25 research outputs found

    Caractérisation et élucidation de la fonction biologique de deux gènes sblA et ppk dont l'interruption a respectivement un effet sur la sporulation et la production d'antibiotiques chez Streptomyces lividans TK24

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    Les Streptomyces sont des bactéries du sol à Gram+ caractérisées par une cycle de différenciation complexe commençant par la germination d'une spore qui donne naissance à un mycélium basal à partir duquel s'érigeront des hyphes aériens qui se différencieront en spores. Les phases tardives du développement du mycélium aérien s'accompagnent de la production de nombreux antibiotiques d'importance industrielle. Nous avons caractérise s deux nouveaux gènes ppk et sblA jouant respectivement un rôle dans la production d'antibiotiques et la sporulation. Une souche ppk est caractérisée par une production accrue des trois antibiotiques produits par S. lividans (actinorhodine, undécylprodigiosine et antibiotique dépendant du calcium) qui est corrélée avec une forte augmentation de la transcription des activateurs spécifiques de ces voies de biosynthèse. ppk code une polyphosphate kinase qui catalyse la polymérisation du phosphate de l'ATP en polymères de phosphate (polyP) ainsi que la régénération de l'ATP à partir d'ADP et de polyP. ppk est transcrit de façon monocistronique à partir d'un promoteur unique et sa transcription est déclenchée par une carence en Pi. Une souche sblA sporule beaucoup plus tôt qu'une souche sauvage. sblA code une protéine possédant une activité phosphoinositide hydrolase/phosphatase. L'expression de sblA est transitoire (durant 6 à 8 h), a lieu principalement durant le développement du mycélium basal et est régulée négativement par deux mécanismes différents. Le premier mécanisme implique une région opératrice, constituée de 9 répétitions directes de la séquence 5'C(C/G)GGAGG(C/T)3', localisée en amont de la région -35 du promoteur et constituant le site de fixation d'un répresseur. Le deuxième mécanisme implique une structure tige-boucle de 23 nt contenant une séquence 5' AGGAGG 3' de type RBS, localisée à 170 pb en deça du codon d'initiation de la traduction et sans doute impliquée dans la régulation de la dégradation spécifique du transcrit sblA.Streptomyces are Gram+ soil bacteria characterized by a complex differentiation cycle beginning by the germination of a spore developing in a substrate mycelium growing within the nourishing medium and giving raise, after a short pause in the growth, to an aerial mycelium that will differentiate into spores. The late stages of this development are accompanied by the biosynthesis of many antibiotics of industrial importance. We have characterized two new genes ppk and sblA affecting respectively antibiotic production and sporulation in S. lividans. An sblA strain sporulates much earlier than the wild type strain. sblA encodes a protein possessing a specific phosphoinositide hydrolase/phosphatase activity. The expression of sblA is transitory (lasting 6 to 8 hours), taking place mainly during the development of the substrate mycelium and being negatively regulated by two different regulatory mechanisms. The first one involves an operator region, constituted by 9 direct repeats of the sequence 5'C(C/G)GGAGG(C/T)3', located upstream of the promoter region and likely to constitute a binding site for a transcriptional repressor. The other one involves a 23nt stem-loop structure containing a RBS-like sequence 5'AGGAGG 3', located 170 bp downstream of the GTG start codon and is thought to play a role in the regulation of the specific degradation of the sblA transcript. A ppk- strain is characterized by an enhanced production of the three antibiotics produced by S. lividans (actinorhodin, undecylprodigiosin and calcium-dependent antibiotic) that correlates with an increased transcription of the specific transcriptional activators of the corresponding biosynthetic pathways. ppk encodes a polyphosphate kinase catalysing the polymerisation of the g phosphate of ATP into polymers of phosphate (polyP) as well as the regeneration of ATP from ADP and polyP. ppk is transcribed as a monocistronic mRNA from a unique promoter sequence and its transcription is triggered by Pi starvation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    Métabolisme énergétique et régulation de la biosynthèse d'antibiotiques chez streptomyces

