Modeling and Statistical Optimization of Culture Conditions for Improvement of Antifungal Compounds Production by Streptomyces albidoflavus S19 Strain of Wastewater Origin.

BACKGROUND: The actinomycetes strains isolated from unexplored ecosystems are a promising alternative for the biosynthesis of novel antimicrobial compounds. Depending on the interesting antifungal activity of the studied strain S19, the statistical method seems to be an effective tool for optimizing the production of anticandidal molecules. INTRODUCTION: This study was conducted in order to optimize the culture parameters (medium nutrients concentrations and initial pH value) affecting the production of antifungal metabolites from S. albidoflavus strain S19 (obtained from wastewater collected in Bejaia region, Algeria) using Response Surface Metho-dology (RSM). The best conditions for anti-Candida albicans compounds biosynthesis were determined. METHODS AND RESULTS: The antimicrobial producer strain S. albidoflavus S19 was identified on the basis of morphological, chemicals characters and physiological characteristics along with 16S rRNA gene se-quencing analysis.Response Surface Methodology by Central Composite Design (CCD) was employed to improve the anti-C. albicans agents production through the optimization of medium parameters. The highest antifungal ac-tivity was obtained by using a mixture of 2g l-1 starch, 4g l-1 yeast extract, 2g l-1 peptone at pH 11. CONCLUSION: The strain S19 isolated from wastewater showed a significant anti-C. albicans activity and this study revealed the effectiveness of RSM and CCD for increasing bioactive compounds production, rising the diameter of inhibition zones from 13 to 34 mm.

Norine: A powerful resource for novel nonribosomal peptide discovery.

Since its first release in 2008, Norine remains the unique resource completely devoted to nonribosomal peptides (NRPs). They are very attractive microbial secondary metabolites, displaying a remarkable diversity of structure and functions. Norine (http://bioinfo.lifl.fr/NRP) includes a database now containing more than 1160 annotated peptides and user-friendly interfaces enabling the querying of the database, through the annotations or the structure of the peptides. Dedicated tools are associated for structural comparison of the compounds and prediction of their biological activities. In this paper, we start by describing the knowledgebase and the dedicated tools. We then present some user cases to show how useful Norine is for the discovery of novel nonribosomal peptides.

Effect of pps disruption and constitutive expression of srfA on surfactin productivity, spreading and antagonistic properties of Bacillus subtilis 168 derivatives.

AIMS: To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. METHODS AND RESULTS: The srfA native promoter was replaced by the constitutive promoter P(repU) in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of P(repU), than that found with P(srfA), led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1.47 g g(-1) of biomass), spreading behaviour and haemolytic activity of the strains. CONCLUSIONS: This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.

Development of a biological test to evaluate the bioavailability of iron in culture media.

AIMS: To develop an easy-to-use and pathogen-free protocol giving reliable information on the bioavailability of iron in a medium. METHODS AND RESULTS: In aerobic conditions, iron bioavailability is very low, and most of its forms cannot be assimilated by micro-organisms. Media with similar iron contents can differ considerably in iron bioavailability, something that is not easily achieved using conventional physicochemical methods. The assay developed in the present work is based on a pyoverdin siderophore release by fluorescent Pseudomonas in response to iron stress. CONCLUSIONS: The test was applied to a complex medium used for the production of diphtheria toxin (DT). A significant difference between the bioavailable iron level and the total chemical concentrations contributed by the various compounds used to make the medium could thus be detected. This can be explained by the formation of salt complexes trapping the iron, which thus cannot be used directly by the micro-organism for its metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay can easily be applied to any medium designed for the production of iron-regulated compounds. This is particularly useful when dealing with processes that use pathogenic strains as was shown in the case based on DT production.

Functional significance of a periplasmic Mn-superoxide dismutase from Aeromonas hydrophila.

AIMS: A better understanding of the role of superoxide dismutases (SODs) from Aeromonas hydrophila and particularly the Mn-SOD which shares a peculiar localization within the bacterial periplasm and is only detected during the stationary phase of growth. METHODS AND RESULTS: A. hydrophila ATCC 7966 can express two distinct SODs: an Fe-SOD and an Mn-SOD. Using insertional mutagenesis, an Mn-SOD-deficient mutant was isolated. After growth of this mutant under conditions leading to the expression of an Mn-SOD, only the Fe-SOD could be detected in nondenaturing PAGE. Study of its response to the oxidative stress showed that the Mn-SOD was not implicated in the protection against intracellular superoxide but defended the bacterial cells against environmental superoxide. CONCLUSIONS: By protecting the bacteria against external superoxide, the role of the Mn-SOD from A. hydrophila is equivalent to that of the Cu/Zn-SOD from the well-studied Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of this Mn-SOD is in agreement with its periplasmic localization and may confer an advantage on the bacteria such as a virulence factor in cases of pathogenicity.

