Am. J. Trop. Med. Hyg., 81(4), 2009, pp. 714–717 doi:10.4269/ajtmh.2009.09-0174 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene
Spatial Variation of Yersinia pestis from Yunnan Province of China Zhikai Zhang,† Rong Hai,† Zhizhong Song, Lianxu Xia, Yun Liang, Hong Cai, Ying Liang, Xiaona Shen, Enmin Zhang, Jianguo Xu,‡ Dongzheng Yu,‡ and Xue-Jie Yu*‡ State Key Laboratory for Infectious Disease Prevention and Control, Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; Department of Pathology, University of Texas Medical Branch, Galveston, Texas; Yunnan Institute for Endemic Disease Control and Prevention, Dali, China
Abstract. Yunnan Province of China is considered the site of origin for modern plague. We analyzed the genotypes of eight Yersinia pestis strains isolated from three counties in Yunnan Province by pulse field gel electrophoresis (PFGE). PFGE showed that strains isolated from the same site were identical regardless of hosts or year of isolation. However, Y. pestis strains isolated from geographically distinct loci were genetically divergent. Whole genome sequences of two strains from two foci in Yunnan showed that the genetic variation of Y. pestis strains was caused by genome rearrangement. We concluded that Y. pestis strains in each epidemic focus in Yunnan were a clonal population and selected by host environments. The genomic variability of the Y. pestis strains from different foci were caused by genome rearrangement, which may provide a positive selective advantage for Y. pestis to adapt to its host environments. previously sequenced were determined by using the SeqBuilder of DNAstar software. A 2-kb FseI fragment that consisted of 1 kb DNA sequence upstream and 1 kb DNA sequence downstream the FseI site was selected from each FseI site in eight Y. pestis genome sequences. Each FseI fragment of strain D106004 was compared with all FseI fragments of each of the remaining seven Y. pestis strains by the Megalign program to determine the counterparts of FseI fragments. Pulse field gel electrophoresis. Pulse field gel electrophoresis (PFGE) was performed following the PulseNet Protocols (http://www.cdc.gov/PULSENET/protocols.htm). Briefly, a bacterial cell suspension (400 μL) was added to 20 μL of Proteinase K (20 mg/mL stock) and 400 μL melted 1% SeaKem Gold agarose containing 1% SDS. The suspension was mixed gently by pipetting and was immediately dispensed into a mold to prepare the plugs. The plugs were placed in a tube and incubated in a 54°C shaking water bath for 1 hour with constant agitation (150–175 rpm) to digest the bacteria. The agarose plugs in each tube were washed sequentially with 15 mL prewarmed (50°C) sterile ultrapure water (2×) and pre-warmed (50°C) sterile TE Buffer (4×). The washings were performed by shaking vigorously in a 50°C water bath for 15 minutes. The plugs were placed in a sterile disposable Petri dish or on a large glass slide and were cut into 2.0- to 2.5-mm-wide slices with a single edge razor blade. The plug slices were submerged into restriction buffer and incubated at 37°C in a water bath for 10 minutes or at room temperature for 15 minutes. After removing the buffer, 200 μL of restriction enzyme mixtures containing 40 U of FseI was added to each tube. The plugs were incubated in a 37°C water bath for 4 hours. DNA was separated for 18 hours by PFGE using a CHEF Mapper (Bio-Rad Laboratories, Hercules, CA). Gels were stained with ethidium bromide, and the images were captured using Gel Doc 2000 (Bio-Rad Laboratories). DNA markers were the 50-kb lambda ladder (Bio-Rad Laboratories) and Salmonella ser. Braenderup H9812 genomic DNA completely digested with XbaI. The PFGE pictures were aligned with each other using strains that had identical patterns as reference using Adobe Photoshop.