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    Les Streptomyces sont des bactéries filamenteuses du sol. Ces bactéries présentent un cycle de différenciation morphologique complexe et produisent de très nombreux métabolites secondaires bioactifs, dont la plupart des antibiotiques connus. La biosynthèse de ces métabolites est contrôlée par un réseau de régulation complexe dont l'étude de mutants sur-producteurs ou non-producteurs permet une meilleure compréhension. Un mutant du gène ppk (polyphosphate kinase) de S. lividans présente une production accrue d'antibiotiques, et l'analyse de ce mutant a suggéré qu' une diminution de la concentration en ATP dans la cellule serait à l'origine de cette sur-production. Cette hypothèse a été testée en exprimant des gènes codant une ATPase chez S. lividans, afin de générer un déficit artificiel en ATP dans la cellule. Il a été démontré que l'expression de l'ATPase stimule la production d'un antibiotique en particulier et que les profils de transcription de certains gènes impliqués dans la biosynthèse d'autres métabolites sont aussi modifiés par la diminution de la concentration en ATP dans la cellule. Un gène dont la sur-expression entraine l'inhibition de la différenciation morphologique et de la biosynthèse d'antibiotiques chez S. coelicolor a également été identifié. Il a été démontré que ce gène code un régulateur transcriptionnel de type TetR dont la caractérisation a été initiée. Enfin, une banque de promoteurs synthétiques de forces variables a été construite pour permettre différents niveaux d'expression de gènes chez Streptomyces et a permis de révéler certaines caractéristiques du gène aphII, utilisé comme rapporteur pour l'analyse de ces promoteurs.Streptomyces are soil-dwelling bacteria characterized by both a complex life cycle and the ability to synthesize numerous bio-active compounds such as antibiotics. Secondary metabolism is controlled by a highly intricate regulatory network and analysis of non-producing or over-producing strains can lead to a better understanding of gene regulation among this network. Deletion of the ppk gene (polyphosphate kinase) in S. lividans leads to antibiotic overproduction. Analysis of the ppk mutant suggested that reduced ATP concentrations in the cell could trigger antibiotic biosynthesis. In order to test this hypothesis, genes encoding an ATPase were expressed in S. lividans. By lowering intracellular ATP concentration, ATPase expression stimulated the biosynthesis of a specific antibiotic compound. It was also shown that transcription profiles of some others biosynthetic genes was also modified by ATP concentration. A gene encoding a negative regulator of both antibiotic biosynthesis and morphological differentiation in S. coelicolor was also isolated. It was shown that this regulator belongs to the TetR transcriptional regulator family and its functional characterization was started. Finally, a library of synthetic promoters of various strengths was engineered in order to allow different levels of gene expression in Streptomyces. This work revealed interesting features of aphII gene used as a reporter in this study.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    Caractérisation et régulation de l'expression de l'opéron phoR/phoP et du gène ppk qui jouent un rôle dans le contrôle de la biosynthèse d'antibiotiques chez Streptomyces lividans

