen)
pGFK24.1	Ap^r	0.3-kb PCR product containing the upstream region of the orf2c operon cloned into pCR2.1(K. Moon, unpublished)
pGFK43.11	Ap^r (Cm^r)	Plasmid containing uidA fused to 300 bp upstream region of the orf2c start codon cloned into the NruI-SphI site of pC-COW (K. Moon, unpublished)
pGFK70.1	Ap^r	1.2-kb PCR product containing RteC_His6 cloned into pCR2.1 (K. Moon, unpublished)
pGFK76.5	Ap^r (Cef^r)	Plasmid containing an in-frame fusion of His₆-tagged rteC to the tetQ promoter (K. Moon, unpublished)
pJPARK5	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −67 and −70 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK7	Ap^r (Em^r)	1.4-kb Sph-SstI fragment from pGFK76.5 containing wild-type pP_Q-rteC; His₆-tagged cloned into SphI-SstI site of pAFD1(this study)
pJPARK18	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −63 and −66 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK19	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −59 and −62 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK20	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −55 and −58 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK21	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −51 and −54 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK22	Ap^r	0.3-kb PCR product containing 20-bp mutated sequence between positions −46 and −65 upstream of the orf2c transcriptional start site cloned into pCR2.1 (this study)
pJPARK26	Kn^r	760-bp PCR product containing wild-type rteC cloned into pCR-Blunt (this study)
pJPARK28	Ap^r	0.3-kb PCR product containing 7-bp mutated sequence between positions −43 and −49 upstream of the orf2c transcriptional start site cloned into pCR2.1 (this study)
pJPARK32	Kn^r	760-bp NdeI-XhoI fragment from pJPARK26 cloned into NdeI-XhoI site of pET28b, creating His₆-tagged rteC (this study)
pJPARK37	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 7-bp mutated sequence between positions −43 and −49 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK38	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 20-bp mutated sequence between positions −46 and −65 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK39	Ap^r Tc^r Cm^r (Cm^r)	uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −35 and −38 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
pJPARK49	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (K174A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK50	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (R178A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK51	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (Y180A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK63	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (D175A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK64	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (E189A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK65	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (K184A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK66	Ap^r (Em^r)	1.2-kb PCR product containing RteC_His6 mutant (K184A) cloned into NcoI-SmaI site of pJPARK7 (this study)
pJPARK76	Ap^r	0.3-kb PCR product containing 5-bp mutated sequence between positions −53 and −57 upstream of the orf2c transcriptional start site cloned into pCR2.1 (this study)
pJPARK78	Ap^r Tc^r Cm^r (Cm^r)	uidA fused 0.3-kb PCR product containing 5-bp mutated sequence between positions −53 and −57 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)

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Parentheses indicate resistances expressed in Bacteroides.

Site-directed mutagenesis.

Site-directed mutagenesis was done using a QuikChange site-directed mutagenesis kit (Stratagene) and iProof high-fidelity DNA polymerase (Bio-Rad). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). Plasmid pGFK24.1 (K. Moon, unpublished), a pCR2.1 vector (Invitrogen) containing the 300-bp region upstream of the orf2c operon, was used as a DNA template in site-directed mutagenesis of the upstream region of the excision operon promoter(P_E). Plasmid pGFK70.1 (K. Moon, unpublished), a pCR2.1 vector (Invitrogen) containing His₆-tagged rteC, was used as a DNA template for site-directed mutagenesis of RteC_His6 for in vivo β-glucuronidase (GUS) assays. The PCR amplicons containing different mutations were transformed into E. coli DH5αMCR competent cells (Bethesda Research Laboratories Inc., Gaithersburg, MD), and cells were grown in Luria broth (LB) agar medium containing ampicillin (100 μg/ml) or kanamycin (50 μg/ml) overnight. Plasmids were isolated and then sequenced to confirm the mutation (UIUC Core DNA Sequencing Facility, Urbana, IL). For site-directed mutagenesis of the upstream region of P_E, the SphI-SmaI restriction fragments were isolated and ligated into the SphI-SmaI site of pMJF2 (6) to create an orf2c::uidA translational fusion, and then the SphI-PvuII fragments containing the orf2c::uidA fusion were cloned into the SphI-NruI site of an E. coli-Bacteroides shuttle vector, pC-COW (9). For site-directed mutagenesis of RteC_His6, the NcoI-SmaI restriction fragments were isolated and ligated into the NcoI-SmaI site of pJPARK7 to replace wild-type RteC_His6 with RteC_His6 mutants. pJPARK7 is a pAFD1 vector containing pP_Q-rteC_His6.

