## 2 Results
### The ppk/pptA/SLI_4382 Operon Belongs to the Pho Regulon  
In order to establish the impact of phosphate availability on the transcriptional regulation of the genes of this operon, RNA was prepared from _wt S. lividans_, its _pho_P mutant and _wt S. coelicolor_ grown for 40 h on solid R2 YE medium with no phosphate added (1 mM final, Pi limitation) or with 4 mM phosphate added (5 mM final, Pi proficiency). Results of RT-qPCR shown in Figure 2d confirmed that the expression of these genes was induced in condition of phosphate limitation and repressed in condition of phosphate proficiency in the _wt_ strain of _S. lividans_, whereas the expression of these genes was 20-fold lower in the _pho_P mutant and at a similar level in both Pi conditions (Figure 2d). This demonstrated that these genes belong to the Pho regulon. The expression of these genes was 4 to 5 fold lower in _S. coelicolor_ than in _S. lividans_, in condition of Pi limitation (Figure 2d), consistently with the previously reported weak expression of genes of the _pho_ regulon in _S. coelicolor_[22]. In condition of Pi proficiency, the expression of these genes was low in the three strains and slightly lower in the _pho_P mutant and in _S. coelicolor_ than in the _wt_ strain of _S. lividans_ (Figure 2d). Interestingly, the expression of the first gene of the operon, _ppk_, was four-fold higher than that of its downstream genes. This intracistronic polarity is well documented in the literature and some studies suggest that it might be due to the degradation of inefficiently translated RNA [23]. The overlap of the 3\({}^{\prime}\) end of _ppk_ and 5\({}^{\prime}\) end of _pptA_ or of the 3\({}^{\prime}\) end of _pptA_ and 5\({}^{\prime}\) end of _SLI_4382_, might reduce the translation efficiency of _pptA_ and _SLI_4382_ making their transcripts more susceptible to degradation. The Disruption of SLI_4383/pptA Conters an Actinorhodin Over-Producing Phenotype to S. lividans TK24  
In order to determine whether the disruption of _SLI_4383_ had an impact on actinorhodin (ACT) production of _S. lividans_, _pptA_ was replaced by antibiotic resistance cassette by PCR-targeting. The cassette was then excised as described in Materials and Methods. A similar disruption was carried out for _SLI_4382_ but the disruption cassette was not excised. On solid R2 YE medium in Pi limitation, the _pptA_ mutant was characterized by an enhanced ACT production, whereas the _SLI_4382:_att3-aac_ mutant had a phenotype identical to that of the _wt_ strain (Figure 4a). The assay of ACT production revealed that the _pptA_ mutant strain produced ACT earlier (60 h versus 72 h) and in 1.5- to 2-fold higher amount than the _ppk_ mutant at 72 h. ACT production of the _pptA_ and _ppk_ mutant strains reached their maximum at 96 h and 108 h, respectively, then remained stable or slightly decreased afterwards, whereas ACT production of _S. coelicolor_ kept increasing and was approximately 2.5-fold higher than that of the _pptA_ mutant at late time points (Figure 4b). The in-frame _SLI_4383/pptA_ deletion mutant was complemented by pMES18, an integrative and conjugative plasmid containing the coding sequence of _pptA_ under the control of its own promoter. The integration of the intact _pptA_ gene at the _attB_ site of the chromosome in the apramycin-resistant ex-conjugants was confirmed by PCR. The complementation of this mutant strain led to a reduction of antibiotic production but did not restore a full non-producing phenotype similar to that of the wild type strain (Figure 4c). This might be due to the fact that the expression of _pptA_ from its own promoter is weaker than when it is expressed from the promoter located upstream of _ppk_. The Total Lipid Content of the SLI 4383/pptA Deletion Mutant Was Lower Than That of the Wild Type and ppk Mutant Strains of S. ividans  
Since we previously demonstrated the existence of a negative correlation between total lipid content and antibiotic production in the _ppk_ mutant of _S. ividans_ and in _S.