Comparative Genomic Analysis of Seven Vibrio alginolyticus Strains Isolated From Shrimp Larviculture Water With Emphasis on Chitin Utilization

Abstract

The opportunistic pathogen Vibrio alginolyticus is gaining attention because of its disease-causing risks to aquatic animals and humans. In this study, seven Vibrio strains isolated from different shrimp hatcheries in Southeast China were subjected to genome sequencing and subsequent comparative analysis to explore their intricate relationships with shrimp aquaculture. The seven isolates had an average nucleotide identity of ≥ 98.3% with other known V. alginolyticus strains. The species V. alginolyticus had an open pan-genome, with the addition of ≥ 161 novel genes following each new genome for seven isolates and 14 publicly available V. alginolyticus strains. The percentages of core genes of the seven strains were up to 83.1-87.5%, indicating highly conserved functions, such as chitin utilization. Further, a total of 14 core genes involved in the chitin degradation pathway were detected on the seven genomes with a single copy, 12 of which had undergone significant purifying selection (dN/dS < 1). Moreover, the seven strains could utilize chitin as the sole carbon-nitrogen source. In contrast, mobile genetic elements (MGEs) were identified in seven strains, including plasmids, prophages, and genomic islands, which mainly encoded accessory genes annotated as hypothetical proteins. The infection experiment showed that four of the seven strains might be pathogenic because the survival rates of Litopenaeus vannamei postlarvae were significantly reduced (P < 0.05) when compared to the control. However, no obvious correlation was noted between the number of putative virulence factors and toxic effects of the seven strains. Collectively, the persistence of V. alginolyticus in various aquatic environments may be attributed to its high genomic plasticity via the acquisition of novel genes by various MGEs. In view of the strong capability of chitin utilization by diverse vibrios, the timely removal of massive chitin-rich materials thoroughly in shrimp culture systems may be a key strategy to inhibit proliferation of vibrios and subsequent infection of shrimp. In addition, transcontinental transfer of potentially pathogenic V. alginolyticus strains should receive great attention to avoid vibriosis.

Keywords: Vibrio alginolyticus; chitin utilization; mobile genetic element; pan-genome; shrimp larviculture.

Figure 1

Average nucleotide identity analysis of the complete genomes of 21 V. alginolyticus strains, together with V. harveyi ATCC 33843 and QT520, and V. parahaemolyticus LVP66 and AM51552.

Figure 2

The pan-genome profiles of the seven V. alginolyticus strains isolated in this study, together with other 14 available genomes of V. alginolyticus strains.

Figure 3

The profiles of pan-genome and core genes of the seven strains (A) and 21 strains (B) of V. alginolyticus, and the profile of increased new genes in the 21 strains (C).

Figure 4

UpSet plot of the intersection of gene clusters in the seven V. alginolyticus strains. The numbers of gene clusters in the core, specific, and intersecting genomes in two or three strains are indicated.

Figure 5

The phylogenomic tree generated based on concatenated alignments of 3854 single-copy core genes among the 21 strains using the ML method. The scale bar indicates the number of substitutions per site (A). The pan-genome phylogeny constructed based on presence/absence of homologous genes with UPGMA method. The scale bar indicates the distance per 100 genes difference (B). ML, maximum likelihood; UPGMA, unweighted pair-group method with arithmetic means; branch labels, bootstrap support values.

Figure 6

Functional enrichment analysis of COGs in the pan-genome of the seven V. alginolyticus strains in terms of the whole-, core-, dispensable- and specific-genes. Designations of functional categories: [J] translation; [A] RNA processing and modification; [K] transcription; [L] replication and repair; [B] chromatin structure and dynamics; [D] cell cycle control and mitosis; [V] defense mechanisms; [T] signal transduction; [M] cell wall/membrane/envelope biogenesis; [N] cell motility; [U] intracellular trafficking and secretion; [O] post-translational modification, protein turnover, chaperones; [C] energy production and conversion; [G] carbohydrate transport and metabolism; [E] amino acid transport and metabolism; [F] nucleotide transport and metabolism; [H] coenzyme transport and metabolism; [I] lipid transport and metabolism; [P] inorganic ion transport and metabolism; [Q] secondary metabolites biosynthesis, transport and catabolism; [R] general function prediction only; [S] function unknown.

Figure 7

The percentages of survival rate of L. vannamei postlarvae after treatment with the seven V. alginolyticus strains.

Figure 8

The phylogenetic tree generated using the neighbor-joining method based on the alignment of 14 single-copy genes involved in chitin-degradation pathway of the seven V. alginolyticus strains. The first nine genes locate on chromosome I, which being separated by symbol “//” from other five genes on chromosomes II. Arrows to the left and to the right indicate genes encoded on the plus and minus DNA strands, respectively. The scale bar indicates the size of genes, while the distances between genes was reduced by 100 times. Gene annotation: endo I_1 and endo I_2: Chitodextrinase; chb, N, N'-diacetylchitobiase; chiP_1: chitoporin; nagE_1/nagE_2, PTS system N-acetylglucosamine-specific EIICBA component; chiD, Chitinase D; chbP, N, N'-diacetylchitobiose phosphorylase; chbG, Chitooligosaccharide deacetylase ChbG; chbA, PTS system N, N'-diacetylchitobiose-specific EIIA component; deaA, chitin disaccharide deacetylase; gbpA, GlcNAc-binding protein A. chiA, chitinase A; cbpD, chitin-binding protein CbpD.

Figure 9

The growth performance of the seven V. alginolyticus strains on medium containing chitin as sole source of carbon-nitrogen.