Triparental matings.

The plasmids containing wild-type RteC, mutant forms of RteC, or mutant forms of P_E were mobilized into Bacteroides strain BT4001 via triparental matings. In each case, the two donors were E. coli DH5αMCR, which contained either mutations in the P_E region or RteC mutants, and E. coli HB101, which contained the IncPα plasmid RP1 (21). RP1 cannot replicate in Bacteroides spp., but it does mobilize E. coli-Bacteroides vectors from E. coli donors to Bacteroides recipients (21). Matings were done on nitrocellulose filters. The plasmids were then mobilized into BT4001, a Bacteroides strain which contains pJPARK7, a pAFD1 vector containing pP_Q-rteC, or BT4001, which contains pGFK43.11, a pC-COW vector containing the P_E orf2c:: uidA fusion.

GUS assay.

The uidA reporter gene on pMJF2 encodes an E. coli β-glucuronidase (GUS). GUS assays were done by the procedure of Feldhaus et al. (6). One unit was defined as 0.01 A₄₁₅ U per min at 37°C. Protein concentrations were determined by the method of Lowry et al. (12).

Overexpression of RteC.

A promoterless rteC gene was amplified from BT4007 using PCR. High-fidelity PCR amplification was done with PfuUltra high-fidelity DNA polymerase (Stratagene). The 670-bp rteC PCR product was cloned into pCR-Blunt (Invitrogen). An NdeI-XhoI fragment was cloned into the NdeI-XhoI sites of pET28b (Novagen), creating His₆-tagged rteC. Escherichia coli BL21 CodonPlus (DE3) RIL (Stratagene) was used as the host strain for wild-type RteC. Cells were grown overnight at 37°C in 10 ml Luria broth (LB) medium with chloramphenicol (50 μg/ml) and kanamycin (30 μg/ml). The overnight cultures were used to inoculate 500 ml of LB medium containing chloramphenicol (50 μg/ml) and kanamycin (30 μg/ml). The culture was incubated with vigorous shaking at 37°C. Cells were grown until the optical density at 600 nm (OD₆₀₀) was 0.3 to 0.4, and isopropyl-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. Cells were grown at 25°C for 3 to 4 h with vigorous shaking. Cells were harvested by centrifugation at 7,000 rpm for 20 min at 4°C. The supernatant fluid was discarded, and the cells were stored at −80°C until use. Overexpression of His₆-tagged RteC protein was confirmed by Western blotting with mouse anti-His antibody (GE Healthcare) as a primary antibody and anti-mouse IgG-horseradish peroxidase (HRP) (Promega) as a secondary antibody. The tagged RteC was tested in Bacteroides strain 4001 containing the P_E orf2c::uidA fusion to ascertain that it activated the orf2c operon similarly to wild-type RteC.

Cell extract preparation and purification of RteC.

The frozen cell pellet was resuspended in 10 ml lysis buffer (50 mM Tris [pH 8.0], 1 M NaCl) to which one Complete Mini EDTA-free protease inhibitor tablet (Roche Applied Science) had been added. The cell suspension was frozen on dry ice and thawed in a 37°C water bath three times. The cells were then lysed by sonication (15 s of sonication and 2 min on ice, six times). The cell lysate was separated by centrifugation at 7,000 rpm for 20 min at 4°C. The supernatant was incubated with 1 ml cobalt resin (Pierce) that had been equilibrated with lysis buffer at 4°C for 1 h and passed over a gravity flow column. The column was eluted with elution buffer (50 mM Tris-HCl [pH 8.0], 1 M NaCl, and 150 mM imidazole). The crude cell extract or the partially purified RteC_His6 was dialyzed overnight in a Slide-A-Lyze cassette (Thermo Scientific) against dialysis buffer (50 mM Tris-HCl [pH 8.0], 1 M NaCl, 1 mm dithiothreitol [DTT], 1 mM EDTA, 20% glycerol) and stored at −80°C until use. The partially purified RteC_His6 was analyzed by both SDS-PAGE and native gel electrophoresis and subjected to Western blotting to determine the molecular weight.

