Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans.

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.

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.

Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor.

Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.

Cloning and characterization of the first actinomycete ß-propeller phytase from Streptomyces sp. US42.

A gene encoding an extracellular phytase was cloned for the first time from an Actinomycete, Streptomyces sp. US42 and sequenced. The sequence of this gene revealed an encoded polypeptide (PHY US42) exhibiting one and six residues difference with the putative phytases of Streptomyces lividans TK24 and Streptomyces coelicolor A3(2), respectively. The molecular modeling of PHY US42 indicated that this phytase belongs to the group of ß-propeller phytases that are usually calcium-dependent. PHY US42 was purified and characterized. Its activity was calcium-dependent and maximal at pH 7 and 65 °C. The enzyme was perfectly stable at pH ranging from 5 to 10 and its thermostability was greatly enhanced in the presence of calcium. Indeed, PHY US42 maintained 80% of activity after 10 min of incubation at 75 °C in the presence of 5 mM CaCl2 . PHY US42 was also found to exhibit high stability after incubation at 37 °C for 1 h in the presence of bovine bile and digestive proteases like of pepsin, trypsin, and chymotrypsin. Considering its biochemical properties, PHY US42 could be used as feed additive in combination with an acid phytase for monogastric animals.

Structure and regulatory targets of SCO3201, a highly promiscuous TetR-like regulator of Streptomyces coelicolor M145.

SCO3201, a regulator of the TetR family, is a strong repressor of both morphological differentiation and antibiotic production when overexpressed in Streptomyces coelicolor. Here, we report the identification of 14 novel putative regulatory targets of this regulator using in vitro formaldehyde cross-linking. Direct binding of purified His6-SCO3201 was demonstrated for the promoter regions of 5 regulators (SCO1716, SCO1950, SCO3367, SCO4009 and SCO5046), a putative histidine phosphatase (SCO1809), an acetyltransferase (SCO0988) and the polyketide synthase RedX (SCO5878), using EMSA. Reverse transcriptional analysis demonstrated that the expression of the transcriptional regulators SCO1950, SCO4009, SCO5046, as well as of SCO0988 and RedX was down regulated, upon SCO3201 overexpression, whereas the expression of SCO1809 and SCO3367 was up regulated. A consensus binding motif was derived via alignment of the promoter regions of the genes negatively regulated. The positions of the predicted operator sites were consistent with a direct repressive effect of SCO3201 on 5 out of 7 of these promoters. Furthermore, the 2.1Å crystal structure of SCO3201 was solved, which provides a possible explanation for the high promiscuity of this regulator that might account for its dramatic effect on the differentiation process of S. coelicolor.

The stringent response is strongly activated in the antibiotic producing strain, Streptomyces coelicolor.

S. lividans and S. coelicolor are phylogenetically closely related strains with different abilities to produce the same specialized metabolites. Previous studies revealed that the strong antibiotic producer, S. coelicolor, had a lower ability to assimilate nitrogen and phosphate than the weak producer, Streptomyces lividans, and this resulted into a lower growth rate. A comparative proteomic dataset was used to establish the consequences of these nutritional stresses on the abundance of proteins of the translational apparatus of these strains, grown in low and high phosphate availability. Our study revealed that most proteins of the translational apparatus were less abundant in S. coelicolor than in S. lividans whereas it was the opposite for ET-Tu 3 and a TrmA-like methyltransferase. The expression of the latter being known to be under the positive control of the stringent response whereas that of the other ribosomal proteins is under its negative control, this indicated the occurrence of a strong activation of the stringent response in S. coelicolor. Furthermore, in S. lividans, ribosomal proteins were more abundant in phosphate proficiency than in phosphate limitation suggesting that a limitation in phosphate, that was also shown to trigger RelA expression, contributes to the induction of the stringent response.

Comparative proteomic analysis of Streptomyces lividans Wild-Type and ppk mutant strains reveals the importance of storage lipids for antibiotic biosynthesis.

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

Crystal structures of free and ligand-bound forms of the TetR/AcrR-like regulator SCO3201 from Streptomyces coelicolor suggest a novel allosteric mechanism.

