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The PhoR/PhoP two-component system of Streptomyces lividans was previously shown to allow the growth of the bacteria at low P i concentrations and to negatively control antibiotic production. The present study focuses on the transcriptional analysis of phoR and phoP, along with the phoU and mtpA genes that are transcribed divergently from the phoRP operon in S. lividans. The effect of phoR, phoP, phoU, and ppk mutations on transcription of these genes was examined under phosphate-replete and phosphate-limited conditions. We demonstrated that phoR and phoP were cotranscribed as a leaderless bicistronic transcript cleaved at discrete sites toward the 3 end of phoR. In addition, phoP could also be transcribed alone from a promoter located at the 3 end of phoR. The phoU and mtpA genes, predicted to encode metal binding proteins, were shown to be transcribed as monocistronic transcripts. The expression of phoR-phoP, phoP, and phoU was found to be induced under conditions of P i limitation in S. lividans TK24. This induction, requiring both PhoR and PhoP, was significantly weaker in the phoU mutant but much stronger in the ppk mutant than in the parental strain. The expression of mtpA was also shown to be up-regulated when P i was limiting but independently of PhoR/PhoP. The induction of mtpA expression was much stronger in the phoU mutant strain than in the other strains. This study revealed interesting regulatory interactions between the different genes and allowed us to propose putative roles for PhoU and MtpA in the adaptation to phosphate scarcity.Phosphate (P i ) is a crucial component of all living organisms, as most of the essential cellular constituents, including ATP, contain P i . Considering the vital importance of P i and its scarcity in the natural world, bacteria have evolved manifold strategies to cope with limited availability of P i . The bacteria first sense P i limitation in the growth medium and then trigger strategies aimed at scavenging and transporting trace amounts of P i that is usually present in the growth medium as metal phosphates (4,35) or at recycling the phosphate present in some phosphate-rich cellular constituents (2, 10). These strategies allow the growth of the bacteria at low P i concentrations. In Escherichia coli or Bacillus subtilis, the two-component systems (PhoB/PhoR and PhoP/PhoR, respectively) were shown to be involved in sensing P i limitation (20,28). An as-yetunknown signal, indicating a P i limitation, triggers autophosphorylation of the sensory kinase on a histidine residue. This phosphoryl group is then transferred to a conserved aspartate residue in the N-terminal receiver domain of a response regulator, increasing the affinity of the latter for its target sites, the pho box, usually present in the promoter regions of genes of the pho regulon (5, 33).Recently, the genes encoding the two-component system of Streptomyces lividans 1326, phoR (encoding a 426-amino-acid [aa]-long sensor kinase) and phoP (encoding a 223-aa-long response regulator), related to analogous sy...

The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the ␥ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In P i sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under P i -limiting conditions was shown to be much higher than that under P i -sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

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Human T-cell lymphotrophic virus type I Rex and p30 are both RNA binding regulatory proteins. Rex is a protein that interacts with a responsive element and stimulates nuclear export of incompletely spliced viral RNAs thereby increasing production of virus particles. In contrast, p30 is involved in the nuclear retention of the tax/rex mRNA leading to inhibition of virus expression and possible establishment of viral latency. How these two proteins, with apparent opposite functions, integrate in the viral replication cycle is unknown. Here, we demonstrate that Rex and p30 form ribonucleoprotein ternary complexes onto specific viral mRNA. Our results explain the selective nuclear retention of tax/rex but not other viral mRNAs by p30. Whereas p30 suppresses Rex expression, it did not affect Rex-mediated nuclear export of RNA containing the Rex response element. In contrast, Rex was able to counteract p30-mediated suppression of viral expression and restore cytoplasmic tax/rex mRNA and Tax protein expression. Together, our data demonstrate a complex regulatory mechanism of antagonizing post-transcriptional regulators evolved by human T-cell lymphotrophic virus type I to allow a vigilant control of viral gene expression. The human T-cell lymphotrophic virus type I (HTLV-I)2 is the causative agent of adult T-cell leukemia/lymphoma and chronic neurological diseases, HTLV-I-associated myelopathy/ tropical spastic paraparesis (1-3). HTLV-I replication relies on the viral trans-activator, Tax, and three 21-bp repeat elements, collectively referred to as the Tax response element, localized in the U3 region of the provirus long terminal repeats (LTRs) (4).Assembly of Tax, CREB (CRE-binding protein), co-activators CBP/p300, and PCAF (p300/CBP-associated factor) onto Tax response element allow Tax-dependent trans-activation and viral gene expression (5-9). Rex is a RNA-binding post-transcriptional regulator that binds specifically to its cis-acting target sequence, the Rex response element (RxRE), present at the 3Ј-end of all viral mRNA (10). Rex possesses a nuclear export signal, which allows it to shuttle between the nucleus and cytoplasm of the host cells in a CRM1-dependent pathway (11). As a result, Rex selectively exports the unspliced (gag/pol) and incompletely spliced (env) viral mRNA from nucleus to the cytoplasm, thereby increasing the expression of structural and enzymatic proteins and formation of progeny virus (12).Two additional viral proteins, HBZ and p30, are involved in negative regulation of virus expression and replication (13-15). The HTLV basic leucine zipper protein, HBZ, is a naturally encoded antisense, which has been shown to interfere with Tax-mediated viral transcription. Studies have shown that p30 interacts with CREB-binding protein CBP/p300 and differentially modulates cAMP-responsive element (CRE) and the Taxresponsive element-mediated transcription (16). HTLV-I p30 is a nuclear/nucleolar resident protein that is directly or indirectly associated with viral RNA, but unlike Rex, it is a nonshuttli...