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Brucellosis is a zoonotic disease, the main cause of livestock abortion and human undulant fever, which is locally epidemic worldwide, and widely epideimic in some countries. Brucella is the causative agent of brucellosis, causing infection in domestic and wild animals. Human brucellosis is caused through contacting with infected animals. The facility of its becoming state of aerosol makes Brucella appealing as biological warfare agents. Therefore, brucellosis is a hazard infectious disease to human health, economic development and social security.According to the host range and biochemical characteristics,Brucella may be divided into six species with 18 biotypes, including B. melitensis (1, 2 and 3), B. abortus (1, 2, 3, 4, 5, 6 and 9), B. suis (1, 2, 3, 4 and 5), B. canis, B. ovis and B. neotomae. The main species epidemic in the worldwide includes B. melitenesis, B. abortus and B. suis. B. abortus mainly infects cattles; B. melitensis infects goat and sheep; B. suis infects several types of animals. Human brucellosis is mainly caused by the three species. Different species of Brucella have different host range, and their virulence for human or animal are different, e.g. some infecting with no symptoms, others showing strongly characteristic symptoms. B. melitensis and B. abortus are the most popularly epidemic strains in China, while in other countries, more incidence of B. abortus infection is reported.Brucella is a conservative bacterium in DNA, which is related with its basic characteristics. Brucella is an intracellular bacterium, having less opportunity to exchange DNA with other organisms. It has two chromosomes, without plasmids, and many exogenous plasmids could not replicate in it, which might cause its limited variation in Brucella genomes. Despite of less variation, Brucella shows DNA polymorphism as proved by DNA hybridization and VNTR.Genome sequencing of bacteria provides a new platform for the research on bacteria, especially when more than one strains have been sequenced, which makes it possible to compare and analyze the whole genome sequence for inferring its genome content and virulence. Since 2001, 10 strains of Brucella including all the six species were sequenced, making it possible to probe the difference of the gene content among different species.In this study, we first downloaded the sequences of the ten Brucella strains, and then analyzed their genome contents. Gene comparison was performed using house made procedures, which resulted in a 0/1 matrix. And then consensus genes and complete genes were analyzed according to their chromosome locations. The results showed that the ten strains are different in chromosome length, coding gene number, tRNA and pseudo-genes. Most of the strains have a nearly 2.1Mb for chromosome I, and 1.2 Mb for chromosome II, while chromosome I of B. suis 23445 is only 1.9Mb, with a large regions of 200kb transferred to chsomosome II.The strains have different number of pseudogenes, B. microti have only 63 pseudo genes, but the strain of 2308 have 316. Accumulation of pseudogene reflects the adaptive evolution of this bacterium, where those genes not necessary are inactivated. Comparison of 10 genome sequences resulted in the finding of 2231 consensus genes and 4815 complete genes, and the genes with differential distribution account for 53.7%, indicating the great differences in Brucella genomes. Most of the differential genes locate on chromosome II, suggesting that most of the differences exist in chromosome II, which is consistent with the point that chromosome II might be from a big plasmid.Genome comparison also identified a number of genes, locating continuously but lost or acquired simultaneously, which are named as differential fragment region (DFR). A total of 46 DFRs were identified, with the gene number from 2 to 41. In order to analyze the distribution of these DFRs in different biotypes from different species, 2 genes of each DFR were selected as representative genes. PCR was used to test the presence or absence of these genes in representative strains. The representative genes of 42 DFRs were identified to be present or absence together, indicating that these genes really compose DFRs. Detailed analysis showed that most of these DFRs present lower GC content than the GC average in the chromosome, and recombinase and tRNA was found adjacent to 16 DFRs. These genetic characteristics indicated that these DFRs might be acquired from other organisms. Cluster analysis of these DFRs suggested that B. melitensis and B. abortus clustered together, and more differences were found among different serotypes of B. suis, which clustered with different species. Functional information and distribution of DFRs among the strains reflected the polymorphism of Brucella genomes, which is related with bacterial biochemical characteristics and virulent phenotypes.Differentiation of Brucella genus is a focus of Brucella evolutionary research. Phylogenesis with whole genome sequences showed that B. ovis is a relatively old species, while B. melitensis and B. abortus are relatively highly-evolved ones. In order to evaluate the change of gene content during Brucella speciation, we analyzed house-keeping gene sequences to infer the phylogenetic tree of representative strains, and analyzed the gene lost and found. 7 house-keeping genes, i.e., aroA, cobQ, dnaK, gap, glk, gyrB, and trpE, were chosen for polymorphism analysis. The results showed that alleles varied from 3 to 7, with least trpE 3 alleles, and glk, 7 alleles. According to the alleles, the 19 strains are divided into 13 STs, of which ST1 (B. abortus 1 and 2), ST2 (B. abortus 5 and 9), ST12 (B. melitensis 2 and 3) have two strains, and ST6 have 3 strains (B. canis, B. suis 3 and 4). SplitsTree analysis clustered the 19 strains into 5 branches: B. abortus, B. suis/B. canis, B. neotamae, B. melitensis and B. ovis. eBURST clonal complex analysis showed that 2 clonal complex, CC1 and CC2, of which CC1 are mainly composed of serotypes of B. abortus, and CC2 composed of B. suis and B. canis. The sequences were concatenated and used by MEGA to infer phylogenetic tree, from which 4 branches were observed, B. ovis, B. melitenesis/B. abortus, B. suis/B. canis and B. neotomae. Based on this phylogenetic tree, gene lost and found were analyzed. No strict rules were observed among these branches except B. ovis, which loss some special DFRs which are present in all other strains. The parallel lost and found of DFRs implied the adaptive evolution of Brucella.In a word, in this study we systematically analyzed the genome sequences of Brucella. The results showed that although Brucella is relatively conservative, great differences are found, and this variation locates mainly on chromosome II. Some DFRs in Brucella genome have the evidence of exogenous acquisition, indicating that these genetic materials are acquired from other organisms. Compared with the phylogenetic tree of house-keeping genes, the lost and found of DFRs did not show consistent changes. However, parallel lost and found of DFRs might be related with the bacterial adaptation to environments.
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