Transcription antitermination by a phosphorylated response regulator and cobalamin-dependent termination at a B₁₂ riboswitch contribute to ethanolamine utilization in Enterococcus faecalis

Abstract

The ability of bacteria to utilize ethanolamine (EA) as a carbon and nitrogen source may confer an advantage for survival, colonization, and pathogenicity in the human intestinal tract. Enterococcus faecalis, a Gram-positive human commensal organism, depends on a two-component signaling system (TCS-17) for sensing EA and regulating the expression of the ethanolamine utilization genes. Multiple promoters participate in eut gene expression in the presence of EA as the sole carbon source and cobalamin (CoB12), an essential cofactor in the enzymatic degradation process. By means of in vivo and in vitro approaches, this study characterized the transcriptional activity identified in the eutT-eutG intergenic region of the E. faecalis eut cluster. Two novel promoters in this region were shown to be active in vivo. The distal P2-1 promoter was associated with a B12 riboswitch that terminated transcription in the presence of CoB12. Transcription elongation from the proximal P2-2 promoter was regulated by antitermination mediated by the phosphorylated form of the response regulator of TCS-17 (RR17). 3'-Rapid amplification of cDNA ends (RACE) analyses of the terminated RNA products allowed precise identification of the hairpin loop structures involved in termination/antitermination. The results uncovered the role of the B12 riboswitch and RR17 in eut gene expression, adding to the complexity of this regulatory pathway and extending the knowledge of possible means of transcription regulation in Gram-positive organisms.

Fig. 1.

Schematic representation of the eut operon in E. faecalis with a magnified view of the eutT-eutG intergenic region. RR and HK indicate the rr17 and hk17 genes (aka eutV and eutW, respectively [15]) encoding the response regulator and histidine kinase, respectively. Shown is the approximate location of the B12 riboswitch, followed by the distal terminator loop, the promoters (bent arrows), and the antiterminator-terminator loops preceding the eutG gene. The contents of the fragments cloned in plasmid pTCVlacSpec and used as templates in the transcription analyses are also illustrated by the lines.

Fig. 2.

Transcription analysis of promoter P2. Time courses of β-galactosidase activity were carried out on E. faecalis V583 carrying the pP2 plasmid. M9HY medium was supplemented with 100 mM EA and 40 nM CoB12 (▪), 100 mM EA (), 40 nM CoB12 (•), or 100 mM glucose, 100 mM EA, and 40 nM CoB12 (▴). A representative growth curve in medium containing EA and CoB12 is shown (□), and the OD₅₂₅ is indicated on the right axis.

Fig. 3.

Transcription analysis of promoter P2-1. (A, C, and D) Time courses of β-galactosidase activity were carried out on E. faecalis V583 carrying plasmids pSP2 (A; squares), pSP3 (C; triangles), and pSP4 (D; circles); dashed lines with open symbols represent cultures grown in 100 mM glycerol; solid lines with closed symbols are results for cultures grown in 100 mM glycerol, 100 mM EA, and 40 nM CoB12; and solid lines with open symbols are results for cultures grown in 100 mM glycerol and 40 nM CoB12. Growth cultures and assays whose results are shown in panels A, C, and D were performed at the same time, but the graphs are separated for clarity. A representative growth curve in medium containing glycerol, EA, and CoB12 is shown in panel A with closed circles, and the OD₅₂₅ is indicated on the right axis. (B) Transcription activities from pSP2 in strain V583 compared to those in the rr17 strain. Growth conditions were as follows: 100 mM glycerol, 100 mM EA, and 40 mM CoB12 (circles); 100 mM glycerol and 40 nM CoB12 (triangles); 100 mM glycerol and 100 mM EA (diamonds); 100 mM glycerol (squares); solid lines with closed symbols represent results for V583; dashed lines with open symbols represent results for the rr17 strain.

Fig. 4.

