## 2 Results
### The ppk/pptA/SLI_4382 Operon Belongs to the Pho Regulon
coelicolor_[5], we assessed the evolution of the lipid content of the _pptA_ mutant throughout growth. Results shown in Figure 5 confirmed that the total lipid content of the _ppk_ mutant was lower than that of the _wt_ strain throughout the cultivation period but steadily increased with time. In contrast, the total lipid content of the _pptA_ mutant that was at a similar level than that of the _ppk_ mutant up to 72 h and decreased afterwards. As previously reported, the total lipid content of _S. coelicolor_ was low and remained low throughout the cultivation period [5]. The lipid content as well as ACT production levels of the _pptA_ mutant was lower than that of the _wt_ strain. The total lipid content of the _pptA_ mutant was lower than that of the _wt_ strain. The total lipid content of the _pptA_ mutant was lower than that of the _ppk_ mutant up to 72 h and decreased afterwards. As previously reported, the total lipid content of _S. coelicolor_ was low and remained low throughout the  
mutant strain, being intermediary those of the _ppk_ mutant and _S. coelicolor_, we concluded that the phosphate/energetic deficit of the _pptA_ mutant was intermediary between that of the _ppk_ mutant and that of _S. coelicolor_. These data confirmed the existence of a negative correlation between total lipid content and antibiotic production [5; 24]. Interestingly, most of ACT produced by the three strains remained intracellular, up to 72 h but after this point most ACT produced by the _ppk_ mutant and _S. coelicolor_ was extracellular, whereas a large proportion of ACT remained intracellular in the _pptA_ mutant even if this proportion decreased with time. To explain these differences, one can propose that ACT remains intracellular as long as it is needed within the cell to fulfill its putative anti-respiratory and anti-oxidant function [5; 25]. In that respect, one cannot exclude than the surprisingly high level of ACT detected in the extracellular medium of _S. coelicolor_ results from altered membrane permeability linked to its altered membrane composition [26] and/or from extensive cell lysis linked to severe phosphate limitation. Proteins of the Pho Region Involved in Pi Supply and Saving Are Up-Regulated in the SLI_4383/pptA Mutant  
Since antibiotic production usually occurs in condition of low phosphate availability, we carried out a comparative proteomic analysis of the _pptA_ mutant with the _wt_ strain of _S. Dividans_ to determine whether the _pptA_ mutant suffers from phosphate stress. The results of this study, shown in Figure 6 a,b, revealed that the expression of the two-component system (TCS) PhoR/PhoP (SLI-4465/SLI-4466) as well as that of the regulatory protein PhoU (SLI_4464) was strongly up-regulated in the _SLI_4383_ mutant as it was in the _ppk_ mutant [16; 17]. This TCS is known to control positively the expression of genes encoding proteins involved in phosphate scavenging, up-take, and saving in condition of Pi limitation [27]. Consistently, proteins belonging to the low affinity ABC phosphate transport system (PstSCAB), SLI_4381 (PstS), SLI_4380 (PstC), SLI_4379 (PtsA), and SLI_4378 (PstB), as well as proteins annotated as phosphatase (SLI_4434) or pyrophosphatase (SLI_3752) were clearly upregulated in the _pptA_ mutant (Figure 6a). Figure 5: FTIR analysis of total lipid/fatty methyl ester (FAME) content of _S. coelicolor_ (white histograms), _S. Dividans_ (light grey histograms), _S. Dividans ppk_ mutant (dark grey histograms), and _S. Dividans SLI_4383/pptA_ mutant (black histograms) at different points throughout the cultivation period carried out on solid R2 YE medium with glucose (50 mM) as the main carbon source and containing 1 mM phosphate (condition of phosphate limitation) at 28 \({}^{\circ}\)C.  
Furthermore, proteins encoded by the neighboring genes of _pptA_, _ppk_, and _SLI_4382_ were also upregulated in the mutant.