Characterization of Multiple Alginate Lyases in a Highly Efficient Alginate-Degrading Vibrio Strain and Its Degradation Strategy.

Alginate is an important polysaccharide in the ocean that supports the growth of marine microorganisms. Many widespread Vibrio species possess alginate lyases and can utilize alginate as a carbon source, but the detailed alginate degradation mechanism in Vibrio remains to be further explored. In this study, we obtained a highly efficient alginate-degrading strain, Vibrio pelagius WXL662, with 11 alginate lyases (VpAly-I to -XI) and further elucidated its molecular mechanism of alginate degradation. Three alginate utilization loci (AUL) were identified in different parts of WXL662's genome, comprising six alginate lyases (VpAly-I, -II, -VIII, -IX, -X, and -XI) and other genes related to alginate degradation. Most of the alginate-degrading genes are strongly induced when alginate is provided as the sole carbon source. Ten alginate lyases (VpAly-I to -X) had been purified and characterized, including six from polysaccharide lyase family 7 (PL7), three from PL17, and one from PL6. These recombinant alginate lyases existing in different cellular locations were active at a wide temperature (10 to 50°C) and pH (4.0 to 9.0) range, with different substrate preferences and diverse degradation products, enabling WXL662 to efficiently utilize alginate in a changing marine environment. Importantly, outer membrane vesicles (OMVs) can act as vectors for alginate lyases (VpAly-II, -V, and -VI) in WXL662. Further investigations of public Vibrio genomes revealed that most alginate-degrading vibrios possess one AUL instead of previously reported "scattered" system. These results emphasize the specific alginate degradation strategy in Vibrio pelagius WXL662, which can be used as a model strain to study the ecological importance of effective alginate-degrading vibrios in the ocean. IMPORTANCE Alginate is an important carbon source in the marine environment, and vibrios are major alginate utilizers. Previous studies focused only on the characteristics of individual alginate lyases in vibrios, but few of them discussed the comprehensive alginate-degrading strategy. Here, we depicted the alginate utilization mechanism and its ecological implications of a highly efficient alginate-degrading Vibrio strain, WXL662, which contained 11 alginate lyases with distinct enzymatic characteristics. Importantly, unlike other vibrios with only one alginate utilization locus (AUL) or the previously reported "scattered" system, three AUL were identified in WXL662. Additionally, the involvement of outer membrane vesicles (OMVs) in the secretion of alginate lyases is proposed for the first time.

Two Highly Similar Chitinases from Marine Vibrio Species have Different Enzymatic Properties.

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45-50 °C and pH 5.0-7.0 for Chi1557, while ~50 °C and pH 3.0-6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

Winogradskyella ouciana sp. nov., isolated from the hadal seawater of the Mariana Trench.

A Gram-staining-negative, strictly aerobic, long-rod shaped with no flagellum and yellow-pigmented bacterium designated strain ZXX205T, was isolated from the hadal seawater at the depth of 7500 m in the Mariana Trench, Pacific Ocean. Phylogenetic analysis based on 16S rRNA gene sequences placed strain ZXX205T within the genus Winogradskyella and strain ZXX205T was most closely related to Winogradskyella flava KCTC 52348T and Winogradskyella echinorum KCTC 22026T with 96.9 % and 96.6 % sequence similarity, respectively. The sequence similarities to all other type strains were 96.3 % or less, and to the type strain Winogradskyella thalassocola LMG 22492T was 94.1 %. Growth occurred in the presence of 0-9.0 % (w/v) NaCl (optimum 3.0 %), at 4-45 °C (optimum 28 °C) and pH 6.0-9.0 (optimum pH 7.5). The sole respiratory quinone was menaquinone 6 (MK-6). The dominant cellular fatty acids (>10 %) of strain ZXX205T were iso-C15 : 0, iso-C15 : 1 G, iso-C16 : 0 3-OH and iso-C16 : 0. The polar lipids profile contained predominantly phosphatidylethanolamine, four glycolipids, four unidentified aminolipids and three unidentified lipids. The genomic DNA G+C content was 35.5 %. The DNA-DNA relatedness (DDH) values between strain ZXX205T and the most closely related species Winogradskyella flava and Winogradskyella echinorum were 21.1 and 20.4 %, respectively. Based on polyphasic taxonomic analysis, strain ZXX205T is considered to represent a novel species in the genus Winogradskyella of the family Flavobacteriaceae, for which the name Winogradskyella ouciana is proposed. The type strain is ZXX205T (=MCCC 1K03851T=JCM 33665T).

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

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.