Gel shift assays.

The 260-bp DNA fragments utilized in the gel shift assay experiments were prepared from plasmids containing the wild-type or mutated P_E region by restriction enzyme digestion (SphI and SmaI) followed by gel extraction. DNA fragments were labeled with [γ-³²P] ATP using PCR, and purified. The DNA fragment (approximately 5 ng) and either partially purified RteC or RteC cell extract were mixed in 10 μl binding buffer and were incubated for 15 min at room temperature. The 5× binding buffer contained 250 mM Tris-HCl (pH 8.0), 5 mM EDTA, 250 mM NaCl, 50% glycerol, and 3.6 μg/ml herring sperm DNA. The binding mixture was loaded onto a 6% DNA retardation gel (Invitrogen), and 0.5× Tris-borate-EDTA buffer (pH 8.0) was used as the running buffer. After electrophoresis, the gels were dried and subjected to autoradiography.

RESULTS

Structural homologs of RteC.

The linear RteC amino acid sequence did not have significant homology to any proteins in the databases (14). Moreover, unlike many DNA binding proteins, which have a high pI, the predicted pI of RteC was 6.9. Accordingly, we decided to look for structural homologs instead of relying on the linear amino acid sequence. To find the possible structural homologs of RteC, we used the protein structure prediction server Robetta (10). The Robetta server uses methods including PSI-BLAST (1) and the Rosetta fragment insertion to generate a hypothetical structure (5, 10). Models can be built by the de novo Rosetta prediction method (1, 5, 10). Based on the Robetta (10) prediction, we found that RteC might have two domains, including a C-terminal domain that contains a winged helix motif (8). A winged helix motif is a subfamily of the helix-turn-helix motif (8). This motif consists of three alpha helices and two beta strands (8). Two antiparallel beta strands make wings, and these wings flank the third helix (8). Typically, the third helix works as a recognition helix and interacts with the major groove of DNA (8). Based on the Robetta structural prediction, compared to structures in the database, human transcriptional factors E2F-4 (29) and DP2 (29) were the closest homologs, with a Z score of 6.46. The E. coli LexA repressor (7) and the E. coli arginine regulator ArgR (11) could also be structural homologs of RteC. The fact that all of these homologs are known DNA binding proteins supported the hypothesis that RteC is a DNA binding protein. E2F-4 forms a heterodimer with the other transcriptional factor, DP2, to efficiently bind DNA, and these two proteins share sequence homology in their DNA binding domains (29). We used ClustalW (25) to align the putative C-terminal domain of RteC, amino acid residues 111 to 217, with DNA binding domains of the human transcriptional factors E2F-4 (29) and DP2 (29), and the secondary structure of the putative RteC DNA binding domain was obtained (Fig. 1). Since helix 3 works as a recognition helix in many winged helix proteins, the putative helix 3 region in RteC (residues 174 to 189) was subject to mutation.

FIG. 1. — Predicted RteC DNA binding domain. The putative RteC C-terminal domain, residues 111 to 217, was deduced based on Robetta prediction results. Based on the Robetta prediction, the putative C-terminal domain might contain a winged helix motif, because the human transcriptional factors E2F-4 and DP2, the closest structural homologs, have winged helix motifs in this region. Amino acid sequences from position 111 to 217 were aligned with the DNA binding domains of these structural homologs of RteC, and the putative secondary structure of the RteC DNA binding domain was obtained. Three alpha helices are represented as H1, H2, and H3 in boxes. Beta strands are shown as arrows labeled s1, s2, and s3.

Alanine substitutions in the putative RteC helix 3.