Transcription analysis of promoter P2-2. (A to C) Time courses of β-galactosidase activity were carried out for E. faecalis strains carrying plasmids pSP (A; squares) and pSP6 (B; circles). Panels A and B show the results obtained with the parental strain V583, and panel C shows the results obtained with the rr17 mutant strain. Dashed lines with open symbols are results for cultures grown in 100 mM glycerol; solid lines with closed symbols are results for cultures grown in 100 mM glycerol, 100 mM EA, and 40 nM CoB12; solid lines with open symbols are results for cultures grown in 100 mM glycerol and 40 nM CoB12. The assays for both strains were performed at the same time, but the graphs are separated for clarity. A representative growth curve in medium containing glycerol, EA, and CoB12 is shown in panel A with closed circles, and the OD₅₂₅ is indicated on the right axis. (D) Results of transcription activity from plasmid pSP carried by the hk17 mutant strain; solid line with closed symbols shows the results for V583; dashed line with open symbols shows the results for the hk17 strain.

Fig. 5.

In vitro termination assay with CoB12. In vitro transcription assays were carried out on the SP4, SP2, P2, and SP3 templates. The results shown were obtained after incubation of the reaction mixtures for 15 min. RNA products: full-length runoff (★); terminated (); CoB12-induced pause (▴); pause product (▾); pause in SP4 and P2 reactions (); RNA from P2-2 (); RNA from P2-1 to terminator in front of eutG (). The open horizontal arrow indicates the ∼500-base product in the P2 reaction mixture that is likely to be the full-length runoff product. The gel with the 120-min reaction products showed essentially identical results (data not shown).

Fig. 6.

Identification of the 3′-end nucleotides of in vitro transcription products from P2-1. The RNA products of the in vitro transcription reactions carried out on the SP4 fragment containing the P2-1 promoter (see Fig. 5) were purified from the gel and used in 3′-RACE reactions as described in Materials and Methods. Underlined nucleotides in the sequencing graphs indicate the dRR17p RNA adapter sequence. Symbols refer to the symbols used in Fig. 5. (A) Runoff RNA (★); shown are nucleotides −168 to −139 upstream from eutG. (B) Terminated RNA (); shown are nucleotides −327 to −295 upstream from eutG. (C) CoB12-induced paused RNA (▴); shown are nucleotides −365 to −333 upstream from eutG. (D) Paused RNA (▾); shown are nucleotides −377 to −345 upstream from eutG. (E) Representation of the B12 riboswitch secondary structure based on base pair predictions by Barrick and Breaker (5). (F) Representation of the B12 riboswitch secondary structure model developed by Fox et al. (15); the boxed hairpin loop is the B12 riboswitch terminator proposed in this study. In panels E and F, red nucleotides indicate the location of the 3′-end nucleotides from panels B to D, and blue nucleotides indicate potential base-paired sequences during termination.

Fig. 7.

In vitro transcription assay of promoter P2-2 in the presence of RR17 and HK17. (A) Reactions were carried out on the SP template containing the P2-2 promoter. Shown are the products of the reaction mixtures incubated for 15 min. RNA products: runoff (RO; ★); terminated (T; ). The gel with the products of the 120-min reactions showed essentially identical results (data not shown). (B) Quantification of RNA products from the in vitro termination assay shown in panel A. The percentages of RNA present in the RO and T bands were calculated as the fraction of the sum of the pixels of the RO and T bands from each reaction (4). Because the products are essentially end labeled (see Materials and Methods), the length of the transcripts is uninfluential in this calculation (4).

Fig. 8.

Identification of the 3′-end nucleotides of the in vitro RNA transcription products obtained from promoter P2-2. The RNA products of the in vitro reactions carried out on the SP template containing the P2-2 promoter (see Fig. 7A) were purified from the gel and used in 3′-RACE reactions as described in Materials and Methods. Underlined nucleotides in the sequencing graphs indicate the dRR17p RNA adapter sequence. Symbols refer to the RNA products in Fig. 7A. (A) Runoff RNA (★); shown are nucleotides +78 to +110 downstream from the translational start of eutG. (B) Terminated RNA (); shown are nucleotides −55 to −23 upstream from eutG. (C) Potential weak antiterminator hairpin. (D) Terminator hairpin. The structures in panels C and D are located upstream from eutG and were predicted by RibEx (1). The energy of the antiterminator and terminator hairpin is expressed in kcal/mol. Nucleotides in blue are shared by the antiterminator and terminator structures. Nucleotides in green are the proposed recognition sequence for RR17.