Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis . In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610 T , G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis .

Bacillus toyonensis is a group of Gram-positive bacteria belonging to the Bacillus cereus group and used in some cases as probiotics or biocontrol agents. To our knowledge, B. toyonensis from the deep sea (depth >1,000 m) has not been documented. Here, we report the isolation and characterization of a B. toyonensis strain, P18, from a deep sea hydrothermal field. P18 is aerobic, motile, and able to grow at low temperatures (4°C) and high concentrations of NaCl (8%). P18 possesses a circular chromosome of 5,250,895 bp and a plasmid of 536,892 bp, which encode 5,380 and 523 genes, respectively. Of these genes, 2,229 encode hypothetical proteins that could not be annotated based on the COG database. Comparative genomic analysis showed that P18 is most closely related to the type strain of B. toyonensis, BCT-7112T. Compared to BCT-7112T, P18 contains 1,401 unique genes, 441 of which were classified into 20 COG functional categories, and the remaining 960 genes could not be annotated. A total of 319 putative virulence genes were identified in P18, including toxin-related genes, and 24 of these genes are absent in BCT-7112T. P18 exerted strong cytopathic effects on fish and mammalian cells that led to rapid cell death. When inoculated via injection into fish and mice, P18 rapidly disseminated in host tissues and induced acute infection and mortality. Histopathology revealed varying degrees of tissue lesions in the infected animals. Furthermore, P18 could survive in fish and mouse sera and possessed hemolytic activity. Taken together, these results provide the first evidence that virulent B. toyonensis exists in deep sea environments.

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.