The Antibacterial Mechanism Of Pseudoalteromonas Flavipulchra JG1

The genus Pseudoalteromonas is ubiquitous in marine environment and could bedivided clearly into pigmented and non-pigmented species clades, and the pigmentedspecies have been shown to synthesize a range of extracellular compounds withantibacterial, antifungal and algicidal activities. A great number of antimicrobialcompounds such as small molecular compounds, proteins and polysaccharides wereisolated from Pseudoalteromonas spp., and the analysis of the Pseudoalteromonasgenomes has been an important way to study the biosynthesis of its variousmetabolites and the adaption to fluctuating marine environments. Studies which focuson the antibacterial mechanisms of Pseudoalteromonas spp. might be conducive tothe discovery of new probiotics or marine drugs and provide extensively applicationson the biocontrol in aquaculture, anti-fouling and red tides. In this study, theantibacterial components have been isolated and purified from Pseudoalteromonasflavipulchra, and their activities also have been explored. Furthermore, we havesequenced and analyzed the genome of P. flavipulchra and expounded the geneticcapabilities which enable the bacterium to reveal a survival advantage in variedmarine environments. This could provide further insight into the synthesis andapplications of the bacterium or its antimicrobial agents.The marine antagonistic bacterium Pseudoalteromonas flavipulchra JG1with agolden-yellow color was isolated from rearing water of healthy turbot (Scophthalmusmaximus) in Qingdao, China. JG1was confirmed to show excellent antibacterialactivities against many fish pathogens, such as Vibrio spp.. After the extraction withpetroleum ether, EtOAc and n-BuOH, the supernatant and the cells of JG1wasseparated into four fractions, petroleum ether, EtOAc, n-BuOH and water extracts.The antibacterial activities against Vibrio anguillarum were observed in the extractsof petroleum ether, EtOAc and water, indicating that the antibacterial substances ofJG1may include small molecular compounds and macromolecules such asantibacterial protein. The antibacterial protein was purified from the extracellular products (ECPs) ofJG1through ammonium sulfate precipitation, SDS-PAGE and electroelution. Afterelectroelution of the gel band with antibacterial activity, the protein was purified toapparent homogeneity, and the antibacterial activity of the single band was alsoobserved by the in-gel antibacterial assay against V. anguillarum. Sequences of twopeptide-fragments from the antibacterial protein were obtained through De novopeptide sequencing. After sequence alignment with the proteome sequence of JG1, acomplete protein sequence and an open reading frame (ORF) were achieved. The ORF,named pfaP, was2,085bases in length. The deduced translation product of pfaP wasa protein of694amino acids with a predicted molecular mass of77.0kDa and atheoretical pI value of4.63. No putative signal peptide was detected with either theSignal P server or the Secretome P server. The PfaP protein was shown to be58%identical to L-lysine oxidase AlpP of Pseudoalteromonas tunicata D2(GenBankAAP73876.1) and54%identical to marinocine antimicrobial protein of Marinomonasmediterranea MMB-1(GenBank AAY33849.1). The inhibitory activity ofextracellular proteins of JG1against V. anguillarum could be abolished in thepresence of catalase, suggesting that the inhibitory effect was mediated by the actionof hydrogen peroxide.The gene pfaP was cloned from the genomic DNA of JG1, inserted into prokaryoticexpression plasmids pET24a(+), pET26b(+), pET32a(+), pBAD/Myc-HisB andpRSFDuet-1, and expressed in Escherichia coli BL21(DE3). However, theantibacterial activities were not observed, which may attribute to the absence ofposttranslational modification of the protein in E. coli BL21(DE3).The EtOAc extract of the P. flavipulchra JG1was subjected to repeated columnchromatography over silica gel, Sephadex LH-20and RP-18to yield five knowncompounds (15). They were identified as p-hydroxybenzoic acid (1), trans-cinnamicacid (2),6-bromoindolyl-3-acetic acid (3), N-hydroxybenzo isoxazolone (4) and2′-deoxyadenosine (5), respectively, by detailed spectroscopic analysis and these datacomparing with the literatures. The antibacterial activities against V. anguillarum ofthe five compounds were measured through TLC bioautography overlay assay.Inhibition zone of each compound was observed, which showed different effectagainst the target strain. MIC values of compounds15were1.25,1.25,0.25,0.25and5mg/ml, respectively. Compound3isolated from JG1was the only brominated compound with a brown color while the other four compounds were non-halogenatedand almost achromatous. In addition, compound3exhibited a yellow color when itwas diluted, so it probably contributes to the yellow colonies of JG1on marine agar2216plate. Compound3showed the greatest antibacterial activity against both Grampositive and Gram negative bacteria, indicating that this pigment could be associatedwith the antibacterial activity of JG1.The genome of P. flavipulchra JG1was sequenced using the Illumina HiSeq2000,and a total of4,913protein encoding genes (4,828,917bp),104tRNA-encoding genesand4rRNA operons were predicted in the draft genome of JG1. Comparing with thegenomes of P. tunicata D2, P. haloplanktis TAC125, P. atlantica T6c and twoPseudoalteromonas strains TW-7and SM9913, a number of COG categories that areoverrepresented in P. flavipulchra JG1, including those for secondary metabolitesbiosynthesis, transport and catabolism, amino acid transport and metabolism as wellas translation, ribosomal structure and biogenesis. The abundance of genes involvedin expression and transport of potential primary and secondary metabolites could beconsistent with the capability of P. flavipulchra to produce various bioactivecompounds. P. flavipulchra shows the second highest proportion of genes assigned todefense mechanisms among Pseudoalteromonas species, giving a hint that JG1could better adapt to the marine environment fluctuation.The genome of P. flavipulchra JG1unveils significant genetic advantages againstother microorganisms, encoding antimicrobial agents as well as abilities to adapt tovarious adverse environments. The antibacterial protein PfaP not only catalyticallyproduces hydrogen peroxide as a bacteriostat but likely also participates in thebiosynthesis of small molecular antibacterial compound (6-bromoindolyl-3-aceticacid). Both the macromolecule and small molecules contribute to the antibacterialactivities of JG1. Besides these already identified chemical structures produced bystrain JG1, a large number of peptide-based secondary metabolites encoded in thegenome still awaits discovery. The identification of various antimicrobial enzymessuch as chitinases, AHL acylases enriches the antagonistic mechanisms of P.flavipulchra JG1and affords several admissible biocontrol procedures in aquaculture.Furthermore, JG1also evolved a range of mechanisms such as genes involved inoxidative and heavy metal stress, antibiotics and other drug resistances to adapt theadverse marine environments or multidrug rearing conditions. The analysis of the genome of P. flavipulchra JG1presented here provides a better understanding of itssurvival advantages against other microorganisms and also an extensive applicationprospect.