We made seven individual alanine substitutions within the putative helix 3 region in RteC. Alanine substitutions were constructed at K174, D175, R178, F179, Y180, K184, and E189 (Fig. 2A, bold). The RteC alanine mutants were then tested for the ability to activate the wild-type orf2c::uidA fusion. We found three amino acid residues that were fully conserved in RteC (residues 174, 178, and 180), E2F-4 (residues 118, 123, and 125), and DP2 (residues 55, 57, and 59). Single-alanine substitution of these amino acids resulted in activity that was decreased to the background level (Fig. 2B). Mutations at D175 and K184 rendered transcription activation of orf2c::uidA defective (Fig. 2B). Mutations at F179 showed 20% decreased activity, while an alanine substitution at E189 did not affect the activity compared to that of the wild type (Fig. 2B). These results show that helix 3 of RteC is important for RteC activity.

FIG. 2. — Alanine substitutions within the putative RteC helix 3. (A) The amino acid sequence of the predicted helix 3 that was subjected to alanine substitutions. Based on sequence alignment with the structural homologs of RteC, fully conserved, strongly conserved, and nonconserved amino acid residues (in bold) in the predicted helix 3 were chosen. Amino acid residues that were changed to alanine are in bold. (B) The RteC alanine substitution mutants were placed under the control of the heterologous *tetQ* promoter and cloned into the *E. coli-Bacteroides* shuttle vector pAFD1. The *orf2c*::*uidA* fusion was provided on a compatible *E. coli-Bacteroides* shuttle vector, pC-COW. The values obtained with wild-type RteC were set to 100%, and the activity of the each RteC mutant is represented as a percentage of the wild-type P_E value. Without RteC, the GUS activity was less than 1 U/mg protein. The experiments were done in triplicate, and the mean values and standard deviations are shown.

Site-directed mutagenesis of the upstream region of P_E.

We also wanted to define the RteC DNA binding site upstream of P_E. Preliminary analysis had suggested that the 20 bp between positions −51 and −70 relative to the orf2c transcriptional start site might be important for activity (14). To identify the important base pairs within this 20-bp region, five different 4-bp mutations were made within this region (Fig. 3A). The changes were complementary to conserve the melting temperature characteristics. The effects of mutations on activity were measured by an in vivo GUS assay. The results for each set of 4-bp mutations within the 20-bp region upstream of P_E on orf2c::uidA are shown in Fig. 3B. Cells containing both the wild-type 20-bp region and wild-type RteC exhibited a 100-fold increase in GUS activity compared to cells without RteC (Fig. 3B). The 4-bp change between positions −67 and −70 did not affect the GUS activity, while the 4-bp mutations of the downstream 16-bp region resulted in more than 2-fold-decreased GUS activity (Fig. 3B). When the entire 20-bp region was changed, the activity was decreased 8-fold (14). The 20-bp region contained a 7-bp inverted-repeat half site. Another inverted-repeat half site was located immediately downstream of the 20-bp sequence, from position −49 to −43 relative to the orf2c transcriptional start site. The fact that mutations in the part of the 20-bp region that contained one inverted-repeat half site resulted in 8-fold-decreased activity (14) but did not decrease GUS activity to the background level may mean that both half sites must be mutated to eliminate gene expression.

FIG. 3. — Site-directed mutagenesis of the upstream region of P_E. (A) In the region upstream of P_E, five sets of 4-bp mutations were created by site-directed mutagenesis. (B) *In vivo* GUS assay results for the mutated constructs. In each case, wild-type P_E or the P_E mutations were fused to the *E. coli uidA* gene, creating a translational fusion, and then cloned into the *E. coli*-*Bacteroides* shuttle vector. His₆-tagged RteC, under the control of the heterologous *tetQ* promoter, was cloned into the other *E. coli*-*Bacteroides* shuttle vector, and then the two plasmids were mobilized into *Bacteroides* strain BT4001. The values obtained with wild-type P_E were set to 100%, and the activity of the each P_E mutant is represented as a percentage of the wild-type P_E value. Without RteC, the GUS activity was less than 1 U/mg protein. The experiments were done in triplicate, and the mean values and standard deviations are shown.

