| The sequencing, splicing and assembling of genomes from three strains of Antarctic marine Cold-adapted petroleum hydrocarbon-degrading bacteria, including the Shewanella sp. NJ49, the Pseudoalteromonassp. NJ289 and the Planococcus sp. NJ41, were performed in this study. The genetic data of the three strains of bacteria was gained through complete genome sequencing. And based on the existing genetic and protein databases, all the obtained complete genomes were annotated with their functions. Moreover, in reference with the metabolic pathways annotations, the metabolic process of the low temperature degrading bacterium were analyzed, along with its related adaptive mechanisms of low temperature resistance, salt tolerance and radiation proof, as well as its degrading enzyme gene. The main research results were summarized as follows:1.The total length of Shewanella sp. NJ49 genome was about 4.7M with an average G+C of 41.57% and four scaffolds. In this genome, there existed three complete prophases and one uncertain prophase; signal peptide was identified in 393 protein-coding genes, accounting for 9.41% of its total number; at least one transmembrane helix regions existed in 4,173 protein-coding genes, 25.23% of its total number. GO annotating result suggested most genes existed in the extracellular fraction, biosynthesis process and cellular component and COG annotating result showed that genes of general functions were in the majority, followed by genes of unknown functions and besides genes involved in the processes of transcription and signal transduction were also in large quantity. It was also indicated that such xenobiotic genes as degradable benzene, naphthalene, dimethylbenzene, styrene, chloralkane, aminobenzoic acid and aromatic compounds were found in Shewanella sp. NJ49.2. The total length of Pseudoalteromonas sp. 289 genome was about 3.5M with an average G+C of 40.83%; neither CRISPRs nor bacteriophage was found in this genome while signal peptide was discovered in 357 proteins, accounting for 9.94% of its total protein number. At least one transmembrane helix region was contained in 853 protein-coding genes, 23.7% of its total number. GO annotating result suggested that most genes existed in the biological process, extracellular nitrogen components and ferritin; COG annotating result showed that the genes of unknown functions were in the majority reaching 28.55%, meaning that there were plenty of unknown functional genes in this bacterium, making it a potential new gene bank. Genes of general functions were in the second place and genes involved in amino acid transport and metabolic process were also in large quantity. KEGG annotating result suggested that metabolism related genes were in the majority with general metabolism related genes at most, followed by amino acid metabolizing genes and next was the carbon metabolizing genes. Phylogenetic analysis indicated that Pseudoalteromonas sp. 289 might be a new kind of bacteria.3. The total length of Planococcus sp. NJ41 genome was 3.1M with an average G+C of 39.69%. Three completed prophages and one uncertain prophage were included in this genome. There were 3,138 coding genes with an overall length of 2,743,959 bp and an average length of 874 bp, and its coding sequence coverage reached 87.17%. In NJ41 genome, there were 127 repetitive sequences in all, among which 74 repetitive sequences were arranged in long terminal repeat(LTR). There were 15 DNA transposons, 24 long interspersed elements(LINE) and 10 short interspersed elements(SINE) in the repetitive sequences, one rolling circle(RC), and three unknown repetitive sequences. In tandem repeat sequences, there were 85 minisatellite DNA and two microsatellite DNA. In non-coding RNA, there were sixty-seven t RNA in all with an average length of 77bp; nine 5s r RNA with an average length of 115bp; nine 16 s t RNA with an average length of 1538bp; nine 23 s r RNA with an average length of 2932bp; two s RNA with an average length of 98 bp. Neither prophage nor CRISPRs was found in NJ41, but there were 7 genomic islands with an average length of 15,429 bp, showing the transverse origins of partial genes, which suggested that the low temperature adaptability or the low temperature degradable functions might have something to do with genes on the genomic island. GO annotating result told that cell and cellular component genes reached a large number of 821, and there were also a great number of genes engaging in biological process and metabolism, which were 1,246 and 1,173 respectively. Among molecular functional large class genes, catalytic activity genes and combining genes were in the majority, reaching the number of 1,319 and 965 respectively. After in-depth analysis, dioxygenase and monooxygenase related genes were found in NJ41 bacteria genome, among which Pcp, nitrilotriacetatemonooxygenase component A and component B, pentachlorophenol-4 monooxygenase and dioxygenase genes-biphenyl dioxygenase, mhq O\tryphtophan-2, 3- dioxygenase, 2-nitromethane dioxygenase and PF00903 protein family were included in monooxygenase genes. COG annotating result indicated that proteins engaged in general metabolism were at most. In the secondary classification, COG class E(amino acid transport and metabolism), J(transduction, ribosome structure and biosynthesis), and P(inorganic ion transport and metabolism) involved more proteins, accounting for 13%, 8% and 8% of the predicted total protein number respectively. Comparing the predicted genome of bacteria NJ41 with KEGG database, among all the 7,205 whole genes, 57%(1790) genes could be matched with genes in KEGG database, and the 1,790 genes were involved in 142 ways, 33 categories to complete metabolism. Annotating result suggested that xenobiotic genes like degradable benzene, naphthalene, dimethylbenzene, styrene, chloralkane, aminobenzoic acid and aromatic compounds were found in bacterial strain NJ41.4. The three strains of bacteria had different low temperature resistance, salt tolerance and radiation proof mechanisms and enzyme degrading systems. In respect of low temperature adaptation mechanism, all three strains of bacteria contained low temperature adaptation molecular chaperone Grp E and HSP, and the difference was that there was plenty of cold shock protein in NJ289 and NJ41 while none in NJ49. However, HSP and HSP transcription factors were abundant and polybasic in NJ49, with its molecular weight varying from 12 K, to 70 K and 10 K, and HSP transcription factors hsf1 annotated as well. Cold shock proteins Csp A, Csp C, and some genes and molecular chaperones genes(Dna J、Dna K、Dna E) similar to cold shock protein have been found in NJ41 bacteria strains. In terms of radiation proof mechanism, all three strains of bacteria depended on the Uvr ABC system to achieve wound repair. The regulatory factor Uvr Y was annotated in bacteria strains NJ49 and Nj289, which was similar to ultra violet DNA repair endonuclease in Schizosaccharomyces bacteria and Bacillus bacteria. In respect of salt tolerance mechanism, all three strains of bacteria included Trk system and Na+/H+ reverse cholesterol transport protein system. The NJ289 also contained prop system while bacterial strain NJ41 included proline biosynthesis genes: transporters i.e., pro A,pro B, pro C and put P. Regarding the degradation of xenobiotics, all three strains of bacteria can degrade certain aromatic hydrocarbons, with their varieties of degrading enzyme systems.5.The three strains of bacteria proved the existence of chromosomal rearrangement of genomic sequence in comparison with other bacteria of the same genus. Phylogenetic analysis indicated that NJ289 might be a new species. Two gene clusters existed within NJ289, which led to some potential in synthesising antibacterial, antitumor or antifungal drugs. And two gene clusters(terpene) were found in NJ41. |
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