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    J'ai étudié au cours de ma thèse les relations existant entre le métabolisme du phosphate et la production d'antibiotiques chez Streptomyces lividans. J'ai plus particulièrement cherché à élucider la dynamique de régulation du gène ppk et du système à deux composants phoR/phoP. On a observé qu' in vivo, Ppk joue un rôle crucial dans la régénération de l'ATP à partir de l'ADP et des polyphosphates. L'expression de ppk est induite en condition de limitation en phosphate d'une manière dépendante de PhoR/PhoP et réprimée en condition d'excès de phosphate et d'ATP par un répresseur putatif. Le phénotype de surproduction d'antibiotiques du mutant ppk est alors dû à un déficit énergétique conduisant à une stimulation des voies métaboliques centrales et à l'accumulation des précurseurs. J'ai étudié par des analyses transcriptionnelles, la régulation de l'expression de phoR/phoP et des gènes divergents phoU et psiA. J'ai démontré que l'expression de phoR/phoP, phoP et de phoU est induite en condition de limitation en phosphate, cette induction est plus importante chez le mutant ppk. J'ai parallèlement déterminé les sites de début de transcription de ces gènes (tsp) et étudié la force relative de leurs promoteurs à l'aide de fusion transcriptionnelle. J'ai démontré également que phoR/phoP, phoP et phoU sont sous le contrôle positif de PhoP. Les mutants phoP (régulateur) et phoR (kinase) sont caractérisés aussi par un phénotype de surproduction d'antibiotiques et de croissance limitée, par contre le rôle de phoU et psiA reste mystérieux. Enfin, j'ai découvert que psiA est aussi induit par la limitation en phosphate de manière dépendante de phoU mais indépendante de PhoR/PhoP.I studied during my thesis the relations existing between the phosphate metabolism and the antibiotic production in Streptomyces lividans. I more particularly sought to elucidate the dynamics of regulation of the ppk gene and of the phoR/phoP two-component system. We have observed that in vivo, ppk play a crucial part in the regeneration of the ATP from the ADP and the polyphosphates. The expression of ppk is enhanced in condition of phosphate limitation in a PhoR/PhoP dependent manner and repressed in condition of phosphate and ATP excess by a putative repressor. The phenotype of antibiotic overproduction in the ppk mutant is then due to an energy deficit leading to a stimulation of the central metabolic pathways and to the accumulation of the precursors. I studied, by transcriptional analysis, the regulation of the phoR/phoP expression and the divergent genes phoU and psiA. I showed that the expression of phoR/phoP, phoP and phoU is induced in condition of phosphate limitation, this induction is more important in the ppk mutant strain. I, in parallel, determined the transcription start points (tsp) of these genes and studied the relative force of the various promoters using transcriptional fusion. I also showed that phoR/phoP; phoP and phoU are under the positive control of PhoP. The mutants phoP (response regulator) and phoR (sensor kinase) are also characterized by a phenotype of overproduction of antibiotics and limited growth. On the other hand the role of phoU and psiA remains mysterious. Lastly, I discovered that psiA is also induced by phosphate limitation in a phoU dependent but PhoR/PhoP independent way.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    Fate of the sblA transcript in Streptomyces lividans and Escherichia coli.

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    In Streptomyces lividans, the tight temporal regulation of the transient expression of the sblA gene was shown to involve an operator-like sequence located on the sblA transcript. This operator-like structure constitutes a stem-loop structure containing a Shine/Dalgarno-like sequence. Its destruction, by site directed mutagenesis, led to an enhancement of sblA expression. This structure thus plays a negative role in the regulation of sblA expression and might be involved in the regulation of the specific degradation of the sblA transcript. In this issue, the fates of the sblA transcript, in S. lividans and in Escherichia coli, were compared. Analysis of the decay of the sblA transcript revealed that, in both species, the sblA transcript was cleaved just behind the stem-loop structure by an RNAse E-like activity. In E. coli, three discrete products resulting from the cleavage of the full-length transcript by the RNAase E at another site, located 282 nucleotides downstream of the stem-loop structure, were detected whereas only one processed product, corresponding to the 5' end of the gene, was detected in S. lividans. These differences in the mode of degradation of the sblA transcript in S. lividans and E. coli are discussed

    Criblage et caractérisation de souches de bactéries Actinomycétales solubilisatrices du phosphate naturel isolées à partir des gisements de phosphate marocains

