## 2 Results
### Sll_4382 and Sll_4383 Amino Acid Sequence Features
The primers are either internal to _ppk_ (A/B, plain black histograms), _pptA_ (E/E, plain white histograms), and _SLI_4382_ (I/J, plain grey histograms) or encompass the _ppk_/_pptA_ junction (C/D, dotted black histograms) or the _pptA_/_SLI_4382_ junction (I/J, dotted grey histograms). **(c)** Determination by RT-PCR of the expression of _pptA_ in _S. lividans_ WT (lane 1) and in the _ppk_ mutant of this strain (lane 2) using primers K and L. Control of DNA amplification using the same primers is shown in lane 3 (**d**) Level of expression of _ppk_ (black histograms), _pptA_ (white histograms), and _SLI_4382_ (grey histograms) determined by RT-qPCR with RNA prepared from _S. coelicolor_, _S. lividans_, and the _phoP_ mutant of _S. lividans_ grown on R2 YE in condition of Pi limitation (1 mM) and proficiency (5 mM). Amplification of fragments, at a similar level, was obtained with primers A and B (internal _ppk_) and primers C and D (_ppk/pptA_ junction) as well as with primers E and F (internal to _pptA_) and primers G and H and primers I and J, corresponding to the _pptA/SLI_4382_ junction and internal to _SLI_4382_, respectively (Figure 2b). These results demonstrated that these three genes constitute an operon. The co-transcription of _ppk_ and _pptA_ led to the prediction that the disruption of the _ppk_ gene by a resistance cassette might have a polar effect on _pptA_ expression. In order to test this hypothesis, RNA was prepared from _S. lividans_ and its _ppk_ mutant grown for 40 h on solid R2 YE medium with no phosphate added (1 mM final, Pi limitation) and primers K and L were used to detect _pptA_ expression. Results shown in Figure 2c indicated that _pptA_ was still expressed in the _ppk_ mutant but at a much lower level than in the _wt_ strain. In consequence, considering the polar effect of the insertion of the hygromycin cassette in _ppk_ on _pptA_ expression, the antibiotic overproducing phenotype of the _ppk_ mutant might be, at least in part, due to the weak expression of _pptA_ in this strain. This residual weak transcription of _pptA_ in the _ppk_ mutant suggested that _pptA_ might also be transcribed alone from its own promoter. In order to test this hypothesis, RACE-PCR experiments were conducted to determine whether a transcriptional start point (TSP) could be detected upstream of the coding sequences of _SLI_4383/pptA_ as well as of _SLI_4382_ (Figure 3a,c). Indeed, a TSP located at a C, 13 nt upstream of the newly proposed GTG start codon of _pptA_, and thus 30 nt upstream of the Ppk TGA stop codon was identified. This allowed the positioning of putative \(-10\) and \(-35\) sequences of this gene at the end of the _ppk_ coding sequence (Figure 3b). Furthermore, a TSP located at a C, 86 nt upstream of the GTG start codon of _SLI_4382_, and thus 37 nt upstream of the _pptA_ TAG stop codon was also identified. This allowed the positioning of putative \(-10\) and \(-35\) sequences of this gene at the end of the _pptA_ coding sequence (Figure 3d). Figure 3: Determination of the transcriptional start point of _SLI_4383/pptA_ and _SLI_4382_ established by 5\({}^{\prime}\) RACE PCR. (**a**,**c**) Chromatograms of 5\({}^{\prime}\) RACE PCR of the _pptA_ and _SLI_4382_ promoter region, respectively. (**b**,**d**) Annotated sequence of the promoter region of _pptA_ and _SLI_4382_, respectively. The bent arrow represents the transcriptional start site, putative \(-10\) and \(-35\) boxes are underlined, the translational start codons (GTG) are in bold letters and underlined, and the translational stop codon of _ppk_ and _SLI_4383_ are in red. The erroneously annotated GTG start codon of PptA in the GenBank entry for the _S. lividans_ genome (GG657756.1) is underlined.