Figures & data
Figure 1. Procedure for sampling and isolation of microorganisms that produce chitinases and form biofilms.
Table 1. Oligonucleotides used in this study.
Figure 2. The clearing zone formed by the isolates and references on the YEM agar plates containing colloidal chitin.
The isolates and references were cultured on the YEM agar plates containing 0.5% colloidal chitin at 30 °C for 5 days. 1, SWSY-3.47; 2, SWSY-3.27; 3, SWCY-1.31; 4, SWSY-3.11; 5, Bacillus circulans WL-12; and 6, Serratia marcescens 2170. B. circulans WL-12 and S. marcescens 2170 were used as references.
Figure 3. Chitinase activity and biofilm formation of the isolates.
Chitinase activity of the isolates produced in the culture supernatants of the YEM medium in the presence of 0.2% colloidal chitin was measured by a modified version of the Schales’ procedure. Biofilm formation of the isolates formed in the 96-well polystyrene microtiter plates containing LB medium was estimated by absorbance at 630 nm using a microtiter plate reader (Bio-Rad, USA). Bar, specific activity (U/mg protein) of chitinases produced by the isolates; triangle, biofilm formation (crystal violet staining [A630]).
Table 2. Sequence analysis of partial 16S rRNA genes from the isolated strains.
Figure 4. Phylogenetic analysis of the isolates (filled rectangle) based on the 16S rRNA gene sequences.
Accession numbers for the available 16S rRNA gene sequences used are given in parentheses behind species and strain names. The phylogenetic tree was calculated and drawn by using the MEGA version 6.0 software after multiple alignments of the data by CLUSTAL W. The tree was constructed using the neighbor-joining method, and the evolutionary distances were computed using the Kimura two-parameter method. The numbers at the branches are bootstrap confidence percentages (%) based on 1000 resampled data sets; only bootstrap confidence percentages > 50% are shown. Bar, 0.02 substitutions per nucleotide position.
Figure 5. Time course of chitinase production in the culture supernatant.
The candidates and reference were cultured in the YEM medium containing 0.5% chitin powder at 30 °C. At certain intervals, a portion of the culture medium was withdrawn, and chitinase activity in the culture supernatants was measured by a modified version of the Schales’ procedure with GlcNAc as a standard. Protein concentration was measured using the BCA Protein Assay Kit (Thermal Scientific, USA) with bovine serum albumin as a standard. (♦, blue), A. lacus SWCS-3.14; (■, pink), A. salmonicida subsp. salmonicida SWSY-1.31; (▲, red), S. plymuthica SWSY-3.47; (●, green), A. salmonicida subsp. salmonicida SWSY-1.411; (∗, violet), S. marcescens 2170. S. marcescens 2170 was used as a reference.
Figure 6. SDS–PAGE and zymography analyses of the chitinase production in the culture supernatant.
The candidates and the reference were grown in YEM medium containing 0.5% chitin powder at 30 °C. The chitinase production was taken from cultured supernatants at day 8 of cultivation and employed for SDS–PAGE and zymography analyses. (A) Protein staining of the polyacrylamide gel with 0.2% Coomassie brilliant blue R-250. (B) Chitinase activity detected on an agar replica of the SDS–polyacrylamide gel. Lane M, size markers; lanes 1 through 4, proteins secreted by A. lacus SWCS-3.14, A. salmonicida subsp. salmonicida SWSY-1.31, S. plymuthica S WSY-3.47, and A. salmonicida subsp. salmonicida SWSY-1.411, respectively; lane 5, proteins secreted by S. marcescens 2170. kDa, Kilodaltons; arrows, protein active bands. 65 μg of protein (130 μg of protein for A. lacus SWCS-3.14) was applied to each lane.
Figure 7. The antagonistic ability of the isolates against the hyphal growth of T. reesei using the dual-culture assay.
The hyphal growth of T. reesei on the PDA:YEM (1:1) plates containing 0.2% glycol chitin in the absence (A) and presence of the candidates (B) at day 3 of cultivation at 30 °C. 1, A. lacus SWCS-3.14; 2, A. salmonicida subsp. salmonicida SWSY-1.31; 3, S. plymuthica SWSY-3.47; 4, A. salmonicida subsp. salmonicida SWSY-1.411. The experiments were performed in triplicate.
Figure 8. The inhibition of hyphal growth of T. reesei treated by the crude proteins.
A suspension of conidia of T. reesei was inoculated onto a paper disk. After 24 h of cultivation at 25 °C, a solution containing 0.4 mg of protein was applied to a well punched at a distance of 15 mm from the plate center. The plates were then incubated at 25 °C for a further 21 h and the inhibition of hyphal growth was evaluated by visual inspection. Control, sterile water. (A) A. lacus SWCS-3.14 crude proteins; (B) S. plymuthica SWSY-3.47 crude proteins; (C) A. salmonicida subsp. salmonicida SWSY-1.31 crude proteins; (D) A. salmonicida subsp. salmonicida SWSY-1.411 crude proteins. 1, The protein solution containing chitinases; 2, the protein solution containing inactivated chitinases by boiling for 10 min. The experiments were performed in triplicate.
Table 3. The growth inhibition of T. reesei by using the crude proteins.
Supplemental material
