Identification And Characterization Of A Bacillus Subtilis Isolate And An Anti-lipopolysaccharide Factor From Deep-seahydrothermal Vent

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 reported a B.subtilis(G7)isolated from the Iheya North hydrothermal field of Okinawa Trough which could cause mortality in fish and mouse.G7 is aerobic,motile,endospore-forming,and requires NaCl for growth.The genome of G7 is composed of one circular chromosome of 4,216,133base 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 GIs,with 42.5%of GI genes being predicted to encode hypothetical proteins.G7 possesses abundant putative virulence genes associated with adhesion,invasion,dissemination,anti-phagocytosis,and intracellular survival.Consistently,G7 was able to cause mortality-leading infection in fish and mice following injection,replicate in mouse macrophages and fish peripheral blood leukocytes,and resist the killing effect of serum complement,in addition to this,we found that G7 adopted different serum resistance strategies towards different animals:G7 can activate the complement system of fish successfully,but its solid cell wall structure can help it resist the killing of complement MAC;G7 can not effectively activate the mouse complement system,and thus avoid the killing.Taken together,our study indicates that G7 is a virulent B.subtilis isolate with unique genetic features,and the results provide the first molecular insight into the virulence of Bacillus species in deep-sea hydrothermal vent.ALF(Anti-lipopolysaccharide factor)is a type of AMPs with a vital role in antimicrobial defense.Although a large amount of ALFs have been identified from neritic and fresh water crustacean species,no functional investigation of ALFs from deep-sea animals have been documented.In the present study,we characterized the immune function of an ALF molecule(named RspALF1)from the shrimp Rimicaris sp.residing in the deep-sea hydrothermal vent in Desmos,Manus Basin.RspALF1shares 51.5%-62.4%overall sequence identities with known shrimp ALFs and contains the conserved LPS binding domain(LBD).Both recombinant RspALF1(rRspALF1)and the LBD-derived peptide(ALF1P1)bound to the cell wall components of Gram-negative and Gram-positive bacteria and killed a wide range of bacteria,especially those from deep-sea hydrothermal field,by damaging bacterial cellular structures.The bactericidal activity of ALF1P1 was optimal and stably maintained from 4°C to 37°C,which is comparable to the ambient temperature range of the habitat of Rimicaris sp.In addition to bacteria,ALF1P1 also exhibited anti-fungal activity.ALF1P1 exhibited high killing efficiencies,which,in terms of MIC values,were ranged between 2?M to 4?M for bacteria and 4?M to 8?M for fungi.When introduced in vivo,both rRspALF1 and ALF1P1 effectively inhibited bacterial infection in shrimp and reduced the dissemination of bacterial and viral pathogens in fish.Together,these results provide the first insight into the biological property of deep-sea ALF and indicate that RspALF1 very likely plays a significant role in immune defense by functioning as a highly effective antimicrobial with a broad target range.