More site-directed mutagenesis was done to determine important base pairs adjacent to the 16-bp region from position −51 to −66. To test our hypothesis that RteC may bind the two inverted-repeat half sites and that the −33 region may be important for activity because it is part of the P_E promoter, four different sets of mutations were made within the upstream region of P_E. One inverted-repeat half-site mutation from position −43 to −49, the other inverted-repeat half-site mutation from position −53 to −57, and the −33 region were changed by site-directed mutagenesis (Fig. 4A). The region from position −35 to −38, called the −33, region was based on sequence similarity with other Bacteroides promoter regions (2). In Bacteroides, there are conserved −7 and −33 regions which are analogous to the −10 and −35 regions in E. coli (2). The 20-bp region from position −45 to −65 relative to the orf2c transcriptional start site was chosen since mutation of 4 bp from position −67 to −70 did not affect the activity (Fig. 4A). Changes in the two inverted-repeat half-site mutations were transition mutations to destroy the inverted-repeat sequence. The 4-bp mutation from position −35 to −38, the −33 region, and the mutations from position −45 to −65 were to complementary sequences to conserve the melting temperature characteristics. When the one inverted-repeat half site from position −43 to −49 was mutated, the activity was decreased about 2-fold compared to that of the wild-type control (Fig. 4B). The other inverted-repeat half-site mutation from position −53 to −57 showed about 12-fold-decreased activity (Fig. 4B). With the mutated −45 to −65 region, the activity was decreased almost to the background level (Fig. 4B). The activity was decreased about 10-fold in the case of the −33 region mutation (Fig. 4B). This result suggested that both inverted-repeat half sites are important for RteC binding.

FIG. 4. — Site-directed mutagenesis of the upstream region of P_E. (A) The region between positions −35 and −65 relative to the *orf2c* transcriptional start site, which contained the inverted half sites and the −33 promoter region, was subject to change. The inverted half sites are indicted by solid-line boxes. The dashed-line box shows the 20-bp region initially targeted. The −33 promoter region is underlined. Four different mutated P_E regions were created by site-directed mutagenesis. Changes in positions −46 to −65 and −35 to −38 were to complementary sequences. The other two P_E mutations, from position −43 to −49 and from position −53 to −57, were transition mutations to eliminate the inverted-repeat sequence. (B) *In vivo* GUS assay results. In each case, wild-type P_E or the P_E mutations were fused to the *E. coli uidA* gene, creating translational fusions, and then cloned into the *E. coli*-*Bacteroides* shuttle vector. His₆-tagged RteC under the control of the heterologous *tetQ* promoter was cloned into the other *E. coli*-*Bacteroides* shuttle vector, and then the two plasmids were mobilized into *Bacteroides* strain BT4001. The values obtained with wild-type P_E were set to 100%, and the activity of the each P_E mutant is represented as a percentage of the wild-type P_E value. Without RteC, the GUS activity was less than 1 U/mg protein. The experiments were done in triplicate, and the mean values and standard deviations are shown.

Effect of P_E region mutations on binding of RteC to DNA.

Our results supported the hypothesis that RteC is a DNA binding protein that binds the upstream region of P_E and activates the expression of excision genes. To confirm that RteC binds DNA and recognizes the inverted-repeat sequence within the upstream region of P_E, gel shift assays were done. Partially purified empty vector (no RteC) was used as a negative control (Fig. 5B, lanes 2, 5, 8, and 11). In the gel shift assays, wild-type RteC binding to wild-type P_E resulted in a supershift (Fig. 5B, lane 3). However, when either inverted-repeat half site or the entire inverted-repeat sequence was disrupted, the supershift no longer occurred (Fig. 5B, lanes 6, 9, and 12). This result showed that RteC can bind DNA in the P_E region and confirmed that both inverted-repeat half sites are important for RteC binding.

FIG. 5. — Effects of P_E mutation on RteC binding. (A) The sequences of three different regions of P_E are shown in boxes. The inverted half sites are indicated by solid-line boxes. The dashed-line box shows the 20-bp region initially targeted. Sequences from position −43 to −49 and from position −53 to −57 relative to the *orf2c* transcriptional start site were changed to disrupt each inverted-repeat half site. The mutations were transition mutations. Sequences from position −46 to −65 were changed to the complementary sequence to disrupt the inverted-repeat sequence. (B) Gel shift analysis was done to test binding of RteC to wild-type and mutated upstream regions of P_E. Reaction conditions were as described in the Materials and Methods section. Lanes 1, 4, 7, and 10, labeled DNA without any protein; lanes 2, 5, 8, and 11, binding mix containing 5 μg of partially purified empty vector (pET28); lanes 3, 6, 9, and 12, binding mix containing 10 μg of partially purified RteC. The supershifted protein-DNA complex is indicated by the arrow. The other shifted band comes from an unknown component of the partially purified RteC preparation. The gel is a representative of three independent experiments.