TetR/AcrR-like transcription regulators enable bacteria to sense a wide variety of chemical compounds and to dynamically adapt the expression levels of specific genes in response to changing growth conditions. Here, we describe the structural characterisation of SCO3201, an atypical TetR/AcrR family member from Streptomyces coelicolor that strongly represses antibiotic production and morphological development under conditions of overexpression. We present crystal structures of SCO3201 in its ligand-free state as well as in complex with an unknown inducer, potentially a polyamine. In the ligand-free state, the DNA-binding domains of the SCO3201 dimer are held together in an unusually compact conformation and, as a result, the regulator cannot span the distance between the two half-sites of its operator. Interaction with the ligand coincides with a major structural rearrangement and partial conversion of the so-called hinge helix (α4) to a 310 -conformation, markedly increasing the distance between the DNA-binding domains. In sharp contrast to what was observed for other TetR/AcrR-like regulators, the increased interdomain distance might facilitate rather than abrogate interaction of the dimer with the operator. Such a 'reverse' induction mechanism could expand the regulatory repertoire of the TetR/AcrR family and may explain the dramatic impact of SCO3201 overexpression on the ability of S. coelicolor to generate antibiotics and sporulate.

Genome Analysis of a Variant of Streptomyces coelicolor M145 with High Lipid Content and Poor Ability to Synthetize Antibiotics.

Streptomyces coelicolor M145 is a model strain extensively studied to elucidate the regulation of antibiotic biosynthesis in Streptomyces species. This strain abundantly produces the blue polyketide antibiotic, actinorhodin (ACT), and has a low lipid content. In a process designed to delete the gene encoding the isocitrate lyase (sco0982) of the glyoxylate cycle, an unexpected variant of S. coelicolor was obtained besides bona fide sco0982 deletion mutants. This variant produces 7- to 15-fold less ACT and has a 3-fold higher triacylglycerol and phosphatidylethanolamine content than the original strain. The genome of this variant was sequenced and revealed that 704 genes were deleted (9% of total number of genes) through deletions of various sizes accompanied by the massive loss of mobile genetic elements. Some deletions include genes whose absence could be related to the high total lipid content of this variant such as those encoding enzymes of the TCA and glyoxylate cycles, enzymes involved in nitrogen assimilation as well as enzymes belonging to some polyketide and possibly trehalose biosynthetic pathways. The characteristics of this deleted variant of S. coelicolor are consistent with the existence of the previously reported negative correlation existing between lipid content and antibiotic production in Streptomyces species.

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

Transcriptional and preliminary functional analysis of the six genes located in divergence of phoR/phoP in Streptomyces lividans.

Streptomyces lividans senses and adjusts to a situation of Pi limitation via the expression of genes of the pho regulon controlled by the two-component system PhoR/PhoP. Interestingly, an in silico analysis of the proteins encoded by the six genes located in divergence of phoR/phoP revealed that the latter bear features often found in metalloproteins involved in the sensing/resistance to oxidative stress. We determined whether genes of this region were belonging to the pho regulon and whether the encoded proteins do play a role in the resistance to oxidative stress. For this purpose, a transcriptional analysis of these genes was carried out on the carbon and nitrogen rich medium R2YE and on a minimal medium (MM). On R2YE, the expression of the genes phoU to sco4225 was much higher than on MM and constant throughout growth. On this medium, the expression of phoU was totally PhoP-dependent whereas the expression of sco4227 and sco4226 was partially PhoP-dependent, taking place from the phoU promoter region. In contrast, on MM, the expression of sco4227 and sco4226 was PhoP-independent whereas that of phoU remained PhoP-dependent and showed, as phoR/phoP, a peak of expression at 48 h. sco4225, sco4224, and sco4223 were transcribed from their own promoter independently of PhoP in both media. The mutants of five out of six genes of the region (Δsco4226 mutant could not be obtained) grew poorly in the presence of exogenous oxidants, suggesting a role of the encoded proteins in the resistance to oxidative stress, especially on the rich medium R2YE.

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner.

