The relative contributions of mutation rate variation, selection, and recombination in shaping genomic variation in bacterial populations remain poorly understood. Here we analyze 3318 Yersinia pestis genomes, spanning nearly a century and including 2336 newly sequenced strains, to shed light on the patterns of genetic diversity and variation distribution at the population level. We identify 45 genomic regions ("hot regions", HRs) that, although comprising a minor fraction of the genome, are hotbeds of genetic variation. These HRs are distributed non-randomly across Y. pestis phylogenetic lineages and are primarily linked to regulatory genes, underscoring their potential functional significance. We explore various factors contributing to the shaping and maintenance of HRs, including genomic context, homologous recombination, mutation rate variation and natural selection. Our findings suggest that positive selection is likely the primary driver behind the emergence of HRs, but not the sole force, as evidenced by the pronounced trend of variation purging within these regions.

ABSTRACT Recent studies revealed that acetylation is a widely used protein modification in prokaryotic organisms. The major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB have been found to be involved in basic physiological processes, such as primary metabolism, chemotaxis, and stress responses, in Escherichia coli and Salmonella . However, little is known about protein acetylation modifications in Yersinia pestis , a lethal pathogen responsible for millions of human deaths in three worldwide pandemics. Here we found that Yp_0659 and Yp_1760 of Y. pestis encode the major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB, respectively, which can acetylate and deacetylate PhoP enzymatically in vitro . Protein acetylation impairment in cobB and yfiQ mutants greatly decreased bacterial tolerance to cold, hot, high-salt, and acidic environments. Our comparative transcriptomic data revealed that the strongly decreased tolerance to stress stimuli was probably related to downregulation of the genes encoding the heat shock proteins (HtpG, HslV, HslR, and IbpA), cold shock proteins (CspC and CspA1), and acid resistance proteins (HdeB and AdiA). We found that the reversible acetylation mediated by CobB and YfiQ conferred attenuation of virulence, probably partially due to the decreased expression of the psaABCDEF operon, which encodes Psa fimbriae that play a key role in virulence of Y. pestis . This is the first report, to our knowledge, on the roles of protein acetylation modification in stress responses, biofilm formation, and virulence of Y. pestis .

The isolation and identification of Yersinia pestis are critical for plague surveillance and diagnosis. This pathogen can be isolated from animals and patients using different kinds of specimens and different cultivation strategies. Yersinia pestis can be identified by its biochemical features, by its susceptibility to bacteriophage lysis, by animal experiments, or via polymerase chain reaction. In this chapter, the techniques used to isolate suspected Y. pestis, as well as the methods to identify this pathogen, are described.

OBJECTIVE:To analyze the plasmid features and geographical distribution characteristics of Yersinia pestis of different plague foci in China.METHODS:A total of 2 213 Yersinia pestis strains were colected from 11 Chinese plague foci separated during 1943 to 2012, and plasmid DNA according to alkali cracking method, and measured the relative molecular mass (Mr) of plasmid DNA based on the standard plasmid contrast method, then analyzed the plasmid profiles by agar gel electrophoresis.RESULTS:A total of 2 213 strains had 16 kinds of plasmids with different Mr, including 4×10(6), 6×10(6), 7×10(6), 13×10(6), 16×10(6), 20×10(6), 22×10(6), 23×10(6), 27×10(6), 30×10(6), 36×10(6), 45×10(6), 52×10(6), 65×10(6), 72×10(6) and 90×10(6). Plasmid were classified into 26 kinds of plasmid profiles. A total of 2 213 Yersinia pestis strains contained 4 large plasmids, 52×10(6), 65×10(6), 72×10(6) and 90×10(6), whose ratio was 22.10% (589/2 213), 75.60% (1 672/2 213), 0.17% (4/2 213), 2.12% (47/2 213), respectively. Among which, strains with plasmid 52×10(6), 65×10(6), 90×10(6) distributed in Qinghai-Tibet plateau Himalayan Marmot natural plague foci, strains with 72×10(6) plasmid only distributed in Inner Mongolia Meriones unguiculatus natural plague foci and Junggar Basin R. opimus natural plague foci, and 65×10(6) plasmid distributed in all the other foci.CONCLUSION:Strains in Chinese 11 plague foci contained 4 kinds of large plasmid, the Mr respectively were 52×10(6), 65×10(6), 72×10(6), 90×10(6), which were classified into 26 kinds of plasmid profiles with other plasmid. These plasmid profiles distributed in relatively independent epidemic focus.

