Biological Characteristics, Phylogeny And Genomic Analysis Of Halobacteriovorax Sp. BALOs9

Bdellovibrio-and-like organisms(BALOs)are a group of motile and obligate predatory Gram-negative bacteria.They have a distinct predatory biphasic lifecycle and are ubiquitous in nature.Since BALOs mainly predate on a diversity of Gram-negative strains,especially most Gram-negative pathogens,they have been applied as potential“living antibiotics”and biocontrol agents in agriculture,animal husbandry and water purification.BALOs currently consist of only 7 genera 10 species.The taxonomic status of the BALOs remains ill defined.In this study,a polyphasic taxonomic characterization and gonomic feature of a new periplasmic BALO were provided.The result as follows:(1)Three prokaryotic predators,BALOs9,BALOs10 and BALOs28,were successfully isolated from Daya Bay seawater by the seawater double-layer plate method with Vibrio alginolyticus as prey.Phylogenetic analyses based on16S rDNA sequences indicated that three strains shared 99.9%similarity to each other,affiliated with the genus Halobacteriovorax in the class Oligoflexia,and composed a same new species.Strain BALOs9(=MCCC 1K03527~T=JCM32962~T)was designated as the type strain,due to its high lysis ability.Cells of the strains were Gram-negative,vibrioid shaped and motile with a single sheathed flagellum(25–28 nm wide).The size of the cells was around 0.3?m×0.5–1.0?m.Three strains were obligate predator that exhibited a biphasic life cycle:a free-swimming attack phase and an intraperiplasmic growth phase within the prey.Bdelloplast was formed.Genome sequencing of strain BALOs9 revealed a genome size of 3.14 Mb and a G+C content of 35.8 mol%.The dDDH(digital DNA-DNA hybridization)estimate values and the gANI(whole genome average nucleotide identity)values between the genome of strain BL9~T and those of BALOs hitherto described were 12.5–19%and 63.49–76.15%,respectively.On the basis of features of the life cycle,physiological,gANI and dDDH data,strain BALOs9 represents a new species within the genus Halobacteriovorax,for which the name Halobacteriovorax vibrionivorans sp.nov.is proposed.(2)The physiological property included the effects of temperature,salinity and pH,vitamins,antibiotics,amino acids and different preservation methods on the growth of BALOs9 were detected.The results showed that strain BALOs9 grew optimally at~37 ~oC,2–4%(w/v)NaCl with pH 7.0–8.0.-80? glycerol was the best preservation method to keep the activity of strain BALOs9.Vitamins thiamine,pyridoxine,ascorbic acid,nicotinic acid,choline chloride,pantothenic accid hemicalcium,4-aminobenzoic acid,riboflavin,folic acid did not affect the growth of strain BALOs9,and fourteen amino acids L-glutamine,L-lysine,L-glutamic acid,L-aspartic acid,L-tyrosine,L-tryptophan;L-phenylalanine,L-isoleucine,L-leucine,L-valine,L-alanine,glycine,L-aspartic acid,L-serine promoted its growth.Strain BALOs9 was resistant to tetracycline hydrochloride and polymyxin B.(3)Seventeen different bacteria strains were choosed to detect prey range of the strain BALOs9.The results showed that the strain BALOs9 had wide prey range,preyed upon the following species:V.alginolyticus,V.parahaemolyticus,Alteromonas macleodii,E.lutimaris,Halomonas meridiana,Marinobacteradhaerens,M.algicola,M.flavimaris,M.hydrocarbonoclasticus,EscherichiacoliandPseudoalteromonas shioyasakiensis.(4)The genome of strain BALOs9 encoded 3011 genes.The KEGG annotation results indicated possible predation-related genes in the BALOs9genome,including chemotaxis,flagella assembly,and type IV pili related genes.Strain BALOs9 had biosynthetic pathways for only five of the amino acids and fifteen amino acid metabolic pathways were missing.Furthermore,the active transport system of amino acids was all missed.It was supposed that strain BALOs9 must grow in a special environment that can provide high amino acid concentration to obtain enough essential amino acids by passive transport pathway.Parasitism was a way to meet this requirement.The above results showed that the new predatory bacteria,BALOs9,with excellent performance had obvious potential useness for subsequent application.In addition,the genomic information of the strain BALOs9provides abundant data for the subsequent study of predation and parasitic mechanisms.