Filamentous microorganisms of the bacterial genus Streptomyces have a complex life cycle that includes physiological and morphological differentiations. It is now fairly well accepted that lysis of Streptomyces vegetative mycelium induced by programmed cell death (PCD) provides the required nutritive sources for the bacterium to erect spore-forming aerial hyphae. However, little is known regarding cellular compounds released during PCD and the contribution of these molecules to the feeding of surviving cells in order to allow them to reach the late stages of the developmental program. In this work we assessed the effect of extracellular sugar phosphates (that are likely to be released in the environment upon cell lysis) on the differentiation processes. We demonstrated that the supply of phosphorylated sugars, under inorganic phosphate limitation, delays the occurrence of the second round of PCD, blocks streptomycetes life cycle at the vegetative state and inhibits antibiotic production. The mechanism by which sugar phosphates affect development was shown to involve genes of the Pho regulon that are under the positive control of the two component system PhoR/PhoP. Indeed, the inactivation of the response regulator phoP of Streptomyces lividans prevented the 'sugar phosphate effect' whereas the S. lividans ppk (polyphosphate kinase) deletion mutant, known to overexpress the Pho regulon, presented an enhanced response to phosphorylated sugars.

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 construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters.

Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the -35 and -10 consensus promoter sequences were completely randomized and some variability was introduced in the -35 (position 6) and -10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 µgml(-1)). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the -10 box, the -10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters.

A Proteomic Analysis Indicates That Oxidative Stress Is the Common Feature Triggering Antibiotic Production in Streptomyces coelicolor and in the pptA Mutant of Streptomyces lividans.

In most Streptomyces species, antibiotic production is triggered in phosphate limitation and repressed in phosphate proficiency. However, the model strain, Streptomyces coelicolor, escapes this general rule and produces actinorhoddin (ACT), a polyketide antibiotic, even more abundantly in phosphate proficiency than in phosphate limitation. ACT was shown to bear "anti-oxidant" properties suggesting that its biosynthesis is triggered by oxidative stress. Interestingly, Streptomyces lividans, a strain closely related to S. coelicolor, does not produce ACT in any phosphate condition whereas its pptA/sco4144 mutant produces ACT but only in phosphate limitation. In order to define the potentially common features of the ACT producing strains, these three strains were grown in condition of low and high phosphate availability, and a comparative quantitative analysis of their proteomes was carried out. The abundance of proteins of numerous pathways differed greatly between S. coelicolor and the S. lividans strains, especially those of central carbon metabolism and respiration. S. coelicolor is characterized by the high abundance of the complex I of the respiratory chain thought to generate reactive oxygen/nitrogen species and by a weak glycolytic activity causing a low carbon flux through the Pentose Phosphate Pathway resulting into the low generation of NADPH, a co-factor of thioredoxin reductases necessary to combat oxidative stress. Oxidative stress is thus predicted to be high in S. coelicolor. In contrast, the S. lividans strains had rather similar proteins abundance for most pathways except for the transhydrogenases SCO7622-23, involved in the conversion of NADPH into NADH. The poor abundance of these enzymes in the pptA mutant suggested a deficit in NADPH. Indeed, PptA is an accessory protein forcing polyphosphate into a conformation allowing their efficient use by various enzymes taking polyphosphate as a donor of phosphate and energy, including the ATP/Polyphosphate-dependent NAD kinase SCO1781. In phosphate limitation, this enzyme would mainly use polyphosphate to phosphorylate NAD into NADP, but this phosphorylation would be inefficient in the pptA mutant resulting in low NADP(H) levels and thus high oxidative stress. Altogether, our results indicated that high oxidative stress is the common feature triggering ACT biosynthesis in S. coelicolor and in the pptA mutant of S. lividans.

Impact of Phosphate Availability on Membrane Lipid Content of the Model Strains, Streptomyces lividans and Streptomyces coelicolor.

