Effect of non-rhizospheric bacterial strains on growth of crop plants.

<div><p>Seeds bacterized with respective bacterial strains (approximately1x10⁷cfu/seed, unless otherwise mentioned) were grown <i>in </i><i>vitro</i> in MS medium. After 30 days of growth, shoot height and root length were measured in centimeters, while fresh weight and dry weight of entire plant were measured in milligrams after 30 days of growth. Data represent the mean of the three independent experiments. The vertical line indicates standard error.</p> <p>(A) Effect of five different bacterial strains on growth of tobacco. Treatments included 1. <i>Bacillus </i><i>cereus</i>, 2. <i>B. subtilis</i>, 3. <i>Paenibacillus </i><i>elgii</i>, 4. <i>Stenotrophomonas </i><i>maltophilia</i>, 5. <i>Serratia </i><i>marcescens</i>, and 6. Control, (n=20). Different letters on each bar represent values that were significantly different (p_0.05). (B) Effect of <i>B. cereus</i> on growth of tobacco, tomato, pigeon pea and groundnut (n=24). Data represents percent increase over control. </p> <p>(C) Colonization of <i>B. cereus</i> on tobacco and groundnut roots. Number of days (d) for tobacco: 10, 20, 30 and 40 days of growth and for groundnut: 5, 10, 15 and 20 days of growth (n=20). Students’ t-test of each growth parameter against control for each crop was performed. ** indicate statistically significant at p<0.01, NS =indicate not significant.</p></div

Representative 2DE gels of <i>B. cereus</i> cell-wall proteome.

<p><i>B. cereus</i> grown in MM media amended with (A) tobacco root exudates or (B) groundnut root exudates. In the first dimension (IEF), 500 μg of protein was loaded on an 18 cm IPG strip with a linear gradient of pH 4-7 and 12.5% SDS-PAGE gels were used in the second dimension. Proteins were visualized by Coomasie blue staining. Arrows point towards the differentially expressed proteins. </p

Kinetic parameters of <i>Sp</i>ChiD fusion chimeras.

<p>Kinetic parameters of <i>Sp</i>ChiD fusion chimeras.</p

Schematic representation of domain organization.

<p><i>Sp</i>ChiD fusion chimeras generated using overlap extension PCR. The designation of chimeras was based on the type of domain fused and its orientation.</p

Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

<div><p>Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from <i>Serratia proteamaculans</i> (<i>Sp</i>ChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. <i>Sp</i>ChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of <i>Sp</i>ChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different <i>Sp</i>ChiD fusion chimeras, two C-terminal fusions <i>viz.</i> ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal <i>Sp</i>ChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.</p></div

Product profiles of the <i>Sp</i>ChiD fusion chimeras.

<p>HPLC quantification profiles for the <i>Sp</i>ChiD fusion chimeras represent the hydrolytic (DP1-DP3) and quantifiable TG (DP5, DP6) products accumulated during the course of reaction with 1 mM DP4 substrate. All the reactions were performed at 40°C, in 50 mM sodium phosphate buffer, pH 8.0. (A) <i>Sp</i>ChiD, (B) ChiD+CBP, (C) CBP+ChiD, (D) ChiD+PKD, (E) PKD+ChiD, (F) CDP and (G) PDC.</p

Degradation of α- or β-chitin by <i>Sp</i>ChiD fusion chimeras.

<p>(A) α-chitin (or) (B) β-chitin (2.5% (w/v)) was incubated with 1μM each of <i>Sp</i>ChiD or its fusion chimeras at 40°C for 1 h, in 50 mM sodium acetate buffer, pH 5.2. The values are based on mean ±SD of three identical experiments. Data were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test. Statistical significance was determined at p≤0.05.</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

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