Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues

Kocuria polaris sp. nov., an orange-pigmented psychrophilic bacterium isolated from an Antarctic cyanobacterial mat sample

Strain CMS 76orT, an orange-pigmented bacterium, was isolated from a cyanobacterial mat sample from a pond located in McMurdo Dry Valley, Antarctica. On the basis of chemotaxonomic and phylogenetic properties, strain CMS 76orT was identified as a member of the genus Kocuria. It exhibited a 16S rDNA similarity of 99·8% and DNA-DNA similarity of 71% with Kocuria rosea (ATCC 186T). Phenotypic traits confirmed that strain CMS 76orT and K. rosea were well differentiated. Furthermore, strain CMS 76orT could be differentiated from the other reported species of Kocuria, namely Kocuria kristinae (ATCC 27570T), Kocuria varians (ATCC 15306T), Kocuria rhizophila (DSM 11926T) and Kocuria palustris (DSM 11025T), on the basis of a number of phenotypic features. Therefore, it is proposed that strain CMS 76orT (=MTCC 3702T =DSM 14382T) be assigned to a novel species of the genus Kocuria, as Kocuria polaris

Sequence alignment and domain organisation for <i>Sp</i> CBPs.

<p>(A) Full-length sequences of <i>Sp</i> CBP21, <i>Sp</i> CBP28, <i>Sp</i> CBP50 and <i>Sm</i> CBP21 (CBP21 from <i>S. marcescens</i>) were aligned using clustalw2. Residues that are thought to be located in the binding surface for chitin present in <i>Sm</i> CBP21, <i>Sp</i> CBP21, <i>Sp</i> CBP50 and not present in <i>Sp</i> CBP28 are shaded in yellow (as derived from the crystal structure of <i>Sm</i> CBP21, as well as mutagenesis studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036714#pone.0036714-VaajeKolstad2" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036714#pone.0036714-VaajeKolstad3" target="_blank">[8]</a>). Residue involved in the chitin-binding and functional properties of <i>Sm</i> CBP21 but also conserved in <i>Sp</i> CBP28 are shaded grey. The arrow indicates the terminal amino acid of the N-terminal signal sequence for respective CBPs. (B) The sequences of <i>Sp</i> CBPs were submitted to SMART domain data base (<a href="http://smart.embl-heidelberg.de/" target="_blank">http://smart.embl-heidelberg.de/</a>). The part indicated in red colour shows the signal peptide and the region Chitin_bind_3 indicates the chitin binding domain.</p

The 3D models of <i>Sp</i> CBP21 and <i>Sp</i> CBP50.

<p>(A and B) The models <i>Sp</i> CBP21 and ChBD region of <i>Sp</i> CBP50 were generated by Modeller9v8 (<a href="http://www.salilab.org/modeller/" target="_blank">http://www.salilab.org/modeller/</a>) using <i>Sm</i> CBP21 (PDB ID: 2BEM) as structure template. Residues important for chitin binding were shown in sticks representation with carbon, oxygen and nitrogen atoms colored light green, red and dark blue, respectively. The figures were prepared using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>), (C and D) Stereo view of the superimposed structure of <i>Sp</i> CBP21 and <i>Sp</i> CBP50 (green) with <i>Sm</i> CBP21 (red), respectively.</p

Details of bacterial CBPs and their binding preferences.

a<p>Details of binding to other substrates not available.</p

β-chitin hydrolysis enhancing effects of <i>Sp</i> CBP21 and <i>Sp</i> CBP50 with <i>Sp</i> ChiD.

<p>Reaction mixture (1 mL) containing 0.25 mg/mL of β-chitin, 0.25 µM/0.50 µM/0.75 µM/1.0 µM <i>Sp</i> ChiD incubated individually with 0.3 µM of <i>Sp</i> CBP21/<i>Sp</i> CBP50 or combining both <i>Sp</i> CBP21 and <i>Sp</i> CBP50 (0.15 µM +0.15 µM/0.30 µM +0.30 µM), in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 24 h at 1000 rpm, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments.</p

Equilibrium adsorption isotherms of <i>Sp</i> CBP21 and <i>Sp</i> CBP50 to α- and β-chitin.

<p>The reaction assay (1 mL) containing 1.0 mg substrates (α- and β- chitin) and varied concentrations of <i>Sp</i> CBP21 and <i>Sp</i> CBP50 starting from 0 to 10.0 µM was incubated (<i>Sp</i> CBP21 with α- and β-chitin, 12 h and 6 h, respectively; <i>Sp</i> CBP50 with α- and β-chitin, 12 h) at 37°C. The reaction mixtures were centrifuged and concentration of bound protein (P_bound) and un-bound free protein (P_free) was determined and plotted to fit into GraphPad Prism software version 5.0. All data sets were fitted to the equation for one-site binding by non-linear regression function, and to calculate <i>B</i>_max and <i>K</i>_d using GraphPad Prism software version 5.0. (A and B) The <i>K</i>_d and <i>B</i>_max values of <i>Sp</i> CBP21 and <i>Sp</i> CBP50 were shown in the inset table.</p

List of primers used for the amplification of genes coding for chitin binding proteins and chitinases from <i>Serratia proteamaculans</i> 568.

a<p>Sequences underlined represent restriction sites.</p

CyanoPhyChe: A Database for Physico-Chemical Properties, Structure and Biochemical Pathway Information of Cyanobacterial Proteins

<div><p>CyanoPhyChe is a user friendly database that one can browse through for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins. We downloaded all the protein sequences from the cyanobacterial genome database for calculating the physico-chemical properties, such as molecular weight, net charge of protein, isoelectric point, molar extinction coefficient, canonical variable for solubility, grand average hydropathy, aliphatic index, and number of charged residues. Based on the physico-chemical properties, we provide the polarity, structural stability and probability of a protein entering in to an inclusion body (PEPIB). We used the data generated on physico-chemical properties, structure and biochemical pathway information of all cyanobacterial proteins to construct CyanoPhyChe. The data can be used for optimizing methods of expression and characterization of cyanobacterial proteins. Moreover, the ‘Search’ and data export options provided will be useful for proteome analysis. Secondary structure was predicted for all the cyanobacterial proteins using PSIPRED tool and the data generated is made accessible to researchers working on cyanobacteria. In addition, external links are provided to biological databases such as PDB and KEGG for molecular structure and biochemical pathway information, respectively. External links are also provided to different cyanobacterial databases. CyanoPhyChe can be accessed from the following URL: <a href="http://bif.uohyd.ac.in/cpc">http://bif.uohyd.ac.in/cpc</a>.</p> </div