In this issue we demonstrated that the phospholipid content of Streptomyces lividans varies greatly with Pi availability being was much lower in Pi limitation than in Pi proficiency whereas that of Streptomyces coelicolor varied little with Pi availability. In contrast the content in phosphate free ornithine lipids was enhanced in both strains in condition of phosphate limitation. Ornithine lipids biosynthesis starts with the N-acylation of ornithine to form lyso-ornithine that is then O-acylated to yield ornithine lipid. The operon sco1222-23 was proposed to be involved in the conversion of specific amino acids into ornithine in condition of phosphate limitation whereas the sco0921-20 operon encoding N- and O-acyltransferase, respectively, was shown to be involved in the biosynthesis of these lipids. The expression of these two operons was shown to be under the positive control of the two components system PhoR/PhoP and thus induced in phosphate limitation. The expression of phoR/phoP being weak in S. coelicolor, the poor expression of these operons resulted into a fivefold lower ornithine lipids content in this strain compared to S. lividans. In the deletion mutant of the sco0921-20 operon of S. lividans, lyso-ornithine and ornithine lipids were barely detectable and TAG content was enhanced. The complementation of this mutant by the sco0921-20 operon or by sco0920 alone restored ornithine lipids and TAG content to wild type level and was correlated with a twofold increase in the cardiolipin content. This suggested that SCO0920 bears, besides its broad O-acyltransferase activity, an N-acyltransferase activity and this was confirmed by the detection of lyso-ornithine in this strain. In contrast, the complementation of the mutant by sco0921 alone had no impact on ornithine lipids, TAG nor cardiolipin content but was correlated with a high lyso-ornithine content. This confirmed that SCO0921 is a strict N-acyltransferase. However, interestingly, the over-expression of the sco0921-20 operon or of sco0921 alone in S. coelicolor, led to an almost total disappearance of phosphatidylinositol that was correlated with an enhanced DAG and TAG content. This suggested that SCO0921 also acts as a phospholipase C, degrading phosphatidylinositol to indirectly supply of phosphate in condition of phosphate limitation.

A Molecule of the Viridomycin Family Originating from a Streptomyces griseus-Related Strain Has the Ability to Solubilize Rock Phosphate and to Inhibit Microbial Growth.

Some soil-borne microorganisms are known to have the ability to solubilize insoluble rock phosphate and this process often involves the excretion of organic acids. In this issue, we describe the characterization of a novel solubilizing mechanism used by a Streptomyces strain related to Streptomyces griseus isolated from Moroccan phosphate mines. This process involves the excretion of a compound belonging to the viridomycin family that was shown to play a major role in the rock phosphate bio weathering process. We propose that the chelation of the positively charged counter ions of phosphate constitutive of rock phosphate by this molecule leads to the destabilization of the structure of rock phosphate. This would result in the solubilization of the negatively charged phosphates, making them available for plant nutrition. Furthermore, this compound was shown to inhibit growth of fungi and Gram positive bacteria, and this antibiotic activity might be due to its strong ability to chelate iron, a metallic ion indispensable for microbial growth. Considering its interesting properties, this metabolite or strains producing it could contribute to the development of sustainable agriculture acting as a novel type of slow release bio-phosphate fertilizer that has also the interesting ability to limit the growth of some common plant pathogens.

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.

Isolation and Characterization of Phosphate Solubilizing Streptomyces sp. Endemic from Sugar Beet Fields of the Beni-Mellal Region in Morocco.

In the course of our research, aimed at improving sugar beets phosphorus nutrition, we isolated and characterized Streptomyces sp. strains, endemic from sugar beet fields of the Beni-Mellal region, which are able to use natural rock phosphate (RP) and tricalcium phosphate (TCP) as sole phosphate sources. Ten Streptomyces sp. isolates yielded a comparable biomass in the presence of these two insoluble phosphate sources, indicating that they were able to extract similar amount of phosphorus (P) from the latter for their own growth. Interestingly, five strains released soluble P in large excess from TCP in their culture broth whereas only two strains, BP, related to Streptomyces bellus and BYC, related to Streptomyces enissocaesilis, released a higher or similar amount of soluble P from RP than from TCP, respectively. This indicated that the rate of P released from these insoluble phosphate sources exceeded its consumption rate for bacterial growth and that most strains solubilized TCP more efficiently than RP. Preliminary results suggested that the solubilization process of BYC, the most efficient RP and TCP solubilizing strain, involves both acidification of the medium and excretion of siderophores. Actinomycete strains possessing such interesting RP solubilizing abilities may constitute a novel kind of fertilizers beneficial for plant nutrition and more environmentally friendly than chemical fertilizers in current use.

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.