INTRODUCTION During the last 2,000 years, three plague pandemics occurred, killing ~200 million people and resulting in social and economic upheavals that are unmatched by any war or other infectious disease pandemic.1 Modern plague probably originated in 1855 in Yunnan Province of China, where the disease rapidly spread to the southern coast of China, reaching Hong Kong and Guangdong in 1894; by 1900, steamships had disseminated the disease all over the world.2–6 Plague is caused by Yersinia pestis, a pathogen that circulates among susceptible rodents and spreads to humans via the bite of infected fleas. Although Yunnan Province of China is considered the site of origin for the most recent pandemic and plague strains spread around much of the world during this event, the genomic structures of Y. pestis isolates from Yunnan have never been molecularly analyzed but should provide useful information with respect to the genomic evolution of Y. pestis. In this report, we describe genomic variability among Y. pestis strains isolated from Yunnan. MATERIALS AND METHODS Determination of the FseI sites in Y. pestis genome. The whole genome sequences of six published strains of Y. pestis were obtained from the NCBI genome database and analyzed for rare cutting restriction enzyme sites in silico using the SeqBuilder of DNAstar software. Among the enzymes that infrequently cut the Y. pestis genome, FseI sites are scattered throughout the entire genome, and the resulting fragment lengths are moderate in size, leading us to choose this enzyme for further restriction digest analyses of the Y. pestis genomic DNA samples used in our study. Identification of the matching FseI sites among eight Y. pestis genomes. Two Y. pestis isolates, D106004 and D182038, from Yunnan were sequenced by shotgun genomic sequencing by us (GenBank accession numbers: CP001585 and CP001589). The FseI site in the two newly sequenced Y. pestis and six strains
* Address correspondence to Xue-Jie Yu, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. E-mail:
[email protected] † These authors contributed equally. ‡ These authors contributed equally.
RESULTS To determine the genomic variation of Y. pestis, we analyzed the genomic patterns of eight strains of Y. pestis isolated
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from three counties including Yulong, Jianchuan, and Midu in Yunnan Province of China. The three counties are adjacent to each other with Yulong and Midu in the north and south of Jianchuan, respectively. Jianchuan County and Yulong County were located in two plateaus and separated by a 50-km mountain area. PFGE showed that strains from the same site were identical in PFGE patterns even though strains were isolated from different hosts at different times (Table 1; Figure 1). Three strains (D106004, strain Z13, and strain Z14) isolated at the same time in 2006 from Yulong County were identical (Figure 1). These strains were isolated from two different rodent species (Rattus nitidus and Apodemus chevrieri; Table 1). Four strains (D182038, 1683, 330, and 332) from Jianchuan County were also identical (Table 1; Figure 1). These strains were isolated from different hosts in a time period that spanned 36 years. Strains 330 and 332 were obtained in 1954, strain D182038 was isolated in 1982, and strain 1683 was isolated in 1990. Except for strain 332 that was isolated from a patient, all other strains were isolated from rodents (Table 1). The PFGE patterns of strains from different sites were varied. Strains from Yulong County were different from Jianchuan County, and they all differed from a single strain (strain 33) from Midu County. Among the bands ≥ 50 kb, only nine bands were common among all the isolates (Figure 1). To determine the molecular basis of the genetic difference, we sequenced the whole genome of strains D106004 and D182038 that were from two foci. Comparing the genomes of strains D106004 and D182038 and six Y. pestis genomes sequenced previously,7–10 we found that all Y. pestis strains had 31 FseI restriction sites except for Chinese strain 91001 and strain Nepal 516 (Table 2). Strain Nepal 516 had 29 sites and the 2 missed FseI sites were located in a pathogenic island that was deleted. Strain 91001 had an extra FseI site (number 13 FseI site). All FseI sites were located in the coding sequences of Y. pestis strains, which may explain the low mutation rate of the FseI site in the genome of Y. pestis. A 2-kb FseI fragment of each FseI site that consisted of DNA sequences 1 kb upstream and 1 kb downstream of each FseI site from D106004 were aligned for all 31 FseI sites to determine the homology of the sequences. The homology of the sequence from all restriction sites in the D106004 chromosome was < 0.220, and the average was 0.193 ± 0.007. This indicated that all 31 FseI restriction sites were in different sequence environments. The sequences of the 2-kb FseI fragments were compared for sequence homology in all strains of Y. pestis whose genomes were sequenced to determine the counterparts of each FseI site in all strains. The results showed that each FseI site was randomly distributed in various positions among the genomes of
Figure 1. PFGE patterns of Y. pestis isolates from Yunnan. The Y. pestis DNA was digested with FseI and separated by PFGE. Lane M1, DNA marker of 50-kb lambda ladder (Bio-Rad Laboratories); Lane M2, XbaI-digested Salmonella ser. Braenderup H9812 genomic DNA. Lanes 1–3, strains D106004, Z13, and Z14 from Yulong County; Lane 4–7, strains D182038, 330, 1683, and 332 from Jianchuan County; Lane 8, strain 33 isolated from Midu County.