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    L objectif de mon travail de thèse était d explorer la capacité des souches d Actinomycètes issues des mines de phosphate marocaines (souches MPM) à solubiliser le Phosphate Naturel (PN, hydroxyapatite) et à limiter la croissance de certains champignons phytopathogènes du sol (Pythium ultimum) afin d utiliser ces souches pour le développement de nouvelles pratiques agricoles moins polluantes que les pratiques actuelles.Ainsi, 55 isolats (18.3%) parmi les 300 isolats MPM testés se sont avérés capables de croître sur un milieu minimum synthétique (SMM) contenant le PN insoluble comme seule source de phosphate (P). Huit souches dont la croissance était la plus active ont été retenues. La capacité de ces 8 souches à solubiliser le PN a ensuite été suivie par des dosages de P soluble dans le milieu SMM liquide et comparée à celle de deux souches de référence Streptomyces lividans et Streptomyces griseus M1323. Une forte hausse de la concentration en P soluble a été observée dans les surnageants des souches issues des MPM et de S. griseus M1323, mais pas dans ceux des cultures de S. lividans. Cette augmentation de la concentration de P dans le surnageant de culture des MPM a été corrélée avec la présence de substances, absorbant à 320 nm et à 430 nm. Ces substances ont été purifiées et il a été confirmé qu elles sont impliquées dans le mécanisme de solubilisation du PN. Elles ont par ailleurs une forte activité antibiotique et anti-fongique. Ces substances sont des sidérophores de type catécholate (appelé Viridomycin G). Ce sont des chélateurs forts du calcium qui détruisent les liens existant entre le calcium et le phosphate du PN, permettant ainsi la libération du phosphate. Elles sont produites quand la quantité de P soluble présente dans le milieu de culture est très faible. Nous avons par ailleurs montré que les souches MPM avaient une aptitude endophytique et avaient une forte capacité de stockage du glucose et du phosphate sous forme notamment de glycogène et polyphosphate. Ces souches produisaient moins de biomasse que la souche de référence S. lividans, mais avaient une plus grande capacité de survie que cette dernière dans des conditions de forte limitation en P soluble (SMM + PN). Toutes ces caractéristiques sont importantes pour une survie de la souche à long terme dans le sol. Par ailleurs, certaines de ces souches étaient également capables de limiter, en conditions de laboratoire, la croissance de Pythium ultimum, principal agent de la fonte de semis des céréales. Cette aptitude est vraisemblablement liée à la capacité démontrée de ces souches à produire des substances antifongiques, des chitinases, des sidérophores et de l Acide Indol Acétique. Par la suite, nous avons testé l efficacité agronomique de ces souches et démontré que certaines avaient la capacité à stimuler la croissance d une plante test (le blé dur) dans un modèle rhizosphérique constitué d un sol riche en PN, d une souche solubilisatrice du PN et de la plante, dans une expérience sous serre. Il s est avéré que la seule présence des souches d Actinomycètes dans le sol utilisé stimulait la croissance de la plante suggérant que ces souches étaient capables de mobiliser, pour la plante, le phosphate naturellement présent sous forme insoluble dans le sol. La présence conjointe dans ce sol des souches d Actinomycètes et du PN pulvérisé stimulait la croissance de la plante, et en particulier celle de son système racinaire, de façon comparable à l addition d engrais phosphaté soluble. De même, ces souches avaient une capacité de protection, contre les effets dévastateurs de Pythium ultimum, comparable à celle du Mycostop®, produit de biocontrôle antifongique commercialisé et constitué de spores de Streptomyces griseoviridis K61. Cette étude a démontré que les Actinomycètes du sol contribuent grandement à la nutrition et donc à la croissance des plantes ainsi qu à leur santé. Cette étude pourrait conduire à la conception/formulation et mise sur le marché de nouveaux produits ayant une activité de bio-engrais phosphaté et bio-fongicide, constitué des souches d Actinomycètes add hoc associées ou non à du PN pulvérisé.The aim of this work was to explore the abilities of Actinomycetes strains originating from the Moroccan phosphate mines (MPM strains) to solubilize natural phosphate (NP, hydroxyapatite) and to limit the growth of some soil-born phytopathogen fungi (Pythium ultimum) in order to use these strains for the development of novel agricultural practices less polluting than the actual ones.Fifty five isolates (18.3%) among the 300 MPM isolates tested were able to grow on a minimal synthetic medium (SMM) containing insoluble NP as sole phosphate (P) source, suggesting that they were able to use the P trapped in the NP for their own growth. Eight strains whose growth was the most active were selected. The efficiency of these strains to solubilize NP was assessed by assaying soluble P in the growth medium and was compared to that of two reference strains Streptomyces lividans and Streptomyces griseus M1323. A sharp increase in the concentration of soluble P was observed in the cultures supernatants