Identification and Characterization of the Alternative σ28 Factor in Treponema denticola

Abstract

FliA (also known as σ²⁸), a member of the bacterial σ⁷⁰ family of transcription factors, directs RNA polymerase to flagellar late (class 3) promoters and initiates transcription. FliA has been studied in several bacteria, yet its role in spirochetes has not been established. In this report, we identify and functionally characterize a FliA homolog (TDE2683) in the oral spirochete Treponema denticola. Computational, genetic, and biochemical analyses demonstrated that TDE2683 has a structure similar to that of the σ²⁸ of Escherichia coli, binds to σ²⁸-dependent promoters, and can functionally replace the σ²⁸ of E. coli. However, unlike its counterparts from other bacteria, TDE2683 cannot be deleted, suggesting its essential role in the survival of T. denticola. In vitro site-directed mutagenesis revealed that E221 and V231, two conserved residues in the σ₄ region of σ²⁸, are indispensable for the binding activity of TDE2683 to the σ²⁸-dependent promoter. We then mutated these two residues in T. denticola and found that the mutations impair the expression of flagellin and chemotaxis genes and bacterial motility as well. Cryo-electron tomography analysis further revealed that the mutations disrupt the flagellar symmetry (i.e., number and placement) of T. denticola. Collectively, these results indicate that TDE2683 is a σ²⁸ transcription factor that regulates the class 3 gene expression and controls the flagellar symmetry of T. denticola. To the best of our knowledge, this is the first report establishing the functionality of FliA in spirochetes. IMPORTANCE Spirochetes are a group of medically important but understudied bacteria. One of the unique aspects of spirochetes is that they have periplasmic flagella (PF, also known as endoflagella) which give rise to their unique spiral shape and distinct swimming behaviors and play a critical role in the pathophysiology of spirochetes. PF are structurally similar to external flagella, but the underpinning mechanism that regulates PF biosynthesis and assembly remains largely unknown. By using the oral spirochete Treponema denticola as a model, this report provides several lines of evidence that FliA, a σ²⁸ transcriptional factor, regulates the late flagellin gene (class 3) expression, PF assembly, and flagellar symmetry as well, which provides insights into flagellar regulation and opens an avenue to investigate the role of σ²⁸ in spirochetes.

Keywords: Treponema; flagella; motility; sigma factors; spirochetes.

FIG 1

Schematic illustration of σs in T. denticola. These factors are annotated in the genome of T. denticola (42). Individual domains in these σ factors were identified by BLAST (Fig. S3) and SMART searches and then aligned to their counterparts (i.e., σ⁷⁰, σ²⁸, and σ⁵⁴) from E. coli and other bacteria (1, 4–6, 8, 10, 90, 91).

FIG 2

TDE2683 is located at a large gene cluster regulated by a σ⁷⁰ promoter. (A) Diagram showing the genes adjacent to TDE2683. Arrows represent the relative positions and orientations of RT-PCR primers that span the intergenic regions between individual ORFs. (B) RT-PCR analysis. For each pair of primers, chromosomal DNA was used as a positive control. The numbers below the primers are predicted sizes of RT-PCR and PCR products. (C) 5′ RACE analysis. The arrow shows the sequencing direction, and an asterisk indicates the transcriptional start site. (D) Sequence comparison between the E. coli σ⁷⁰ promoter and the promoter sequence identified upstream of TDE2687 (designated P_TDE2687).

FIG 3

TDE2683 is a homolog of FliA. (A) Sequence alignment of FliA homologs. TDE2683 (WP_002667112) is aligned with E. coli FliA (NP_416432), S. Typhimurium FliA (NP_460909), and B. subtilis SigD (WP_187704716). Only partial alignment is shown. Asterisks represent three invariable residues in the family of FliA. The residues in red are to be mutated. The alignment was analyzed using Clustal Omega. (B) Homology modeling shows that TDE2683 (FliA_Td) has structural topology similar to that of the FliA proteins (σ²⁸) of E. coli and S. Typhimurium. The figure was generated by the Swiss-Model server using FliA_Ec (PDB ID 1SC5) (8) as a template.

