Transcriptional regulation of MG_149, an osmoinducible lipoprotein gene from Mycoplasma genitalium

Abstract

Transcriptional regulation remains poorly understood in Mycoplasma genitalium, the smallest self-replicating cell and the causative agent of a spectrum of urogenital diseases. Previously, we reported that MG_149, a lipoprotein-encoding gene, was highly induced under physiological hyperosmolarity conditions. In this study we further analysed MG_149 transcription with a focus on the identification of promoter elements and regulatory mechanisms. We established MG_149 as a genuine osmoinducible gene that exhibited the highest transcript abundance compared with other lipoprotein genes. Using genetic approaches, we demonstrated that the -10 region of the MG_149 promoter was essential for osmoinduction. Moreover, we showed that MG_149 osmoinduction was regulated by DNA supercoiling, as the presence of novobiocin decreased MG_149 expression in a dose-dependent manner. Taken together, these results indicate that DNA supercoiling participates in controlling MG_149 expression during in vivo-like conditions.

Figure 1

Induction of MG_149 by hyperosmolarity conditions. M. genitalium cells were grown to exponential phase and stressed with NaCl for 1 h prior to RNA isolation. Northern blot analysis was performed to determine the relative levels of MG_149 transcripts. For each sample, 3 μg of total RNA were loaded and separated on 1% agarose gel by electrophoresis. Ribosomal RNA (rRNA) stained with ethidium bromide served as loading control. After capillary transfer, nylon membranes were hybridized with ³²P-labeled MG_149 probe generated as described in Experimental procedures. A). Induction of MG_149 by various concentrations of NaCl (indicated on top). B). Reversibility of MG_149 induction in the absence of osmotic stress. Medium swap, stressed M. genitalium cells were returned to medium without NaCl addition and the growth was continued for 1 h.

Figure 2

Abundance of MG_149 transcripts compared with other osmoinducible lipoprotein genes. M. genitalium cells were grown to exponential phase and treated with or without 0.2 M NaCl for 1 h prior to RNA isolation. cDNAs were prepared with 1.5 μg of total RNA using 1 μl of a mixture containing reverse primers from six lipoprotein genes (2 μM each). cDNA templates were diluted 50 X, and the quantities of transcripts for these selected genes were measured by qRT-PCR as described in Experimental procedures. Values represent the means ± standard error (SE) from two independent experiments assayed in triplicate. A statistically significant difference in the expression between control and NaCl treated cultures was observed for all five genes (P<0.001, two-tailed Student t test).

Figure 3

Determination of MG_149 transcriptional start site (TSS). The TSS was determined with PE and 5′ RACE. Total RNAs were isolated from M. genitalium grown in the presence or absence of 0.2 M NaCl for 1 h. Shown is the separation of PE products (arrows) on 6% sequencing gel alongside DNA ladders (A, T, C and G) generated with the same PE primer. Due to 5′ phosphorylation of the radiolabelled PE primer, the mobility of PE products is shifted about 1 nt downward relative to the sequencing ladder. The TSS +1, the predicted -10 and -35 regions, and the start codon of MG_149 are bolded and marked. PE1, M. genitalium treated with 0.2 M NaCl; PE2, control culture.

Figure 4

Generation of M. genitalium mutants with truncation/modification in MG_149 promoter region. A). Schematic representation of the strategy for generation of promoter truncation/modification mutants by homologous recombination. Short bars represent flanking primers (PDF5′ and PDF3′) used for confirming mutants by PCR amplification. B). PCR confirmation of mutants. All mutants exhibited products ~2 kb larger than that of the wild-type strain because of tetM438 insertion. A representative gel image is shown. WT, wild type; PD2, mutant.

Figure 5

Deletion analysis of MG_149 promoter. Exponential phase cultures of wild type (WT) strain and MG_149 promoter deletion mutants (PD1 to PD6) were grown in the presence or absence of 0.2 M NaCl for 1 h. Northern blot analysis was performed to determine the relative levels of MG_149 transcripts as described in Fig. 1. PD1 contains the full intergenic region upstream of MG_149. The sequences from tetM438 are shown in italicized lowercase. The EcoRI cleavage sites (GAATTC) are also highlighted. The additional lower molecular weight bands below the major one, prominently observed in PD3 mutant, could be degradation products of MG_149 mRNA produced during RNA isolation.

Figure 6

Mutation analysis of MG_149 promoter. Exponential phase cultures of M. genitalium wild-type strain (WT) and mutants with site mutation(s) in -10 or -35 regions of MG_149 promoter were grown in the presence or absence of 0.2 M NaCl for 1 h. Northern blot analysis was performed to determine the relative levels MG_149 transcripts as described in Fig. 1. Site mutation(s) introduced in each mutant are bolded.

Figure 7

Analysis of the -10 region of MG_149 promoter by transcriptional fusion. Two M. genitalium mutants were generated with the 100 bp sequence upstream MG_186 being removed (ΔP) or replaced with the -10 region of MG_149 promoter (_-10). Wild-type (WT) strain and mutants were grown in the presence or absence of 0.2 M NaCl for 1 h, and MG_186 transcript levels were determined by qRT-PCR. 16S rRNA was used as normalizer. Values represent the means ± SE from three biological repeats assayed in triplicate. A statistically significant difference in expression of MG_186 between control and NaCl treated cultures was observed for WT and (_-10) mutant (P<0.001, two-tailed Student t test).

Figure 8

Regulation of MG_149 by DNA supercoiling. A). Novobiocin reduces the osmoinduction of MG_149. Exponential phase M. genitalium cells were treated as indicated (top) for 1 h. Northern blot analysis was performed to determine the relative levels of MG_149 transcripts as described in Fig. 1. B). Expression kinetics of MG_149 and MG_122. M. genitalium cells were grown to exponential phase and treated with 0.2 M NaCl (NaCl) or 0.2 M NaCl plus 8 μg ml⁻¹ novobiocin (NaCl + Nov). Cultures were grown for 20, 40 and 60 min, and RNAs were harvested at each time point for analysis. Transcript levels of each gene were measured with qRT-PCR using 16S rRNA as normalizer. Values represent the means ± SE from two independent experiments assayed in triplicate. The addition of novobiocin reduced the osmoinduction of MG_149 (P ≤ 0.001, two tailed Student t test) and inhibited the expression of MG_122 (P < 0.001). C). Cultures of PD4 mutant were treated with 0.2 M NaCl or 0.2 M NaCl plus novobiocin as indicated. Northern blot analysis was performed as described above.

Figure 9

Discriminator region (DR) is not responsible for the osmoinduction of MG_149. Exponential phase cultures of M. genitalium wild-type strain (WT) and mutant with site mutations in the DR (DR_m) were treated with 0.2 M NaCl and novobiocin as indicated for 1 h. Northern blot analysis was performed to determine the relative levels of MG_149 transcripts. The DR is bracketed and the position of site mutations introduced in DRm is bolded.