| Yersinla pestis, the causative agent of plague, causes at least 160,000,000 people's death during the three plague pandemics in the world: the Justinian plague, the Black Death and the modern plague. With the increasing incidents of human plague, appearance of multi-drug resistant strains, its potential usage as an agent of biological warfare and ambiguous nosogenesis, it's very important to study how Y. pestis works. In order to understand the secrets of this pathogen better, we carried out genomic and comparative genomic analysis of Y. pestis, identified potential new toxin proteins, furthermore, discussed one of secretion signals in type â…¢ secretion system which exists in many gram negative bacteria, type â…¢ secretion chaperones' signals mechanism, based on motifs' and structure s' conservation.Complete genome sequencing and annotation of Y. pestis strain 91001While the complete genome sequence of Y. pestis strain CO92 and KIM which is virulent to human being had been finished, we sequenced the complete genome of Y. pestis strain 91001,a strain avirulent to human being, and compared its genome sequence with CO92 and KIM, in order to understand the nosogenesis at DNA sequence level better, then, find out new toxin factors. We used "whole genome shotgun" approach and got a single circular chromosome and four plasmids, named pPCP1,pCD1,pMT1 and pCRY, with length of 9,609bp, 106,642bp, 70,159bp, 21.742bp respectively. First three plasmids are similar to refrence strain CO92 and KTM; the last one is new and never reported, maily encode type â…£ secretion system genes independently. There are 4,037 CDSs in 91001 chromosome, including 100 genes of translation ribosomal structure and biogenesis, 204 genes of cell motility and secretion, 296 genes of DNA replication recombination and repair, 205 genes of transcriptional regulatory, 141 pseudogenes, and others. Plasmid pCD1 predicts 98 genes, most of them are type â…¢ secretion system gene clusters; Plasmid pMT1 predicts 129 genes,most of them are hypothetical proteins. The preliminary proteomics research uncovered 1193 proteins by different methods, which count for 28.7% of all the predicted CDSs in 91001 genome.Comparative genomics anlysis of Yersinia pestisBy comparison of genome of Y. pestis strain CO92, KIM and 91001, we found that there are lots of Insertion sequence (IS). Due to the rearrangements mediated by IS, the structure of 91001 chromosome shows dramatic differences compared with CO92 and KIM. 91001 and CO92 have their special DNA sequence while KIM doesn't. Highly virulent strain CO92 and KIM have 93 genes except pseudogenes which are lost in 91001, they are 4 genes related to virulence, 6 genes of regulation, 20 genes related to enzyme and metabolism, 29 genes of phage, 3 genes of flagellum, 8 genes of membrane, 1 gene of lipoprotein, 21 hypothetical genes including 8 conserved hypothetical genes. Known virulent genes and pathogenicity islands (for example, type III secretion system, type IV secretion system) are complete without deletion or pseudogene with homology alignment. We search all the genes shared in CO92 and KIM but lost in 91001 by the methods of suquence alignment and motif searching, and identified 6 potential new virulent genes and 3 genes related to drug resistance.Type III secretion chaperone have conserved characterized motifs although less similar in sequenceType III secretion system is one of the important pathogencity islands in Y. pestis. Many gram-negative pathogens utilize type III secretion system to inject toxins into the cytosol of eukaryotic cell; some effector proteins (toxins) are secreted by their cognate chaperones through type III secretion. Although Cheng etc. presented in 2000 that the common amino acid sequence or secretion signal might be conserved among these chaperones, previous studies indicated that there was little amino-acid sequence similarity among these chaperones. However, we analyzed 35 type III secretionchaperones by motif search, found out 4 characterized motifs, give a functional classification of type III secretion chaperones based on characterized motifs, identified conservative sites in the same motif family, and predicted 70 potential new TTSC. In addition, we presented a new hypothesis based on Birtalan's hypothesis and results above: the same functional TTSC family, not all TTSC, tends to shoe-horned into a single structure related to secretion signal.Type III secretion chaperones of the same functional family possibly have the same structureChaperone SicP% CesT> SigE^ SycE(Class IA) had been identified to have similar crystal structure in PDB database. Although chaperone Spal5 (Class IB) has the conserved fold of Class IA chaperones, it forms a different dimer. We used Dali to do structure alignment of SycN, SycH and AvrPphF with CesT. The results showed that these 3 TTSC obviously have the similar structure. Unknown structures of other TTSCs have no homolog in PDB database by BLAST. We predicted these TTSCs' structure by threading and found out that TTSCs in 2nd motif family and 7th motif family have similar structure fold, TTSCs 3rd in motif family have similar structure fold, TTSCs 1st in motif family have similar structure fold, another 4 TTSCs which can't be found any characterized motifs have different structure fold. This result supports our hypothesis.Discussion of TTSC's secretion mechanism based on structure conservationThere are three hypotheses on type III secretion system's signal: N-terminal, TTSC and mRNA hypotheses. Now it tends to think that all the TTSC forms a similar structure and functional classification, which mostly related to secretion signals. But, Based on analysis of characterized motifs and prediction of structure folding, it seems that all TTSC in the same functional family have a similar structure, not all the TTSC classified into 3 class. According to different function and number of substrate, there are most possibly more than three TTSCs' secretion signals, which can be recognized...
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