Y. pestis strains (Table 2). It suggested that the different positions of each FseI site among the strains were caused by the genome rearrangement of Y. pestis. The two Yunnan strains (D106004 and D182038) were closest to each other and next to CO92, an isolate from the United States (Table 2). It suggested that the Yunnan strains and the US strains share a quite recent common ancestor as suggested previously.11 We constructed the FseI physical maps using the 2-kb FseI fragments for strains 106004 and D182038 (Figure 2). Comparing the physical maps of D106004 and D182038 showed that the most remarkable features of their genomes were inversions and/or transpositions of certain genomic fragments. Thirteen of the 31 FseI sites between D106004 and D182038 were in different orientations because of inversion of genomic sequences. Even the corresponding FseI sites in the same orientation in some FseI restriction sites were in very different positions (transposition) such as FseI sites 15 and 16. Less than one half of the FseI sites were in the same position and orientation between these genomes. These results suggested that genome rearrangement occurred in the Y. pestis genome isolated in Yunnan and the PFGE pattern difference between the Y. pestis strains from Yunnan was related to extensive genome rearrangement (Figure 2). DISCUSSION We showed the spatial variability of Y. pestis isolates from Yunnan, i.e., the isolates from each endemic focus is different. The isolates were obtained from three endemic foci in a mountain area in Yunnan Province, and the distance between
Table 1 Yersinia pestis strains from Yunnan Province and their genotypes Strain
Year
Host
Location
Genotype
D106004 Z13 Z14 D182038 1683 330 332 33
2006 2006 2006 1982 1990 1954 1954 1952
Rattus nitidus Apodemus chevrieri Apodemus chevrieri Apodemus latronum Apodemus latronum Human Rattus flavipectus Rattus flavipectus
Yulong County Yulong County Yulong County Jianchuan County Jianchuan County Jianchuan County Jianchuan County Midu County
I I I II II II II III
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Table 2 FseI restriction sites in the genomes of Y. pestis strains D106004*
D182038
CO92
91001
Antiqua
Nepal 516
Kim
Pestoides F
1† 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
1 2 3 5 4 7 6 13 12 11 10 9 8 17 14 15 16 18 19 20 21 22 25 24 23 26 27 28 29 30 31
1 3 2 4 5 6 13 7 8 9 10 11 12 14 17 16 15 18 19 20 21 22 24 25 23 26 27 28 29 30 31
28 26 27 20 5 6 14 7 9 8 10 11 12 15 18 17 16 19 4 3 1 2 31 30 29 21 24 23 22 25 32
26 4 5 6 7 8 15 9 10 11 12 13 14 16 19 18 17 20 21 22 23 24 25 28 29 27 3 2 1 30 31
5 24 23 22 21 20 12 19 18 17 – – 11 13 16 15 14 10 9 8 7 6 27 26 25 4 3 2 1 28 29
29 28 27 26 25 24 17 23 22 21 20 19 18 16 13 14 15 12 11 10 9 8 3 2 1 7 6 5 4 30 31
24 4 5 14 20 13 6 12 11 10 9 8 7 15 18 17 16 19 21 22 23 25 1 2 3 30 27 28 29 26 31
* Strain name. † FseI restriction site of strain D106004 and its counterpart of other strains were listed in each row. – = FseI site missing in the strain.