DISCUSSION

RteC does not have primary amino acid sequence homology to any known proteins in the databases. To get past this issue, we used the protein structure prediction program Robetta (10) to predict the structure of RteC in the hopes of finding other proteins with structural homology. We chose Robetta to predict the structure of RteC because it can generate a model for an entire sequence without sequence homology to proteins of known structure (5, 10). Previously, we used the PSIPRED protein structure prediction server (13). The secondary structure prediction showed that RteC might contain many α coils and β sheets, but no clear motifs stood out. Thus, the Robetta program (10) has been the most informative. Based on the Robetta prediction, we found four structural homologs of RteC, and all were involved in transcriptional regulation. Those homologs were E2F-4 (29) and DP2 (29), the E. coli LexA repressor (7), and the E. coli arginine regulator ArgR (11). These proteins all contain winged helix motifs in their DNA binding domains. Guided by this, we could see that RteC also appears to have such a motif. The pI of RteC was predicted to be 6.9, and the putative structural homologs of RteC, human transcriptional factor E2F-4 (29) and DP2 (29), and E. coli LexA (7) also exhibited neutral pI values. However, the DNA binding domains of these proteins have higher pI values. In RteC, the predicted pI of the C-terminal domain is 9.1. This suggested that RteC amino acid residues 111 to 217 might be involved in DNA binding. All four structural homologs of RteC work as dimers, and electrophoretic analysis under native condition confirmed that RteC forms a tetramer (Fig. 6B, lane 2). Since E2F-4 (29) and DP2 (29) were the closest structural homologs of RteC, based on the Robetta prediction, these two transcription factors were chosen for comparison to RteC. The putative secondary structures of RteC DNA binding domains were obtained by aligning DNA binding domains of E2F-4 and DP2 with the C-terminal domain of RteC. Since helix 3 works as a recognition helix in most winged helix proteins (8), the putative helix 3 of RteC was subjected to alanine substitutions. Western blot analysis confirmed that RteC mutants were stable and expressed at the same level as the wild type.

FIG. 6. — Electrophoretic analysis of RteC. (A) Electrophoresis under denaturing conditions. Lane 1, partially purified RteC on SDS gel; lane 2, Western blot analysis of RteC with anti-His antibody. RteC monomer (26 kDa) is indicated by the arrow. (B) Electrophoresis under native conditions. Lane 1, partially purified RteC on native gel; lane 2, Western blot of RteC with anti-His antibody. The location of RteC monomer (26 kDa) is indicated by the filled arrow. The open arrow indicates RteC tetramer (104 kDa).

Based on sequence alignment by ClustalW (25), three amino acid residues in the putative helix 3, K174, R178, and Y180, are fully conserved. Both E2F-4 (29) and DP2 (29) use a conserved Arg-Arg-XXX-Tyr-Asp motif to contact half of the palindromic sequence CGCGCG. (29). In E2F-4 and DP2, three amino acid residues in an RRXYD motif, two arginines and a tyrosine, directly contact the DNA (29), and both R178 and Y180 in the putative RteC helix 3 were aligned with two of these three residues that are responsible for DNA contact. The alanine mutation at D175 resulted in about 10-fold-decreased activity. In E2F-4 and DP2, the aspartate in RRXYD motif makes charged hydrogen bonds to both arginines and appears to stabilize this arrangement (29). Both aspartates in the RteC putative helix3 might stabilize the other amino acids that contact DNA bases, and this might explain the more than 10-fold-decreased activity. Taken together, our results support the hypothesis that RteC is a winged helix-type DNA binding protein and that the predicted DNA binding region, helix 3, is important for binding.

