Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

We already reported the use of a long synthetic signal peptide (LSSP) to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b). This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203

Souches de micro-organismes pour la mise en oeuvre d'un procede de surproduction de metabolites, notamment a activite antibiotique ou enzymatique

The subject of the present invention is the use of strains of microorganisms producing compounds of interest, in which the ppk gene coding for polyphosphate kinase is inactivated, for the implementation of a process for stimulating the production of these compounds of interest by said transformed strains

The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans

International audienc

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

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

Souches de micro-organismes pour la mise en oeuvre d'un procede de surproduction de metabolites, notamment a activite antibiotique ou enzymatique

The subject of the present invention is the use of strains of microorganisms producing compounds of interest, in which the ppk gene coding for polyphosphate kinase is inactivated, for the implementation of a process for stimulating the production of these compounds of interest by said transformed strains

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

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

Highly efficient production of the staphylococcal nuclease reporter in <em>Lactobacillus bulgaricus</em> governed by the promoter of the <em>hlbA</em> gene

International audienc

Transposition in <em>Lactobacillus delbrueckii</em> subsp. <em>bulgaricus</em>: identification of two thermosensitive replicons and two functional insertion sequences

International audienceIn this report, it is shown that the rolling circle replicon pG(+)host and the theta replicon pIP501 are thermosensitive in Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus). Using a pIP501 derivative as a delivery vector for six insertion sequences originating from lactic acid bacteria, it is shown that IS1223 and IS1201 transpose in L. bulgaricus

Secretion of cyclodextrin glucanotransferase in <i>E. coli</i> using <i>Bacillus subtilis</i> lipase signal peptide and optimization of culture medium

72-79The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/ml) obtained for CGTase activity was very close to the predicted value (9.27 U/ml)