of the MPM strains and of S. griseus M1323, but non in those of S. lividans. This increase in the P concentration correlated with the presence of substances absorbing at 320 nm and 430 nm. These substances were purified and I confirmed that these substances, that had also strong antibiotic and anti-fungi activities, were involved in the NP solubilisation process. They are siderophores of the catecholate family (called Viridomycin G) acting as strong calcium chelators destroying the links between calcium and P in the NP and thus allowing the release of P. They were excreted when the concentration of P in the growth medium was really weak. I have also shown that these strains have the abilities to colonize the plant root system (endophytic property) and have very high glucose and phosphate storage abilities, (accumulating glycogen and polyphosphate) compared to the reference strain S. lividans. These strains produce less biomass but survive better than the latter in condition of very low soluble P availability (SMM + NP). All together, these characteristics are very important for the long term survival of these bacteria in the soil. Furthermore, some of these strains were also able to limit the development, in laboratory conditions, of Pythium ultimum, the main agent of damping off of cereals. This property is likely to be linked to the demonstrated ability of these strains to excrete anti-fungal substances, chitinases, siderophores and Indol Acetic Acid. Subsequently, I have tested the agronomic efficiency of these strains and demonstrated that they had the ability to stimulate wheat plant growth in a rhizospheric model constituted by a soil rich in insoluble P, a NP solubilising strain and the plant, in greenhouse experiments. I demonstrated that the presence of a MPM Actinomycetes strain, on its own, was stimulating wheat plant growth suggesting that this strain was able to mobilize for the plant good, the insoluble P naturally present in the soil. The joined presence in this soil of the Actinomycetes strain and of the pulverised NP was stimulating wheat plant growth (specially roots growth) at a level comparable (only 10% lower) to that achieved by the addition of soluble phosphate amendment. Furthermore, these strains had the ability to protect the plant against the devastating effects of Pythium ultimum, at a level comparable (only 14% lower) to that achieved by Mycostop®, a commercial antifungal biocontrol product constituted by spores of Streptomyces griseoviridis K61. This study demonstrated that the soil-born filamentous and sporulating Actinomycetes greatly contribute to the feeding and the fitness of the plants and can be of great value for agriculture. This study is expected to lead to the conception/formulation and commercialization of novel bio-phosphate fertilizers and bio-fungicides constituted by the ad hoc Actinomycetes strains associated or not to pulverize NP. The availability of such products could contribute to the development of a sustainable agriculture more respectful of the environment and of human health than the today agriculture.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24

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    Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RTPCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis

    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

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    Number: 1 Publisher: Multidisciplinary Digital Publishing InstituteSome 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

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

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    International audienceA 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 β-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

    Structural and Biochemical Analysis of a Phosin from Streptomyces chartreusis Reveals a Combined Polyphosphate- and Metal-Binding Fold

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    International audienceX-ray crystallographic analysis of a phosin (PptA) from Steptomyces chartreusis reveals a metal-associated, lozenge-shaped fold featuring a 5–10 angström wide, positively charged tunnel that traverses the protein core. Two distinct metal-binding sites were identified in which the predominant metal ion was Cu2+. In solution, PptA forms stable homodimers that bind with nanomolar affinity to polyphosphate, a stress-related biopolymer acting as a phosphate and energy reserve in conditions of nutrient depletion. A single protein dimer interacts with 14–15 consecutive phosphate moieties within the polymer. Our observations suggest that PptA plays a role in polyphosphate metabolism, mobilisation or sensing, possibly by acting in concert with polyphosphate kinase (Ppk). Like Ppk, phosins may influence antibiotic synthesis by streptomycetes

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

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    International audienceStreptomyces 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 ÎĽgml(-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
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