each site was < 50 km. The variation of Y. pestis suggested that Y. pestis in each endemic focus were clonal populations. We further compared the genomic sequences of two strains from Yunnan to determine the mechanism of the Y. pestis variation. We showed that different PFGE patterns between strains of Y. pestis are caused by genome rearrangement. The Y. pestis genome rearrangement phenomenon was discovered when genome sequence of Kim was compared with that of Y. pestis CO92.7 A previous study showed that the genome rearrangement of Y. pestis is mediated by insertion sequences (IS).12 By comparing the closely related strains, we found that the genome rearrangement phenomenon in Y. pestis is extremely active. In the genome sequence analysis of D106004 and D182038, we found that, in fact, the Y. pestis genome is composed of many relatively independent segments. These segments themselves were highly conserved, which led to the conservative genome composition character. However, the order and orientation of the segments were different between the two strains. This suggests that the segments can be reshuffled during the replication of the genome. The Y. pestis genome seems to be unified, but the positions and orientations of independent segments within the genomes can vary without obvious changes in the biology and pathogenicity of this bacterium, as long as all segments are present. We also showed that there is no temporal variability among Y. pestis isolates in a single endemic focus spanning a period of almost four decades by PFGE. Because PFGE only identifies major genetic variation, minor genetic variation may exist among these strains, which need to be further studied using
Figure 2. FseI physical maps of D106004 and D182038. The outer circle represents D106004 and the inner circle is D182038. The FseI sites on D106004 (pointed outward) were numbered clockwise from 1 to 31, and the counterpart of each FseI restriction site on D182038 (pointed inward) was labeled same number regardless of its position. The segments separated by the FseI sites were numbered clockwise from 1 to 31 in D106004; the counterpart of each segment on the D182038 was marked same number regardless of its position. Open box, the FseI sites on both ends of the segment were same in position and orientation between the two strains; red box, one end or both ends of the segment had different FseI sites between the two strains; green box, two FseI sites on the ends of the segment were inverted between the two genomes; yellow box, both FseI sites on the end of the segment were the same in sequence and orientation, but different in position between the two strains. B4–b13, the segments corresponding to Bands 4–13 from top to bottom on PFGE of D106004, which were present in all isolates of Y. pestis from Yunnan. This figure appears in color at www.ajtmh.org.
rapidly evolving markers such as single-nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), and IS analyses.13–15 Received April 3, 2009. Accepted for publication July 14, 2009. Acknowledgments: The authors thank Yan Liu for discussion of the manuscript and assistance in preparation of figures and Dr. Jere McBride (University of Texas Medical Branch at Galveston) for reviewing the manuscript. Financial support: This study was supported by a grant from the Special Fund for Health Sector, the People’s Republic of China (Award 200802016) and a grant from the ministry of science and technology of the People’s Republic of China (Award 2004BA718B07). Authors’ addresses: Zhikai Zhang, Rong Hai, Lianxu Xia, Hong Cai, Ying Liang, Xiaona Shen, Enmin Zhang, Jianguo Xu, and Dongzheng Yu, State Key Laboratory for Infectious Disease Prevention and Control, Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, PO Box 5, Changping, Beijing 102206, People’s Republic of China. Zhizhong Song and Yun Liang, Yunnan Institute for Endemic Disease Control and Prevention, 5 Wenhualu, Dali 671000, Yunnan Province, Peoples’s Republic of China. Zhikai Zhang and Xue-jie Yu, Department of Pathology, University of Texas Medical Branch, Galveston, TX, 775550609.
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