pseudotuberculosis YpIII strain, RT-PCR (using an sraG-specific oligo in reverse transcription) was used to examine SraG RNA level at different growth phases. As shown in Fig. 1, compared with the expression patterns in
the sraG deletion mutant and the SraG complementing strains, the transcription levels of SraG are invariable under all tested growth stages. A secondary structure of SraG was predicted (Fig. S1) by RNAstructure software (Reuter & Mathews, 2010). To investigate the targets of SraG, we next performed 2D gel analysis to compare the whole-cell protein patterns of WT with ΔsraG from cultures grown to exponential phase. Expressions of 16 proteins having more than 1.5-fold changes between ΔsraG and WT (Table 1 and HSP assay Fig. S2). Among these proteins, seven were down-regulated and nine were up-regulated. We next performed semi-quantitative RT-PCR to compare the mRNA levels of these candidate targets. Among these potential targets, only pnp and YPK_1205 (encoding an hypothetical protein) showed significantly different mRNA levels (Fig. S3). To confirm the different expression level of YPK_1205 in WT and ΔsraG (Fig. 2a), we constructed a single-copy translational fusion of YPK_1205 with lacZ (named 1205zST). β-Galactosidase activities
were tested when isogenic strains were grown to mid-log phase. Expression of 1205zST in the ΔsraG strain was 2.6-fold higher than that in WT (Fig. 2b). Western blotting also confirmed higher YPK_1205 protein level in ΔsraG (Fig. 2c). Mitomycin C mw To further confirm that YPK_1205 mRNA is negatively regulated by SraG, we next performed RT-PCR and observed higher levels of YPK_1205 transcript (based on cDNA level generated by reverse transcription from total RNA) in ΔsraG than in either WT or the SraG complemented strain (Fig. 2d). These results are consistent with the result observed by 2D gel analysis and indicate that SraG negatively regulates YPK_1205 mRNA. The YPK_1205 gene is located downstream of YPK_1206. Inverse RT-PCR was used to examine whether YPK_1206 and YPK_1205 were cotranscribed (there is a 57-bp intergenic
region between them, Fig. 3a). As shown in Fig. 3(b), a region including both the YPK_1206 and YPK_1205 fragment was amplified from one cDNA template, these indicating that the two genes are indeed in one operon. Similar experiments confirmed that YPK_1206-1205 is not cotranscribed with YPK_1204 or YPK_1207 (data not shown). The next question was whether expression of YPK_1206 is also regulated by SraG. We therefore constructed a translational fusion of YPK_1206 with lacZ (1206zST), which was transconjugated into both WT and ΔsraG. Expression of 1206zST was 1.6-fold higher in ΔsraG than in WT (Fig. 3c, columns 1 and 2), indicating that YPK_1206 expression is also negatively regulated by SraG. RT-PCR was used to determine the YPK_1206 mRNA level in WT, ΔsraG and the complementary strains.