Preliminary evidence suggested that RteC might bind somewhere in the 20 bp upstream of P_E (positions −51 to −70). To narrow down the region that is important for activity, we made five sets of 4-bp mutations within this 20-bp region and found the mutation of the first 4 bp (positions −67 to −70) did not have any effect (Fig. 3B). Since changing the entire 20 bp resulted in an 8-fold decrease in activity but did not decrease activity to the background level, we hypothesized that there might be another region important for activity. A closer look at upstream region of P_E revealed an inverted-repeat sequence, one half of which overlapped the 20-bp regions (positions −51 to −70). We made four different sets of mutations in the upstream region of P_E (Fig. 4A). The activity was decreased when each inverted-repeat half site was mutated, and it decreased to the background level when the entire inverted repeat was disrupted. Mutation of the −33 region resulted in 10-fold-decreased activity, as expected if this region is part of the promoter (Fig. 4B). These results suggest that RteC may recognize and bind two inverted-repeat half sites and that the −33 region is also important for full activity. In vitro binding of RteC to this region, as indicated by the ability to shift the DNA, confirmed this result and added further support to the hypothesis that RteC is a DNA binding protein. The partially purified RteC contained nonspecific binding proteins that were observed in gel shift assays. Therefore, we decided to do mutagenesis analysis instead of in vitro footprint assays.

In summary, we have confirmed that RteC is a DNA binding protein and have identified important amino acid residues that might be involved in DNA binding. A possible DNA target sequence on the DNA was also localized.

Acknowledgments

This work was supported by a grant (AI/GM 22383) from the National Institutes of Health.

Footnotes

^▿

Published ahead of print on 29 October 2010.

REFERENCES

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23.Stevens, A. M., N. B. Shoemaker, and A. A. Salyers. 1990. The region of a Bacteroides conjugal chromosomal tetracycline resistance element which is responsible for production of plasmidlike forms from unlinked chromosomal DNA might also be involved in transfer of the element. J. Bacteriol. 172:4271-4279. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Stevens, A. M., N. B. Shoemaker, L. Y. Li, and A. A. Salyers. 1993. Tetracycline regulation of genes on Bacteroides conjugative transposons. J. Bacteriol. 175:6134-6141. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
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27.Wang, Y., N. B. Shoemaker, and A. A. Salyers. 2004. Regulation of a Bacteroides operon that controls excision and transfer of the conjugative transposon CTnDOT. J. Bacteriol. 186:2548-2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Whittle, G., N. B. Shoemaker, and A. A. Salyers. 2002. The role of Bacteroides conjugative transposons in the dissemination of antibiotic resistance genes. Cell Mol. Life Sci. 59:2044-2054. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Zheng, N., E. Fraenkel, C. O. Pabo, and N. P. Pavletich. 1999. Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP. Genes Dev. 13:666-674. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r1] 1.Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r2] 2.Bayley, D. P., E. R. Rocha, and C. J. Smith. 2000. Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure. FEMS Microbiol. Lett. 193:149-154. [DOI] [PubMed] [Google Scholar]

[r3] 3.Cheng, Q., B. J. Paszkiet, N. B. Shoemaker, J. F. Gardner, and A. A. Salyers. 2000. Integration and excision of a Bacteroides conjugative transposon, CTnDOT. J. Bacteriol. 182:4035-4043. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r4] 4.Cheng, Q., Y. Sutanto, N. B. Shoemaker, J. F. Gardner, and A. A. Salyers. 2001. Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon. Mol. Microbiol. 41:625-632. [DOI] [PubMed] [Google Scholar]

[r5] 5.Chivian, D., D. E. Kim, L. Malmstrom, P. Bradley, T. Robertson, P. Murphy, C. E. Strauss, R. Bonneau, C. A. Rohl, and D. Baker. 2003. Automated prediction of CASP-5 structures using the Robetta server. Proteins 53(Suppl. 6):524-533. [DOI] [PubMed] [Google Scholar]

[r6] 6.Feldhaus, M. J., V. Hwa, Q. Cheng, and A. A. Salyers. 1991. Use of an Escherichia coli beta-glucuronidase gene as a reporter gene for investigation of Bacteroides promoters. J. Bacteriol. 173:4540-4543. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r7] 7.Fogh, R. H., G. Ottleben, H. Ruterjans, M. Schnarr, R. Boelens, and R. Kaptein. 1994. Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy. EMBO J. 13:3936-3944. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r8] 8.Gajiwala, K. S., and S. K. Burley. 2000. Winged helix proteins. Curr. Opin. Struct. Biol. 10:110-116. [DOI] [PubMed] [Google Scholar]

