Genomic analysis and chitinase characterization of Vibrio harveyi WXL538: insight into its adaptation to the marine environment

Abstract

Chitin, the most abundant bio-polymer in seawater, may be utilized by various microorganisms as a carbon source. Vibrios have been regarded as one of the main groups of chitin consumers in the marine carbon cycle and chitinase producers. The organisms are widely distributed in the aquatic environment. However, the co-working mechanism between their chitinases, and whether the chitinase's diversity contributes to their adaption to the environment, needs to be further elucidated. Here, we obtained a chitinolytic strain, Vibrio harveyi WXL538 with eight putative chitinase-coding genes. Five of the genes, i.e., Chi4733, Chi540, Chi4668, Chi5174, and Chi4963, were overexpressed and validated, in which Chi4668, Chi4733 and Chi540 were purified and characterized. The result of Chi4668 was described in our previous study. Endo-chitinase Chi4733 degraded colloidal chitin to produce (GlcNAc)₂ and minor (GlcNAc)₃. The enzymatic activity of Chi4733 was 175.5 U mg^-1 and Kcat/Km was 54.9 s^-1 M^-1. Chi4733 had its maximum activity at 50°C and pH 4-6, activated by Sr²⁺, Co²⁺, Ca²⁺, and Mg²⁺ and inhibited by Al³⁺, Zn²⁺, Cu²⁺, Ni²⁺, and SDS. Exo-chitinase Chi540 degraded colloidal chitin to (GlcNAc)₂. The enzymatic activity of Chi540 was 134.5 U mg^-1 and Kcat/Km was 54.9 s^-1 M^-1. Chi540 had its maximum activity at 60°C and pH 6-8, was activated by Sr²⁺, Ca²⁺, and Mg²⁺ but inhibited by K⁺, Ba²⁺, Zn²⁺, Cu²⁺, Ni²⁺, SDS and urea. Whole genome analysis of V. harveyi WXL538 and characterization of its chitinase can provide a better understanding of its adaptability to the changing marine environment.

Keywords: Vibrio harveyi; adaptation; chitinase characterization; genome analysis; marine environment.

Figure 1

Growth and chitin degradation of Vibrio harveyi WXL538 under different conditions. (A) Conditions varied at temperature. (B) Conditions varied at pH.

Figure 2

Comparison of (GlcNAc)₂ operon and Nag operon in Vibrio harveyi WXL538 and V. cholerae O1 N16961. (A) (GlcNAc)₂ operon. (B) Nag operon. The percentage referred to the identity of amino acid sequences.

Figure 3

Neighbor-joining tree based on amino acid sequences of putative chitinases in WXL538 and other known chitinases. A hexosaminidase from GH20 was used as an outgroup. Bold Font indicated the chitinases were from V. harveyi WXL538. Numbers at nodes are the levels of bootstrap support (%). Scale bar, 0.2 substitutions per amino acid position.

Figure 4

Multiple-sequence alignment of amino acid sequences of GH18 chitinases in strain WXL538 and referential experimentally valid chitinases from other species within the GH18 domain. The referential chitinases are 4TX8_A from Chromobacterium violaceum, MnChi460 from Moritella marina (4HMC), 122–588 aa of ChiA_PSEO7 (P32823) from Pseudoalteromonas piscicida, 309–784 aa of chitodextrinase (P96156) from V. furnissii, 160–544 aa of ChiA_SERA (P07254) and ChiB_SEMR (Q54276) from Serratia marcescens. Multiple-sequence alignment was performed by the MAFFT. Conserved amino acids are shaded in black (90% conservation or more) or in grey (70 to 90% conservation). Red boxes, three conserved domains of the GH18 family [SXGG, DXXDXDXE, and Y(D/N)].

Figure 5

Effect of temperature and pH on Chi4733 and Chi540. (A) Optimal temperature and thermal stability of Chi4733. (B) Optimal temperature and thermal stability of Chi540. (C) Optimal pH of Chi4733. (D) pH stability of Chi4733. (E) Optimal pH of Chi540. (F) pH stability of Chi540.

Figure 6

Hydrolysis property of Chi4733 and Chi540. (A–C) The degradation products of Chi4733 for 1% (w/v) colloidal chitin (GlcNAc)₃ and (GlcNAc)₄. (D–F) The degradation products of Chi540 for 1% (w/v) colloidal chitin, (GlcNAc)₃ and (GlcNAc)₄. Purified Chi4733 and substrates were incubated at 50°C, and Chi540 were at 60°C for different time intervals.