The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans.

In most Streptomyces species, antibiotic production is triggered in a condition of phosphate limitation, a condition that is known to be correlated with a low intracellular ATP content compared to growth in a condition of phosphate proficiency. This observation suggests that a low ATP content might be a direct trigger of antibiotic biosynthesis. In order to test this hypothesis, we introduced into the model strain Streptomyces lividans, a functional and a non-functional ATPase cloned into the replicative vector pOSV206 and expressed under the control of the strong ErmE* promoter. The functional ATPase was constituted by the α (AtpA), ß (AtpB) and γ (AtpD) sub-units of the native F1 part of the ATP synthase of S. lividans that, when separated from the membrane-bound F0 part, bears an ATPase activity. The non-functional ATPase was a mutated version of the latter, bearing a 12 amino acids deletion encompassing the active site of the AtpD sub-unit. S. lividans was chosen to test our hypothesis since this strain hardly produces any antibiotics. However, it possesses the same biosynthetic pathways of various specialized metabolites as S. coelicolor, a phylogenetically closely related strain that produces these metabolites in abundance. Our results demonstrated that the over-expression of the functional ATPase, but not that of its mutated version, indeed correlated with the production of the bioactive metabolites of the CDA, RED and ACT clusters. These results confirmed the long known and mysterious link existing between a phosphate limitation leading to an ATP deficit and the triggering of antibiotic biosynthesis. Based on this work and the previous published results of our group, we propose an entirely novel conception of the nature of this link.

The Phosin PptA Plays a Negative Role in the Regulation of Antibiotic Production in Streptomyces lividans.

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145.

The Streptomyces genus is well known for its ability to produce bio-active secondary metabolites of great medical interest. However, the metabolic features accompanying these bio-productions remain to be defined. In this study, the comparison of related model strains producing differing levels of actinorhoddin (ACT), showed that S. lividans, a weak producer, had high TriAcylGlycerol (TAG) content indicative of a glycolytic metabolism. In contrast, the strong producer, S. coelicolor, was characterized by low TAG content, active consumption of its polyphosphate (PolyP) stores and extremely high ATP/ADP ratios. This indicated highly active oxidative metabolism that was correlated with induction of ACT biosynthesis. Interestingly, in conditions of phosphate limitation, the ppk mutant had TAG content and ACT production levels intermediary between those of S. lividans and S. coelicolor. This strain was characterized by high ADP levels indicating that Ppk was acting as an Adenosine Di Phosphate Kinase. Its absence resulted in energetic stress that is proposed to trigger an activation of oxidative metabolism to restore its energetic balance. This process, which is correlated with ACT biosynthesis, requires acetylCoA to fuel the Krebs cycle and phosphate for ATP generation by the ATP synthase coupled to the respiratory chain, resulting in low TAG and polyP content of the ACT producing strains.

Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity.

Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [3H]phosphatidic acid (PA) released from [3H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.

Phosphate Homeostasis in Conditions of Phosphate Proficiency and Limitation in the Wild Type and the phoP Mutant of Streptomyces lividans.

Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphorylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore, short polyP was shown to be present outside the cell, tightly bound to the mycelium via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.

Repression of antibiotic production and sporulation in Streptomyces coelicolor by overexpression of a TetR family transcriptional regulator.

The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its -35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches.

Regulation of ppk expression and in vivo function of Ppk in Streptomyces lividans TK24.

The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the gamma 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 Pi 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 Pi-limiting conditions was shown to be much higher than that under Pi-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.

Identification of five novel mutations in the factor XI gene (F11) of patients with factor XI deficiency.

Factor XI (FXI) deficiency is an inherited autosomal recessive disorder associated with bleeding of variable severity. However, many cases of dominant disease transmission have been recently described. This disorder is rare in the general population, whereas it is commonly found in individuals of Ashkenazi Jewish ancestry. This study reports the molecular genetic analysis of FXI deficiencies in 11 unrelated families of different origin. Five novel mutations have been identified. Severe FXI deficiency of two unrelated patients resulted from two novel mutations: one deletion (960-961delGT) in exon 9 predicting a frameshift, and a Ser-4Leu mutation located in the signal peptide. In addition, three novel missense mutations associated with partial FXI deficiency have been identified: Cys122Tyr, Glu297Lys and Glu579Lys.

The 62-kb upstream region of Bombyx mori fibroin heavy chain gene is clustered of repetitive elements and candidate matrix association regions.

We sequenced an 80 kb DNA region containing the complete sequence of the silkworm Bombyx mori fibroin gene and its flanking, especially the upstream, regions (-62 kb). About 30% of the 62 kb upstream region is composed of repetitive elements including short interspersed elements Bm1, long interspersed elements L1Bm and mariner-like elements Bmmar1 which are widespread over the silkworm genome. This 62 kb region is also enriched of commonly considered matrix association region (MAR) motifs. A total of 25 individual MAR recognition signatures (MRSs) were identified, with 24 at the upstream and one at the downstream region. Combining two newly developed MAR prediction programs (MAR-finder and Chrclass), ten candidate MARs were predicted, with five containing MRS and seven related to the repetitive elements. The wide distribution of nested repetitive elements, candidate MARs, DNase I hypersensitive sites and other potential regulatory factors recognition sites indicates this region is probably a unique huge cis-acting element contributing to the regulation of the spatial and temporal specificity and efficiency of fibroin gene expression.