## 2 Results  
### Sll_4382 and Sll_4383 Amino Acid Sequence Features  
At the Sanger center, SLI_4382 is described as a 142 AA long protein belonging to the nudix hydrolase family and bearing similarities with enzymes involved in the cleavage of diadenosine hexaphosphate (Ap6A) into two molecules of ATP. On its side, SLI_4383/PptA is described as a 388 AA long protein bearing a TTA leucine codon, possible target for _bldA_ regulation [21] and possessing a conserved histidine alpha-helical domain (\"CHAD\" domain), considered as a polyphosphate binding module [19; 20]. Interestingly, a comparison of the predicted protein product of _SLI_4383/_pptA_ (GenBank entry EFD67904.1) to putative orthologues from other _Streptomyces_ species revealed the presence of N-terminal extensions of various length whose extremity is highly conserved in almost all orthologues but which is missing in the protein of _S. lividans_ as it is annotated at the Sanger center (Figure 1). In the recently determined crystal structure of _S. chartreusis_ PptA [20], this N terminal region comprises part of the first \(\alpha\)-helix of the protein fold. Since in the _S. lividans_ genome (GenBankank entry GG657756.1) a highly similar sequence is present immediately upstream of the designated _pptA_ translational start codon, it seems likely that this codon was erroneously assigned to an internal GTG (Val) codon. Predictions using various programs listed in Materials and Methods further confirmed this hypothesis and indicated that the most probable in frame GTG start codon is located 68 codons upstream of that provided at the Sanger center (GenBankank entry EFD67904.1). _S. lividans_ PptA would thus be 456 AA long, with a coding region 204 nucleotides longer than anticipated and overlapping with the C-terminal region of _ppk_, the GTG start codon of _pptA_ being located 17 nt upstream of the TGA stop codon of _ppk_. Figure 1: Multiple sequence alignment of the N-terminal region of PptA-like proteins from various _Streptomyces_. The crystal structure of _S. chartreusis_ PptA (PDB entry 6 RN5) is schematically shown above the alignment. Dashed line represent structurally disordered areas, whereas the rectangle in solid lines represent the N-terminal \(\alpha\)-helix (\(\alpha\)1). Asterisks under the alignment indicate strongly conserved hydrophobic residues expected to play key roles in the stability of the protein fold. The red bar above the alignment indicates the region that is missing in the current database entry for PptA (SLI_4383) of _S. lividans_ (GenBank entry EFD67904.1) due to incorrect assignment of the translation start site (M). Ppk, pptA and SLI_4382 Are Co-Transcribed But pptA and SLI_4382 Can Also Be Transcribed Alone from Their Own Promoter  
In order to establish the transcriptional situation of the genes _ppk_, _pptA_, and _SLI_4382_ that are located just upstream of the genes encoding the high affinity phosphate transporter (PstSCAB), RNA was prepared from _S. lividans_ grown for 40 h on solid R2 YE medium with no phosphate added (1 mM final, Pi limitation) and specific primers, internal to each gene or between two adjacent genes, were designed to assess their transcriptional organization by RT-qPCR (Figure 2a). Figure 2: Transcriptional situation of the contiguous _ppk_, _pptA_, and _SLI_4382_ genes. **(a)** Schematic representation of the genetic map of the region encompassing genes _ppk_, _pptA_, and SLI_4382_ represented by black arrows. Primers used to establish the transcriptional situation are represented by thin vertical arrows on a line above the genetic map. Deduced transcriptional situation is represented by dotted lines below the genetic map (**b**) Level of expression of _ppk_, _pptA_, and _SLI_4382_ and their junctions determined by RT-qPCR with RNA prepared from _S. lividans_ and its _phoP_ mutant grown in conditions of Pi limitation. Position of the primers used is shown in **(a)** and sequences of the primers are listed in Table S3.