Comparative Genomic Analyses of the Genus Photobacterium Illuminate Biosynthetic Gene Clusters Associated with Antagonism

Abstract

The genus Photobacterium is known for its ecophysiological versatility encompassing free-living, symbiotic, and pathogenic lifestyles. Photobacterium sp. CCB-ST2H9 was isolated from estuarine sediment collected at Matang Mangrove, Malaysia. In this study, the genome of CCB-ST2H9 was sequenced, and the pan-genome of 37 Photobacterium strains was analysed. Phylogeny based on core genes showed that CCB-ST2H9 clustered with P. galatheae, forming a distinct clade with P. halotolerans, P. salinisoli, and P. arenosum. The core genome of Photobacterium was conserved in housekeeping functions, while the flexible genome was well represented by environmental genes related to energy production and carbohydrate metabolism. Genomic metrics including 16S rRNA sequence similarity, average nucleotide identity, and digital DNA-DNA hybridization values were below the cut-off for species delineation, implying that CCB-ST2H9 potentially represents a new species. Genome mining revealed that biosynthetic gene clusters (BGCs) involved in producing antimicrobial compounds such as holomycin in CCB-ST2H9 could contribute to the antagonistic potential. Furthermore, the EtOAc extract from the culture broth of CCB-ST2H9 exhibited antagonistic activity against Vibrio spp. Intriguingly, clustering based on BGCs profiles grouped P. galatheae, P. halotolerans, P. salinisoli, P. arenosum, and CCB-ST2H9 together in the heatmap by the presence of a large number of BGCs. These BGCs-rich Photobacterium strains represent great potential for bioactive secondary metabolites production and sources for novel compounds.

Keywords: Photobacterium; antagonism; comparative genomics; pan-genome analysis; secondary metabolites.

Figure 1

Phylogenetic relationship of Photobacterium. The maximum likelihood tree was constructed based on 92 bacterial core genes with UBCG. Vibrio cholerae ATCC 14035 was used as an outgroup, and support values of more than 50 are indicated by dots on nodes.

Figure 2

Average nucleotide identity (ANI) between Photobacterium genomes. The colour bar represents the value of ANI.

Figure 3

Core and pan-genome analyses of Photobacterium genus. (a) Barplot of the pan-genome matrix. (b) Plot of the estimated pan-genome size with Tellelin fit. (c) Cloud, shell, soft-core, and core clusters of the pan-genome. (d) Plot of the estimated core genome size with Tellelin and Willenbrock fit.

Figure 4

(a) Distribution of COG functional categories for cloud, shell, soft core, and core Photobacterium pan-genome clusters. (b) COG functional classification of strain CCB-ST2H9 specific genes. A: RNA processing and modification, C: energy production and conversion, D: cell cycle control and mitosis, E: amino acid metabolism and transport, F: nucleotide metabolism and transport, G: carbohydrate metabolism and transport, H: coenzyme metabolism, I: lipid metabolism, J: translation, K: transcription, L: replication and repair, M: cell wall/membrane/envelop biogenesis, N: cell motility, O: post-translational modification, protein turnover, chaperone functions, P: inorganic ion transport and metabolism, Q: secondary structure, S: function unknown, T: signal transduction, U: intracellular trafficking and secretion, V: defense mechanisms, W: extracellular structures, and Z: cytoskeleton.

Figure 5

Heatmap representation of Photobacterium biosynthetic gene clusters (BGCs). The column dendrogram represents hierarchical clustering of bacteria by shared BGCs numbers, and the colour code represents the number of BGCs.

Figure 6

Comparison of holomycin biosynthetic gene cluster in Photobacterium, S. clauvuligerus, Y. ruckeri, and Pseudoalteromonas sp. Similarity (%) of genes in CCB-ST2H9 to those of P. galatheae are indicated in brackets. Light blue indicates oxidative gene. Orange indicates biosynthesis gene. Magenta indicates MFS transporter genes. Green indicated transcription regulator gene. Grey indicates gene with unknown function.

Figure 7

Disk diffusion assay of EtOAc extract on seven Vibrio species and control strains. Scale bars indicate 2 cm.