FIG 4

FliA_Td substitutes for the function of FliA_Ec in E. coli. (A) Detection of FliA_Td in E. coli by immunoblotting analysis. For this experiment, equivalent amounts (~10 μg) of whole-cell lysates of three E. coli strains were analyzed by SDS-PAGE and then probed with specific antibodies against E. coli GroEL (αGroEL) and T. denticola FliA (αFliA_Td). GroEL was used as a loading control. WT, E. coli DH5α; ΔfliA_Ec, fliA deletion mutant of E. coli; cfliA_Td, engineered E. coli strain in which the entire fliA_Ec gene was replaced in frame with fliA_Td. (B) Visualization of E. coli flagella. E. coli cells were first stained with Ryu as previously described (82) and then visualized under dark-field illumination at ×100 magnification using a Zeiss Axiostar Plus microscope. (C) Swimming plate assay. This assay was carried out on swimming plates containing 1% tryptone, 1% NaCl, and 0.4% Bacto agar, as previously documented (87). The plates were incubated at 37°C overnight.

FIG 5

EMSA shows that FliA_Td binds to two σ²⁸-dependent promoters. (A) EMSA using E. coli fliC promoter (P_fliC) as a DNA probe. For this assay, biotin-labeled P_fliC (Bio-P_fliC) was incubated with increasing amounts of wild-type FliA_Td recombinant protein (rFliA_Td) and titrated with increasing concentrations of unlabeled P_fliC (Unl-P_fliC). (B to D) EMSA using T. denticola flaB2 promoter (P_flaB2) as a DNA probe. Biotin-labeled P_flaB2 (Bio-P_flaB2) was incubated with increasing amounts of rFliA_Td (B) or two mutated recombinant proteins, rFliA^E221D (C) and rFliA^V231E (D), and competed out with increasing concentrations of unlabeled P_flaB2 (Unl-P_flaB2).

FIG 6

Characterizations of two fliA_Td site-directed mutants of T. denticola. (A) Swimming plate assay of WT, FliA_Td^E221D, and FliA_Td^V231E strains. This assay was carried out on 0.35% agarose plates containing TYGVS medium diluted 1:5 with PBS. The plates were incubated anaerobically at 37°C for 5 days. A nonmotile Δtap1 mutant was used as a control to determine initial inoculum sizes. (B) Immunoblotting analysis of WT, FliA_Td^E221D, and FliA_Td^V231E strains. Equivalent amounts of whole-cell lysates were analyzed by SDS-PAGE and then probed with specific antibodies to DnaK, FlaA, and FlaBs. DnaK was used as a loading control. (C to F) qRT-PCR analysis of WT, FliA_Td^E221D, and FliA_Td^V231E strains. For this experiment, the levels of four flagellar filament genes (flaA, flaB1, flaB2, and flaB3), two chemotaxis genes (cheA and cheY), two flagellar genes (flgE and fliG), TDE2682 (fapA), and TDE1346 (σ⁷⁰) were measured by qRT-PCR, as previously described (33). The dnaK gene transcript was used as an internal control to normalize the readouts of qRT-PCR. The results are expressed as the level of individual gene transcripts in two mutants relative to that in WT. *, P < 0.05.

FIG 7

Whole-cell cryo-ET analysis of T. denticola wild type and two mutants. (A) Projection image of a WT cell; (B to F) projection images of FliA_Td^E221D and FliA_Td^V231E mutant cells. PF originating from one end are colored in yellow, and those from the opposite end are in orange. The boxed images in panel E and F show the detailed structure of PF and flagellar protrusions. PF, periplasmic flagella; OM, outer membrane; IM, inner membrane. Each panel was generated by combining multiple high-resolution cryo-ET images; the image contrast is different among these data.