Study Of Adaptation Mechanism Of Deep Sea Bacterium Shewanella Piezotolerans WP3 To Extreme Environment-the Role And Regulation Of Fatty Acid System And Deep Sea Phage SW1 In Various Environmets

Deep-sea is a special ecosystem for its permanent coldness, high pressure and darkness. The study towards the deep-sea microbes will be helpful to the understanding of the origin of life, the character of biodiversity and to the exploitation of microbial resources in deep sea. The metabolic and ecological study of life on extreme conditions will give some insights on evolution and adaptation mechanism of extremophiles in response to stress conditions and will pave a new way for the development of anti-stress agricultural and animal husbandry products and for health of human beings.The deep sea bacteria used in this study was isolated from deep-sea sediments of 1914m depth in west Pacific. The phylogenetic analysis and molecular study shows that the bacterium is a psychrophilic and piezotolerant microbe with the term as Shewanella piezotolerans WP3, which will be an ideal material to study the adaptation mechanism of extremophiles to the extreme environments. The aim of this study is to investigate the role and regulation of fatty acid system in Shewanella response to different growth conditions. Additionally, we have isolated and identified a novel filamentous phage SW1 from WP3, which is the first filamentous phage isolated from Shewanella genus. Furthermore, we have found this phage can be induced by low temperature and some other low temperature related characters, which will give the basis for investigating the role of phage in deep-see environment.Shewanella inhibits in various environments and it is the most microbes isolated from deep sea. The Shewanella genus is capable of synthesis various types of low melting fatty acids, so it is the idea material for investigating the role and regulation of fatty acid system in adaptation of deep-sea environment. The genome sequences of 15 Shewanella strains were searched and compared for genes involved in fatty acid synthesis. All genes supposed to be involved in typical typeⅡfatty acid biosynthesis pathway could be easily identified from the genomes and the typeⅡfatty acid biosynthesis pathway of Shewanella genus was constructed. Complete EPA synthesis gene cluster was found in all of the Shewanella genomes although only few of them were found to produce EPA. The roles and regulation of fatty acids synthesis in Shewanella was further elucidated in Shewanella piezotolerans WP3 response to different temperatures and pressures. WP3 increased significantly the contents of EPA and BCFA when grown under low temperature and /or high pressure. EPA, but not MUFA was determined to be crucial for its growth at low temperature and high pressure.The microarray of WP3 was used to do the transcriptome analysis of WP3 in response to different growth temperatures (4℃and 20℃) and pressures (0.1Mpa and 20Mpa). Among the genes involved in the fatty acid synthesis, only a gene cluster for branched chain amino acid ABC transporter (swp3487-3492, LIV-I) was up regulated by low temperature. Quantitative RT-PCR clearly verified the induction of these transporter genes by low temperature. To determine if this gene cluster is responsible for the BCFA increment at low temperature, mutations were constructed on orfswp3487, which encodes the only putative substrate-binding protein of this ABC transporter as described in the materials and methods. The LIV-I deficient mutant was named WP3^ΔLIV. The fatty acid profiles of WP3^ΔLIV grown under different conditions were characterized. Interestingly, its fatty acid profile was very similar with that of the wild type strain when they grown at 20℃and 0.1Mpa. However, the mutant strain WP3^ΔLIV almost lost the low-temperature and high-pressure dependent BCFA regulation ability. By using the isotopic tracer method, we further verified that the regulation of BCFA synthesis is controlled by the transporter efficiency.Genome sequencing of the deep-sea bacterium Shewanella piezotolerans WP3 suggests that it may harbor a functional filamentous phage (named as SW1). SW1 has 7718 nucleotides, contains 9 open reading frames (ORFs) encoding putative proteins which are homologous to that of previously known filamentous phages. Interestingly, a putative ssDNA binding protein (ORF104) of the phage, which is determinant protein for phage particle assembly, was found over-expressed at 4℃versus 25℃by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). According to this clue, the active phage particles were isolated from WP3; it has a single-stranded DNA (ssDNA) genome, able to integrate into the chromosome with a single copy, and also exists as extra chromosomal plasmid. The host range of SW1 was tested using Shewanella oneidensis, Shewanella marinintestina, Shewanella violacea, Shewanella psychrophila WP2 and E. coli. SW1 can only infect WP2 and form phage plaques on plates when culture temperature was equal to or lower than 15℃. It implied that the SW1 production may be regulated by temperature. To testing its temperature stability, 70℃about 10min will totally inactivate its activity, which is 10℃lower than other filamentous phage's maximum temperature. Reverse-transcription quantitative PCR (RT-QPCR) analysis further revealed that the transcription of the key genes of SW1 including the putative repressor gene orf116 was significantly induced (3-12 times) at low temperature (4℃). Followed, phage particles were isolated from WP3 cultures incubated at different temperatures (4℃, 10℃, 15℃, 20℃, and 25℃). The quantity of the phage particles were enumerated by counting the phage plaques formed on WP2 cell lawns on the agar plates. No or few active phage particles were isolated from cultures of 20, 25℃as no phage plague was observed. Active phage particles were isolated from the cultures of WP3 at 4℃, 10℃and 15℃. The WP3 cell culture at 4℃yields higher number of phage particles than that from 10℃and 15℃. This data clearly demonstrated that the phage production is temperature regulated, and low temperature can induce the phage production in WP3.SW1 is also induced by Mitomycin C and UV irradiation, indicating that the production of the phage depends on the cellular SOS response. However, the transcription of orf116 is not induced by MMC and UV, which is different from that at low temperature, implying that the mechanism of low temperature inducement may be not the same as the SOS response inducement.