| Phenylobacterium zucineum is a novel facultative intracellular bacterium which is recently discovered in a kind of tumor cell lines by our group. It is not only a novel bacterium with possible significance, but also different from other known bacteria. Because of parasitizing in tumor cells, it is most possible related to pathogensis since most known intracellular bacteria are pathogens. Because of the facultative intracellular character, P. zucineum has two different modalities in and out of cells respectively. So that studies on mechanism of invasion into human host cells are important. Because of our studies on the bacterium' s invasion via a "Zipper" entry mechanism, which is that after expressing some surface proteins and reacting with surface receptors of eukaryotic cells, combination of receptors and ligands induces alteration and redistribution of the cell skeleton structure, extension of cell membrane, parceling the bacteria, and invading into the host cells, it is a pivot to find out the expressed proteins and their reaction mechanism. Because of there is no bacterium similar to P. zucineum, it is a novel and controversial discovery. So that it is important to explore some untraceable evidences for potential significances. To resolve the problems above, we need to discover what the bacterium really is, in order to show the lights to explore potential significances. Whole genome sequencing is a essential method, for we can predict important genes and proteins, find out their functions and possible mechanism, and promote discovery of biological significances.Whole genome sequencing of bacteria genomes is a successful technology nowadays. Since DOE opened microbial project in 1994, development ofgenome sequencing after 2000 has pushed the bacteria genome projects to rise at a 50% rate per year. There are 343 bacteria genomes on NCBI up to May 2006.Bacteria genome sequencing is widely used. In use of studying on pathogensis of bacteria, diseases prevention and therapy, we can identify pathogenic genes, produce vaccines and new antibiotics. In use of biotechnology applications, we make use of bacteria to anti-radiate, utilize nitrogen resource, biodegrade and produce heat-resistant enzymes for PCR technology, which induced a revolution in Molecular Biology. In study of microbial evolution, we can explore the evolutionary relationships because many genes transfer between bacteria, or even between prokaryotic organisms.Using a "whole-genome-shotgun" method, we sequenced P. zucineum' s genome. At the step of library construction, three DNA libraries in size of 2. 0-2. 5Kb>2. 5-3. 0Kb and 3-4Kb, were established with pUC18 as carriers. At the step of sequencing, we added lycine, increased annealing temperature and prolonged time for extension, in order to optimize sequencing reaction based on Dideoxy Sanger Sequencing and P. zucineum' s high GC content. Besides, different kinds of dye and sequencing equipments were used in different libararies. At the step of assembly, Phred-Phrap-Consed was used to recognize and assemble, and finally consensus sequences and contigs were attained. At the step of annotation, "Glimmer" was used to predict ORFs (open reading frame), and "blast" was used to search homologous sequences in non-redundant database of NCBI, in order to predict functions of every CDS (coding sequence).66,540 valid sequencing reactions (Phred Q20, >50bp) were obtained at last, and the total length sequenced is 17.8Mb, which is as redundantas 4 X. We sequenced its 4, 379, 231bp genome, including a 3, 996, 255bp ring chroraosom and a 382, 976bp plasmid. The chromosome encodes 5, 029 primarily-predicted ORFs (Open Reading Frame). Among them, 2,483 (49.4%) are classified according to homology to other documented proteins with known functions, 1, 626 (32. 3%) have homology to other hypothetical proteins, and the rest, 920 (18.3%) are functionally unknown, and there are 512 predicted ORFs in plasmid. It is possible that there exit new genes with new functions because of a lot of sequences with unknown functions. On the other hand, there is GC skew in genome of P. zucineum. GC skew is a feature which only exits in prokaryotic organisms, and it shows that the number of G in leading strand is bigger than the number of C in lagging strand.One of the interesting features of the P. zucineum has the high GC content of 71. 10%, which is the fourth highest in 343 gonome-sequenced bacteria. The feature prefigures the complex secondary structure in the genome and possible relationship between actinobacteria which also have high GC content. Actinobacteria is the most important bacterium to produce antibiotics, and there are 56% of more than 2000 antibiotics all over the world produced by actinobacteria. Besides, actinobacteria is a transitional type between bacteria and epiphyte.There are 25 COGs in NCBI and P. zucineum contains 20 of them. Among them, the most is "function unknown" group. But 4 of the 20 COGs are more important because of the characters of facultative intracellular bacterium and its "zipper" invasion. They are Cell wall/membrane/envelope biogenesis;Cell cycle control, cell division, chromosome partitioning;Signal transduction mechanisms;Intracellular trafficking, secretion, and vesicular transport.Sequences of P. zucineum have the highest homology to genome of C. crescentus, a single-cell model system to study cellular differentiation, asymmetric division, and their coordination with cell cycle progression. One cell of C. crescentus can divide into two types of cells. And this bacterium has many ilk genes with pathogens. Its master regulator of cell cycle is CtrA gene, which controls one quarter of cell cycle regulators and participates in many progresses in cell cycle. CtrA codes a key response regulator of two-component signal-transduction system, an important system in recognition, invasion, pathogensis and symbiosis of pathogens to host. A sequence named CtrZ in P. zucineum has 93% homology to CtrA. After blasting genes regulated by the two, 34 in 45 genes directly controlled by CtrA have homology to sequences in P. zucineum. At the same time, biological experiments prove that CtrZ has the same function with CtrA, and CtrZ gene is a master cell cycle regulator in P. zucineum. |
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