Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum

Abstract

In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with the green fluorescent protein reporter gene (GFP), and promoter activity was measured both as fluorescence emission from Escherichia coli cultures transformed with the different plasmid constructs and using quantitative RT-PCR. For tprJ, G and E-derived clones, fluorescence was significantly higher with constructs containing eight Gs or fewer, while plasmids containing the same promoters with none or more Gs gave modest or no signal above the background. In contrast, tprF/I-derived clones induced similar levels of fluorescence regardless of the number of Gs within the promoter. GFP mRNA quantification showed that all of the promoters induced measurable transcription of the GFP gene; however, only for Subfamily II promoters was message synthesis inversely correlated to the number of Gs in the construct.

Fig. 1

Schematic representation of the tprJ, tprI, tprG, tprF and tprE loci in the Nichols strain of Treponema pallidum. Location of the TSSs and poly-Gs is indicated along with the position of the primers used for promoter amplification (for promoter sequence see Fig. 2, for primer sequence see Fig. 2 or Table 1). As previously reported (Giacani et al., 2005a) transcriptional analysis of these loci showed that promoters upstream of tprJ and tprG (P.J and P.G in the figure) generate messages encompassing both tprJ/I and tprG/F sequences, while alternative promoters upstream of tprF and tprI (P.F and P.I in the figure) may generate suboperonic messages. The tprE TSS has not been identified yet using in vitro transcription assay or 5′-RACE (rapid amplification of cDNA ends) as for tprJ, I, G and F (Giacani et al., 2005a); therefore, its TSS is hypothetical. However, it has been mapped using RT-PCR analysis within the first 250 nucleotides upstream of the tprE start codon. The 3′ boundaries of these transcription units are still unknown.

Fig. 2

tprE, G, J and F/I promoter sequences cloned into the pGlow-TOPO vector and used for the GFP reporter assay. Regulatory elements are in bold. Underlining indicates the primers used to amplify the promoter regions. Numbers to the right of the homopolymeric repeats represent the number of G residues tested for each tpr promoter. As explained in Fig. 1, the tprE TSS (+1) is hypothetical and based on analogy with the other Subfamily II tprs. +1: transcriptional start site (experimentally determined); RBS: ribosomal binding site (putative); tpr SC: tpr gene start codon; GFP ORF: green fluorescent protein ORF. Amino acids (Lys–Gly–Gln–Phe–Cys–Arg–Ser–Arg) between the tpr putative SC and the GFP ORF start are vector-encoded.

Fig. 3

Distribution of the number of G residues within the homopolymeric repeats located upstream of the tprE (a), tprG (b), tprJ (c), tprF (d) and tprI (e) ORFs. DNA was extracted from treponemal strains harvested from rabbits at peak orchitis and promoter regions amplified (using primers schematically represented in Fig. 1 and reported in Table 1). Twenty clones were sequenced for each tpr for each strain (Nichols, Chicago, Bal 73-1 and Sea 81-4) to analyze the range of variability of the poly-G tracts. For clarity, some symbols were slightly offset to prevent overlap.

Fig. 4

Strength of the tpr promoters containing different numbers of G residues measured with the GFP reporter assay. Data represent fluorescence readings (expressed in arbitrary units, Ar.U) normalized to the OD of the culture after subtraction of background fluorescence. Subfamily II tprE and G promoters containing eight Gs show the highest activity (a, b). No promoters with seven Gs have been identified yet. tprJ promoter with seven Gs induces higher fluorescence than the same promoter with eight Gs (c). Subfamily I tprF/I promoter activity is not influenced by the number of Gs (d). tprF and tprI promoters are identical. Fluorescence signal induced by the lac promoter, always >27 000 Ar.U/OD_{600 nm}, is not shown to allow visualization of sample bars. Means ± SE of quadruplicate values are represented.

Fig. 5

Strength of the tpr promoters containing different numbers of G residues measured by GFP quantitative real-time RT-PCR. This analysis shows for Subfamily II promoters (tprE, tprG, and tprJ) a decrease in the amount of GFP mRNA as the number of G residues increases. Similar low levels of GFP mRNA (note values on y-axis) are induced by Subfamily I promoters (tprF and tprI) regardless of the number of Gs. Values represent the means ± SE of quadruplicate measurements. Where the bars are not visible, the mean values are shown as numbers. Positive control is not shown (value reported) so that tprF/I values could be visualized. Negative and positive controls were RNA extracted from Escherichia coli cultures transformed with a promoterless pGlow-TOPO vector and lac promoter/pGlow-TOPO vector, respectively.

Fig. 6

Real time quantitative RT-PCR analysis of Subfamily II tpr message levels in treponemes harvested at peak orchitis from rabbits experimentally infected with the Nichols, Sea 81-4, Chicago and Bal 73-1 strains of Treponema pallidum. Message levels were correlated with the distribution of G residues within tprE, G and J promoters reported in Fig. 3. Reported values represent the mean ± SE of triplicate assays for each cDNA sample; number of copies of tpr transcript are expressed per 100 copies of the 47 kDa lipoprotein message. The Sea 81-4 tprJ locus harbors a tprG/J hybrid molecule, which required specific primers (Table 1).