Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan

<p>Abstract</p> <p>Background</p> <p>Chitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (<it>csnN106</it>) in <it>Streptomyces lividans</it>.</p> <p>Results</p> <p>Two <it>S. lividans </it>strains were used for induction experiments: the wild type strain and its mutant (<it>ΔcsnR)</it>, harbouring an in-frame deletion of the <it>csnR </it>gene, encoding a negative transcriptional regulator. Comparison of chitosanase levels in various media indicated that CsnR regulates negatively the expression of the heterologous chitosanase gene <it>csnN106</it>. Using the <it>ΔcsnR </it>host and a mutated <it>csnN106 </it>gene with a modified transcription operator, substantial levels of chitosanase could be produced in the absence of chitosan, using inexpensive medium components. Furthermore, chitosanase production was of higher quality as lower levels of extracellular protease and protein contaminants were observed.</p> <p>Conclusions</p> <p>This new chitosanase production system is of interest for biotechnology as only common media components are used and enzyme of high degree of purity is obtained directly in the culture supernatant.</p

Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments

AbstractOligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of d-glucosamine oligosaccharides ((GlcN)n, n=3,4,5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (Tm) of E22Q was found to be 44.4°C at pH 7.0. However, the Tm increased upon the addition of (GlcN)n by 1.3°C (n=3), 2.5°C (n=4), 5.2°C (n=5), or 7.6°C (n=6). No appreciable change in Tm was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)n on the thermal stability was examined using the other protein, RNaseT1, but the oligosaccharide did not affect Tm of the protein. Thus, we concluded that the stabilization effect of (GlcN)n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in Tm induced by (GlcN)6 binding were 2.7°C for E22D and 4.2°C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft

NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases

Detection of prokaryotic promoters from the genomic distribution of hexanucleotide pairs

BACKGROUND: In bacteria, sigma factors and other transcriptional regulatory proteins recognize DNA patterns upstream of their target genes and interact with RNA polymerase to control transcription. As a consequence of evolution, DNA sequences recognized by transcription factors are thought to be enriched in intergenic regions (IRs) and depleted from coding regions of prokaryotic genomes. RESULTS: In this work, we report that genomic distribution of transcription factors binding sites is biased towards IRs, and that this bias is conserved amongst bacterial species. We further take advantage of this observation to develop an algorithm that can efficiently identify promoter boxes by a distribution-dependent approach rather than a direct sequence comparison approach. This strategy, which can easily be combined with other methodologies, allowed the identification of promoter sequences in ten species and can be used with any annotated bacterial genome, with results that rival with current methodologies. Experimental validations of predicted promoters also support our approach. CONCLUSION: Considering that complete genomic sequences of over 1000 bacteria will soon be available and that little transcriptional information is available for most of them, our algorithm constitutes a promising tool for the prediction of promoter sequences. Importantly, our methodology could also be adapted to identify DNA sequences recognized by other regulatory proteins

(1RS,2SR,5SR)-9-Benzyl-2-[(1RS)-1-hy­droxy­benz­yl]-9-aza­bicyclo­[3.3.1]nonan-3-one from synchrotron data

In the crystal structure of the racemic title compound, C22H25NO2, solved and refined against sychrotron diffraction data, the hy­droxy group and the carbonyl O atom participate in the formation of O—H⋯O hydrogen bonds between pairs of enanti­omers related by a crystallographic centre of symmetry

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and kcat, while Km− was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity

DNA Data Visualization (DDV): Software for Generating Web-Based Interfaces Supporting Navigation and Analysis of DNA Sequence Data of Entire Genomes

Data visualization methods are necessary during the exploration and analysis activities of an increasingly data-intensive scientific process. There are few existing visualization methods for raw nucleotide sequences of a whole genome or chromosome. Software for data visualization should allow the researchers to create accessible data visualization interfaces that can be exported and shared with others on the web. Herein, novel software developed for generating DNA data visualization interfaces is described. The software converts DNA data sets into images that are further processed as multi-scale images to be accessed through a web-based interface that supports zooming, panning and sequence fragment selection. Nucleotide composition frequencies and GC skew of a selected sequence segment can be obtained through the interface. The software was used to generate DNA data visualization of human and bacterial chromosomes. Examples of visually detectable features such as short and long direct repeats, long terminal repeats, mobile genetic elements, heterochromatic segments in microbial and human chromosomes, are presented. The software and its source code are available for download and further development. The visualization interfaces generated with the software allow for the immediate identification and observation of several types of sequence patterns in genomes of various sizes and origins. The visualization interfaces generated with the software are readily accessible through a web browser. This software is a useful research and teaching tool for genetics and structural genomics

production of chitooligosaccharides from Rhizopus oligosporus NRRL2710 cells by chitosanase digestion

The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglu- cosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS 13C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25 m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN–GlcNAc, (GlcN)2–GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosan- ase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact

CLUSS: Clustering of protein sequences based on a new similarity measure

<p>Abstract</p> <p>Background</p> <p>The rapid burgeoning of available protein data makes the use of clustering within families of proteins increasingly important. The challenge is to identify subfamilies of evolutionarily related sequences. This identification reveals phylogenetic relationships, which provide prior knowledge to help researchers understand biological phenomena. A good evolutionary model is essential to achieve a clustering that reflects the biological reality, and an accurate estimate of protein sequence similarity is crucial to the building of such a model. Most existing algorithms estimate this similarity using techniques that are not necessarily biologically plausible, especially for hard-to-align sequences such as proteins with different domain structures, which cause many difficulties for the alignment-dependent algorithms. In this paper, we propose a novel similarity measure based on matching amino acid subsequences. This measure, named SMS for Substitution Matching Similarity, is especially designed for application to non-aligned protein sequences. It allows us to develop a new alignment-free algorithm, named CLUSS, for clustering protein families. To the best of our knowledge, this is the first alignment-free algorithm for clustering protein sequences. Unlike other clustering algorithms, CLUSS is effective on both alignable and non-alignable protein families. In the rest of the paper, we use the term "<it>phylogenetic</it>" in the sense of "<it>relatedness of biological functions</it>".</p> <p>Results</p> <p>To show the effectiveness of CLUSS, we performed an extensive clustering on COG database. To demonstrate its ability to deal with hard-to-align sequences, we tested it on the GH2 family. In addition, we carried out experimental comparisons of CLUSS with a variety of mainstream algorithms. These comparisons were made on hard-to-align and easy-to-align protein sequences. The results of these experiments show the superiority of CLUSS in yielding clusters of proteins with similar functional activity.</p> <p>Conclusion</p> <p>We have developed an effective method and tool for clustering protein sequences to meet the needs of biologists in terms of phylogenetic analysis and prediction of biological functions. Compared to existing clustering methods, CLUSS more accurately highlights the functional characteristics of the clustered families. It provides biologists with a new and plausible instrument for the analysis of protein sequences, especially those that cause problems for the alignment-dependent algorithms.</p