Distinctive microbial community and genome structure in coastal seawater from a human-made port and nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean

Abstract

Pollution in human-made fishing ports caused by petroleum from boats, dead fish, toxic chemicals, and effluent poses a challenge to the organisms in seawater. To decipher the impact of pollution on the microbiome, we collected surface water from a fishing port and a nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean. By employing 16S rRNA gene amplicon sequencing and whole-genome shotgun sequencing, we discovered that Rhodobacteraceae, Vibrionaceae, and Oceanospirillaceae emerged as the dominant species in the fishing port, where we found many genes harboring the functions of antibiotic resistance (ansamycin, nitroimidazole, and aminocoumarin), metal tolerance (copper, chromium, iron and multimetal), virulence factors (chemotaxis, flagella, T3SS1), carbohydrate metabolism (biofilm formation and remodeling of bacterial cell walls), nitrogen metabolism (denitrification, N2 fixation, and ammonium assimilation), and ABC transporters (phosphate, lipopolysaccharide, and branched-chain amino acids). The dominant bacteria at the nearby offshore island (Alteromonadaceae, Cryomorphaceae, Flavobacteriaceae, Litoricolaceae, and Rhodobacteraceae) were partly similar to those in the South China Sea and the East China Sea. Furthermore, we inferred that the microbial community network of the cooccurrence of dominant bacteria on the offshore island was connected to dominant bacteria in the fishing port by mutual exclusion. By examining the assembled microbial genomes collected from the coastal seawater of the fishing port, we revealed four genomic islands containing large gene-containing sequences, including phage integrase, DNA invertase, restriction enzyme, DNA gyrase inhibitor, and antitoxin HigA-1. In this study, we provided clues for the possibility of genomic islands as the units of horizontal transfer and as the tools of microbes for facilitating adaptation in a human-made port environment.

Fig 1. Map of the seawater sampling sites.

(A) The sampling sites (in green pentagon) are in northern Taiwan facing the Northwestern Pacific Ocean. Sampling points in a fishing port (Badouzi Fishing Port in orange circles) and nearby offshore island (Heping Island in blue boxes) are shown (map was made from open source https://github.com/Wesely/Taiwan-Python-Map). (B) Principal component analysis (PCA) based on OTU level for the microbiota composition of all samples (PC1 = 76.9%, PC2 = 16.8%). Heping Island (HPisland) and Badouzi Fishing Port (BDZfishP) are indicated in blue boxes and orange circles, respectively.

Fig 2. Composition of OTU and LDA analyses.

(A) Venn diagrams showing the shared and unique OTUs of the microbiota in the costal seawater of Heping Island and Badouzi Fishing Port. (B) Linear discriminant analysis effect size (LEfSe) cladogram of the microbial composition in seawater of Heping Island and Badouzi Fishing Port. Green and red nodes/shades specify taxa significantly higher in relative abundance in Heping Island and Badouzi Fishing Port, respectively. The taxa abundance is proportional to the circle size. The threshold of the linear discriminant analysis (LDA) score was 4.0. (C) Histogram of the LDA scores showing differential abundances of microbial taxa between Heping Island and Badouzi Fishing Port.

Fig 3. Microbial composition (in relative abundance) in Badouzi Fishing Port and Heping Island.

The distribution of major contributing families (top ten) based on the analysis of (A) 16S rRNA gene amplicon sequencing and (B) whole-genome shotgun sequencing.

Fig 4. Heatmaps showing the relative abundances of genes of metabolisms.

(A) nitrogen cycling metabolism, (B) carbohydrate metabolism, and (C) ABC transporters. Samples were collected from three sites in Badouzi Fishing Port (BDZfishP1, BDZfishP2, BDZfishP3) and Heping Island (HPisland1, Hpisland2, Hpisland3).

Fig 5. Heatmaps showing the relative abundances of genes conferring resistance to antibiotics and metals and encoding virulence factors.

(A) Resistance to 20 categories of antibiotics and (B) Metal tolerance to 13 categories of metals and (C) Virulence factors. Samples were collected from three sites in Badouzi Fishing Port (BDZfishP1, BDZfishP2, BDZfishP3) and Heping Island (HPisland1, Hpisland2, Hpisland3).

Fig 6. 19 metagenome assembled genomes (MAGs) discovered in the coast seawater of Heping Island and Badouzi Fishing Port.

(A) Phylogenetic analysis of MAGs. Color codes indicate the taxonomic assignment of “class”. (B) Heatmap showing the abundance of 19 MAGs. Samples were collected from three sites in Badouzi Fishing Port (BDZfishP1, BDZfishP2, BDZfishP3) and Heping Island (HPisland1, HPisland2, HPisland3). Accompanied table includes statistics of MAG including total length (bp), N50 (bp) and GC (%).

Fig 7. Heatmaps showing the relative abundance of genes with metabolic functions associated with 19 MAGS discovered in the coast seawater of Heping Island.

(A) Top ten metabolic functions of high abundance. (B) Other metabolic functions. Color codes indicate the taxonomic assignment of “class”.

Fig 8. Heatmaps showing the relative abundance of genes with metabolic functions associated with 19 MAGS discovered in the coast seawater of Badouzi Fishing Port.

(A) Top ten metabolic functions of high abundance. (B) Other metabolic functions. Color codes indicate the taxonomic assignment of “class”.

Fig 9. GC contents and direct repeat sequences on the tRNA structures of two genomic islands in the genome of Phaeobacter italicus discovered in the costal seawater of Badouzi Fishing Port.

(A, B) GC content as a percentage, employing a scanning window of 3000 bps. (C, D) tRNA secondary structures and the direct repeat sequences on one side of the genomic islands (perfect repeat nucleotides are colored in red circles, and imperfect repeat nucleotides are colored in orange). The tRNAs in C and D belong to the genomic islands shown in A and B, respectively. (A) GI_2 (B) GI_4 (see S13 Table in S2 File).

Fig 10. Genomic islands in the genome of Phaeobacter italicus discovered in the costal seawater of Badouzi Fishing Port.

Gene clusters in the genomic island are shown, and sequence comparisons of the genomic island (upper) with the corresponding genome of Phaeobacter italicus available in NCBI (lower) are shown in (A) GI_2 (B) GI_4 (see S13 Table in S2 File). tRNA genes are colored in gray. “DR” (in red circles) indicates the direct repeat on the two flanking sides of the genomic islands.

Fig 11. Microbial community network in the costal seawater of Heping Island and Badouzi Fishing Port.

The color of the network edges denotes positive (blue) and negative (red) correlation, and the edge width is proportional to the correlation coefficient. Only network edges with an absolute value of the correlation coefficient larger than 0.8 and bootstrap p value smaller than 0.01 were retained. Family names of bacteria are designated.

Fig 12. A conceptual figure for major discoveries of the microbiome from the costal seawater of Heping Island and nearby Badouzi Fishing Port.

Dominant species in two sampling sites are demonstrated. Specific functions of genes encoding for the nitrogen metabolism, carbohydrate metabolism, antibiotics resistance, and virulence factors are summarized.