Chitinolytic and Fungicidal Potential of the Marine Bacterial Strains Habituating Pacific Ocean Regions

Abstract

Screening for chitinolytic activity in the bacterial strains from different Pacific Ocean regions revealed that the highly active representatives belong to the genera Microbulbifer, Vibrio, Aquimarina, and Pseudoalteromonas. The widely distributed chitinolytic species was Microbulbifer isolated from the sea urchin Strongylocentrotus intermedius. Among seventeen isolates with confirmed chitinolytic activity, only the type strain P. flavipulchra KMM 3630^T and the strains of putatively new species Pseudoalteromonas sp. B530 and Vibrio sp. Sgm 5, isolated from sea water (Vietnam mollusc farm) and the sea urchin S. intermedius (Peter the Great Gulf, the Sea of Japan), significantly suppressed the hyphal growth of Aspergillus niger that is perspective for the biocontrol agents' development. The results on chitinolytic activities and whole-genome sequencing of the strains under study, including agarolytic type strain Z. galactanivorans Dji^T, found the new functionally active chitinase structures and biotechnological potential.

Keywords: antifungal activity; chitin-degrading enzymes; chitinase activity; functional genomics; marine chitinolytic bacteria; whole-genome sequencing.

Figure 1

The degradation of colloidal chitin, supplemented into LB agar medium, by the marine bacterial strains: clearing zones were around colonies of Microbulbifer thermotolerans on the 4th–6th days; Vibrio sp. Sgm 5 on the 4th day; Vibrio sp. 14G-20 and Sgm 22 on the 6th day; both Pseudoalteromonas sp. B530 and Pseudoalteromonas flavipulchra KMM 3630^T on the 6th day; Aquimarina muelleri and Zobellia galactanivorans Dji^T on the 11th day, caused by their putative chitinase activity.

Figure 2

Chitinase activity in the culture supernatant of Microbulbifer thermotolerans, Vibrio spp., Aquimarina muelleri, and Zobellia galactinovorans. Reactions were carried out for 15 min, at 37 °C, using colloidal chitin (0.1%) as a substrate, and 20 mM sodium phosphate buffer, pH 6.0. The experiments were performed in triplicate and standard deviations are shown as error bars.

Figure 3

SDS–PAGE analysis of the culture supernatants: lane kDa, protein size markers (BioRad); lanes C—control culture supernatants grown without colloidal chitin, lanes CC—culture supernatants grown in the presence of colloidal chitin. The lanes under square brackets 1—Microbulbifer thermotolerans Sgf 25; square brackets 2–6—M. thermotolerans Sh 1–5; square brackets 7—M. thermotolerans JCM 14709^T; square brackets 8—M. thermotolerans Sgm 25/1; square brackets 9—M. thermotolerans 14G-22; square brackets 10—Vibrio sp. Sgm 5; square brackets 11—Vibrio sp. 14G-20; square brackets 12—Vibrio sp. Sgm 22; square brackets 13—Pseudoalteromonas sp. B530; square brackets 14—Pseudoalteromonas flavipulchra KMM 3630^T; square brackets 15—Aquimarina muelleri V1SW 51; square brackets 16—A. muelleri V1SW 49^T; square brackets 17—Zobellia galactanivorans Dji^T. Predicted induced chitin-degrading enzymes compared to control are underlined by squares with broken line. Protein was concentrated to 30 µg with 10% trichloroacetic acid and applied into the well.

Figure 4

Chitinase activity of the recombinant proteins: 531 Sgf 25 GH18, 509 Sgf 25 GH18, 571 Sgf 25 GH18, and 1036 Sgf 25 GH18 from Microbulbifer thermotolerans Sgf 25; 440 DjiT GH18, 547 DjiT GH23, and 321 DjiT GH23 from Zobellia galactanivorans Dji^T. The crude extract of E. coli Rosetta DE3 transformed by the plasmid pET40 b(+) without a target gene insertion was used as the negative control. The chitinolytic activity was determined by measuring the amount of reducing ends liberated from enzymatically hydrolysed colloidal chitin.

Figure 5

Antifungal activity of the isolates against the hyphal growth of Aspergillus niger. 1, Pseudoalteromonas sp. B530; 2, Pseudoalteromonas flavipulchra KMM 3630^T; 3, Vibrio sp. Sgm 5. The inhibition of mycelial extension was determined by visual inspection on agar plates (Difco Marine Broth, bacteriological agar-agar, 0.2% colloidal chitin) and shown by arrows. The experiments were performed in triplicate.