Occurrence of two superoxide dismutases in Aeromonas hydrophila: molecular cloning and differential expression of the sodA and sodB genes.

Aeromonas spp., considered as emerging opportunistic pathogens, belong to the family Vibrionaceae. Among the criteria currently used for their classification is the presence of a single FeSOD (iron-containing superoxide dismutase), which distinguishes them from Enterobacteriacea. In this paper the cloning of the sodA and sodB genes encoding two different SODs in Aeromonas hydrophila ATCC 7966 is reported. The sodB gene encoded an FeSOD (196 amino acids, 21.5 kDa), was constitutively expressed and showed 75% homology with the E. coli FeSOD. The sodA gene encoded a protein of 206 amino acids (22.5 kDa) with MnSOD (manganese-containing SOD) activity and showed 55% homology with the Escherichia coli MnSOD. The MnSOD of A. hydrophila was detected only during the stationary phase of growth under high aeration or when induced by lack of iron. Nevertheless, paraquat had no detectable effect on its production. The amino-terminal part of the Mn-containing protein contained a putative signal sequence which could permit a periplasmic localization.

News & notes: diversity of superoxide-dismutases among clinical and soil isolates of Streptomyces species.

Comparison of the nature, activity, and cellular localization of superoxide-dismutases (SOD) from soil and clinical isolates of Streptomyces species was investigated to identify possible factors that could account for the pathological role of the strains isolated from human lesions. Results showed that all of the studied strains possessed a cytoplasmic Ni-SOD. This particular SOD, found in isolates from patients, could be a new taxonomic criterion to identify Streptomyces species with greater precision. A second minor SOD, assimilated to an Fe/Zn-SOD, was detected in some strains, but no relationship was established between the presence of this enzyme and the clinical origin of the strains.

Binding to DNA, cellular uptake and biological activity of a distamycin-ellipticine hybrid molecule.

A hybrid molecule which conjugates the minor groove binding agent distamycin and an ellipticine derivative was synthesized and evaluated for cytostatic and cytotoxic activities against L1210 leukaemia cells in vitro. The binding of the hybrid molecule, named 'Distel', to a range of natural DNAs and synthetic polynucleotides with different base pair arrangements was studied by electric linear dichroism. The interaction with DNA simultaneously implicates binding of the distamycin part in the minor groove and intercalation of the ellipticine chromophore. The drug binds to DNA without any apparent preference for AT or GC polynucleotides, and can accommodate both homopolymeric and co-polymeric sequences as a binding site. However, the geometry of the drug-DNA complex varies depending on the targeted sequence. The lower activity of the hybrid as compared to the ellipticine derivative cannot be explained in terms of DNA binding. Taking advantage of the fluorescence of the pyridocarbazole chromophore, fluorescence microscopy was used to map cellular uptake of the hybrid molecule compared to the ellipticine derivative. Both the conjugate and the ellipticine derivative preferentially accumulate in the nuclei of HeLa cells rather than in the cytoplasm. Nuclei of ellipticine derivative-treated cells appear markedly more fluorescent than those of cells treated with the hybrid, which seems to be preferentially located in the nucleoli. Therefore, we consider the possibility that the difference in cytotoxicity between the two ellipticine-containing drugs is due to different intranuclear concentrations of these two compounds.

Comparison between E1A gene from oncogenic and non-oncogenic adenoviruses in cellular transformation (Ad E1A conserved region).

All adenoviruses transform primary BRK cells in vitro, but only cells transformed by oncogenic adenoviruses are tumorigenic for immunocompetent animals. The transforming E1 regions of human Ad 2 and Ad 12 also differ from each other in the frequency in which they can transform BRK cells. We have investigated these properties which can be assigned to the specific domain of the E1A region. For this purpose, chimeric E1A regions between Ad 2 and Ad 12 have been constructed. The efficiency of cell transformation appeared to be determined by the encoding region. The promoter sequences were not important for an efficient cellular transformation although the E1B region cis activated in E1A transcription in both cell transformation and transient expression. We show that sequences located in the E1B promoter were responsible for this effect. In the encoding region the CR 1 domain was essential for the cell transformation frequency.