[r9] 9.Gardner, R. G., J. B. Russell, D. B. Wilson, G. R. Wang, and N. B. Shoemaker. 1996. Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-d-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4. Appl. Environ. Microbiol. 62:196-202. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r10] 10.Kim, D. E., D. Chivian, and D. Baker. 2004. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32:W526-W531. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.Larsen, R., G. Buist, O. P. Kuipers, and J. Kok. 2004. ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis. J. Bacteriol. 186:1147-1157. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. [PubMed] [Google Scholar]

[r13] 13.McGuffin, L. J., K. Bryson, and D. T. Jones. 2000. The PSIPRED protein structure prediction server. Bioinformatics 16:404-405. [DOI] [PubMed] [Google Scholar]

[r14] 14.Moon, K., N. B. Shoemaker, J. F. Gardner, and A. A. Salyers. 2005. Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT. J. Bacteriol. 187:5732-5741. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r15] 15.Moore, W. E., and L. V. Holdeman. 1974. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl. Microbiol. 27:961-979. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r16] 16.Salyers, A. A., A. Gupta, and Y. Wang. 2004. Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol. 12:412-416. [DOI] [PubMed] [Google Scholar]

[r17] 17.Salyers, A. A., N. B. Shoemaker, and L. Y. Li. 1995. In the driver's seat: the Bacteroides conjugative transposons and the elements they mobilize. J. Bacteriol. 177:5727-5731. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r18] 18.Salyers, A. A., N. B. Shoemaker, A. M. Stevens, and L. Y. Li. 1995. Conjugative transposons: an unusual and diverse set of integrated gene transfer elements. Microbiol. Rev. 59:579-590. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Salyers, A. A., N. B. Shoemaker, A. Cooper, J. D'Elia, and J. A. Shipman. 1999. Genetic methods for Bacteroides species. Methods Microbiol. 29:229-249. [Google Scholar]

[r20] 20.Salyers, A. A., G. Whittle, and N. B. Shoemaker. 2004. Conjugative and mobilizable transposons, p. 125-143. In R. V. Miller and M. J. Day (ed.), Microbial evolution: gene establishment, survival, and exchange. ASM Press, Washington, DC.

[r21] 21.Shoemaker, N. B., and A. A. Salyers. 1987. Facilitated transfer of IncP beta R751 derivatives from the chromosome of Bacteroides uniformis to Escherichia coli recipients by a conjugative Bacteroides tetracycline resistance element. J. Bacteriol. 169:3160-3167. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Shoemaker, N. B., H. Vlamakis, K. Hayes, and A. A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl. Environ. Microbiol. 67:561-568. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r23] 23.Stevens, A. M., N. B. Shoemaker, and A. A. Salyers. 1990. The region of a Bacteroides conjugal chromosomal tetracycline resistance element which is responsible for production of plasmidlike forms from unlinked chromosomal DNA might also be involved in transfer of the element. J. Bacteriol. 172:4271-4279. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r24] 24.Stevens, A. M., N. B. Shoemaker, L. Y. Li, and A. A. Salyers. 1993. Tetracycline regulation of genes on Bacteroides conjugative transposons. J. Bacteriol. 175:6134-6141. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r25] 25.Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r26] 26.Wang, Y., E. R. Rotman, N. B. Shoemaker, and A. A. Salyers. 2005. Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT. J. Bacteriol. 187:2673-2680. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r27] 27.Wang, Y., N. B. Shoemaker, and A. A. Salyers. 2004. Regulation of a Bacteroides operon that controls excision and transfer of the conjugative transposon CTnDOT. J. Bacteriol. 186:2548-2557. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r28] 28.Whittle, G., N. B. Shoemaker, and A. A. Salyers. 2002. The role of Bacteroides conjugative transposons in the dissemination of antibiotic resistance genes. Cell Mol. Life Sci. 59:2044-2054. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r29] 29.Zheng, N., E. Fraenkel, C. O. Pabo, and N. P. Pavletich. 1999. Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP. Genes Dev. 13:666-674. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Characterization of the Bacteroides CTnDOT Regulatory Protein RteC^▿

Jiyeon Park

Abigail A Salyers

Abstract