The deep sea is one of the largest biomes on Earth, encompassing 95% of the Earth’s oceanic volume and reaching depths of over 10,000 m [1,2]. It is a unique environment that is characterised by low temperatures, high hydrostatic pressure—which increases by one atmosphere with every 10 m increase in water depth—low oxygen, and no sunlight beyond 1000 m . These conditions are relatively constant, with little impact from ocean currents and seasonality . However, it is estimated that less than 0.001% of the deep sea has been explored, and relatively little is known about its microbial inhabitants compared to those of the terrestrial world . The deep sea is, perhaps, one of the last frontiers of biodiscovery and, as such, has been gaining attention as a source of potential novel microbial compounds.
Deep-sea microorganisms are uniquely adapted to the extreme conditions of their environmental niche. Notably, deep-sea microbial proteins are often optimally active at low temperatures  and can have unusual amino acid structures , leading to an increased tolerance to high pressure [7,8].
Some recently described antimicrobials are produced by novel deep-sea bacteria isolated from deep-sea sediment. These include marthiapeptide A, a cyclic peptide produced by Marinactinospora thermotolerans ; lobophorins E, F and K, antibiotics from Streptomyces spp. [10,11]; and anthracimycin, an anti-anthrax (Bacillus anthracis) antibiotic produced by Streptomyces sp. CNH365 . Other such antimicrobials have also been found in deep seawater, for example, brainimycins B and C (macrolides) produced by Pseudonocardia carboxydivorans . Deep-sea bacteria found in association with higher organisms are also now gaining attention as producers of novel antimicrobials, particularly those from deep-sea coral  and sponges .
Bacteriocins are small (<10 kDa), ribosomally synthesised antimicrobial peptides produced by bacteria that target other bacteria, and to which the producer has immunity. Bacteriocins can be divided into two main classes: class I (lantibiotics or lanthipeptides) and class II (non-lanthionine-containing bacteriocins) . Lanthipeptides can be further subdivided into five subclasses, I-V, based on the biosynthetic enzyme(s) involved in their modification . The class II bacteriocins are subdivided into four groups: the anti-listerial pediocin-like peptides (class IIa), two-component peptides (class IIb), cyclic/circular bacteriocins (class IIc) and the linear and non-pediocin-like peptides (class IId) . Bacteriocins are emerging as alternatives to antibiotics due to their spectrum of activity—which can be broad or narrow—heat stability and their capacity for bioengineering to generate derivatives with value-added properties . This is of particular importance, given the significant and growing threat that antibiotic-resistant organisms pose in the medical and food industries. According to a 2019 report by the Centres for Disease Control and Prevention (CDC), almost three million infections, including foodborne illness, caused by antibiotic-resistant organisms occur each year in the United States of America alone .
To date, very few studies have explored the production of bacteriocins from deep-sea fish microbial isolates. One example is BaCf3, a bacteriocin produced by Bacillus amyloliquefaciens BTSS3, isolated from the intestine of a deep-sea shark. This highly thermostable bacteriocin was initially identified through in vitro screening methods and showed inhibitory activity against pathogenic bacteria, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus and Salmonella enterica Typhimurium [20,21].
Previous work carried out in our laboratory generated metagenome-assembled genomes (MAGs) from shotgun metagenomic sequencing of intestinal microbiomes samples from deep-sea fish from the Atlantic Ocean . The fish were collected as part of a groundfish survey. Analysis of the MAGS revealed information on the taxonomic and functional diversity of these samples, such as a predominance of Pseudomonadota (Proteobacteria), as well as a large abundance of genes involved in DNA repair, protein folding and motility. Bacterial isolates were recovered from the intestines and skin of a subset of these deep-sea fish and were preliminary screened for bioactivity against the Gram-positive target organism, Lactobacillus delbrueckii subsp. bulgaricus. L. bulgaricus is commonly used in the assessment of antimicrobial activity due to its low-pH resistance and sensitivity to a wide range of bacteriocins [23,24,25]. In this study, a cohort of these bacterial isolates were reassessed for bioactivity against the target strain, Lactobacillus delbrueckii subsp. bulgaricus, and were subsequently screened against a range of various target strains.
The aim of this study was to determine the bacteriocinogenic (ability to produce bacteriocins) potential of deep-sea fish microbial isolates (n = 65) through in vitro screening against various target strains, including pathogenic bacteria, and through in silico mining of the genomes of a selection of these isolates (n = 36) for putative bacteriocin biosynthetic gene clusters. We also looked for the presence of other secondary metabolite gene clusters and antimicrobial resistance (AMR) genes.