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Certain bacterial species produce antimicrobial compounds only in the presence of a competing species. However, little is known on the frequency of interaction-mediated induction of antibiotic compound production in natural communities of soil bacteria. Here we developed a high-throughput method to screen for the production of antimicrobial activity by monocultures and pair-wise combinations of 146 phylogenetically different bacteria isolated from similar soil habitats. Growth responses of two human pathogenic model organisms, Escherichia coli WA321 and Staphylococcus aureus 533R4, were used to monitor antimicrobial activity. From all isolates, 33% showed antimicrobial activity only in monoculture and 42% showed activity only when tested in interactions. More bacterial isolates were active against S. aureus than against E. coli. The frequency of interaction-mediated induction of antimicrobial activity was 6% (154 interactions out of 2798) indicating that only a limited set of species combinations showed such activity. The screening revealed also interaction-mediated suppression of antimicrobial activity for 22% of all combinations tested. Whereas all patterns of antimicrobial activity (non-induced production, induced production and suppression) were seen for various bacterial classes, interaction-mediated induction of antimicrobial activity was more frequent for combinations of Flavobacteria and alpha- Proteobacteria. The results of our study give a first indication on the frequency of interference competitive interactions in natural soil bacterial communities which may forms a basis for selection of bacterial groups that are promising for the discovery of novel, cryptic antibiotics.
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Improved Catalytic Efficiency and Active Site Modification of 1,4-β-D-Glucan Glucohydrolase A from Thermotoga neapolitana by Directed Evolution*
Thermotoga neapolitana 1.4-β-D-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 °C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The Vmax for the mutant was 108 ± 3 units mg-1, whereas that of the WT was 75 ± 2 units mg-1. The Km for both proteins was nearly the same. The kcat for the new enzyme increased by 31% and its catalytic efficiency (kcat/Km) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.
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The transcriptional response of Arabidopsis to genotoxic stress – a high‐density colony array study (HDCA)
A genome‐wide transcription profiling of Arabidopsis upon genotoxic stress has been performed using a high‐density colony array (HDCA). The array was based on a library of 27 000 cDNA clones derived from Arabidopsis cells challenged with bleomycin plus mitomycin C. The array covers more than 10 000 individual genes (corresponding to at least 40% of Arabidopsis genes). After hybridisation of the HDCA with labelled cDNA probes obtained from genotoxin‐treated (bleomycin plus mitomycin C, 6 h) and untreated seedlings, 39 genes revealed an increased and 24 genes a decreased expression among the 3200 highly expressed clones (representing approximately 1200 individual genes because of redundancy of the cDNA library). Of the 4900 clones with a low transcriptional level, the expression of 500 clones was found to be altered and 57 genes with increased and 22 genes with decreased expression were identified by sequence analysis of 135 identified clones. The HDCA results were validated by real‐time PCR analysis. For about 80% of genes (34 out of 42), alteration in expression was confirmed, indicating the reliability of the HDCA for transcription profiling. DNA damage and stress‐responsive genes encoding, for instance transcription factors (myb protein and WRKY1), the ribonucleotide reductase small subunit (RNR2), thymidine kinase (TK), an AAA‐type ATPase, the small subunit of a DNA polymerase and a calmodulin‐like protein were found to be strongly upregulated. Also, several genes involved in cell cycle regulation revealed significant alteration in transcription, as detected by real‐time PCR analysis, suggesting disturbance of cell cycle progression by mutagen treatment.
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