Objective To analyze the epidemiological characteristics of plague occurred between 1956 and 2012 in Xinghai County of Qinhai Province,in order to provide a scientific basis for formulating proper control measures.Methods Investigation and monitoring reports,work summaries of epidemic processing of natural foci of plague in Xinghai County between 1956 and 2012 were analyzed using descriptive epidemiology.Results Between 1956 and 2012,33 Yersinia pestis strains were separated from various animals and other media in Xinghai County,among 12 cases were separated in Marmot Himalayan,accounted for 36.36％.The main foci was detected in Ziketan Town where a total of 27 cases were detected accounted for 81.82％(27/33).During the past 57 years,human plague occurred 3 times,with 33 cases infected and among them 11 dead,the fatality rate was 33.33％.Human plague occurred mostly in Ziketan Town from July to August; most of the cases were pneumonic plague and infected by peeling and eating marmots,contacting with infected sheep dogs or patients.Conclusions Marmot Himalayan plague foci in Xinghai County is still in the active phase,monitoring and pre-warning should be strengthened,and various control measures should be properly implemented,in order to reduce the incidence of human plague.

OBJECTIVE:To study the pathogenic ecology characteristics of plague in Qinghai plateau.METHODS:Applied molecular biology techniques, conventional technologies and geographic information system (GIS) to study phenotypic traits, plasmid spectrum, genotype, infected host and media spectrum etc.of 952 Yersinia pestis strains in Qinghai plateau plague foci, which were separated from different host and media in different regions during 1954 to 2012.RESULTS:The ecotypes of these strains were Qingzang plateau (91.49%, 871/952),Qilian mountain (6.41%, 61/952) and Microtus fuscus (1.26%, 12/952).83.6% (796/952) of these strains contained all the 4 virulence factors (Fr1, Pesticin1,Virulence antigen, and Pigmentation), 93.26% (367/392) were velogenic strains confirmed by virulence test.725 Yersinia pestis strains were separated from Qinghai plateau plague foci carried 9 kinds of plasmid, among which 713 strains from Marmot himalayan plague foci carried 9 kinds of plasmid, the Mr were 6×10(6), 7×10(6), 23×10(6), 27×10(6), 30×10(6), 45×10(6), 52×10(6), 65×10(6) and 92×10(6) respectively. 12 Yersinia pestis strains were separated from Microtus fuscus plague foci carried only 3 kinds of plasmid, the Mr were 6×10(6), 45×10(6), 65×10(6). Meanwhile, the strains carrying large plasmid (52×10(6), 65×10(6) and 92×10(6)) were only distributed in particular geographical location, which had the category property. The research also confirmed that 841 Yersinia pestis strains from two kinds of plague foci in Qinghai plateau had 11 genomovars. The strains of Marmot himalayan plague foci were given priority to genomovar 5 and 8, amounted to 611 strains, genomovar 8 accounted for 56.00% (471/841), genomovar 5 accounted for 23.07% (194/841). Besides, 3 new genomovars, including new 1(62 strains), new 2(52 strains), new 3(48 strains) were newly founded, and 12 strains of Microtus fuscus plague foci were genomovar 14.CONCLUSION:The main host and media of Qinghai plateau plague foci directly affected the spatial distribution regularities of plague epidemic and the pathogens characteristics, meanwhile the polymorphism of plague ecological geographic landscape leds to the complexity of Yersinia pestis' genotype.

Objective To identificate and analyze the source of Yersinia pestis (Y.pestis) from a human plague death case in Huangyuan County,Qinghai Province.Methods The organs,lymph and blood samples of this human plague death case in Huangyuan County,Qinghai Province in 2011 were collected for laboratory tests including isolating and culturing Y.pestis,biochemical identification,toxicity identification and genes' different region(DFR) analysis.Results Y.pestis was separated from organ samples,and it was identified as Qinghai-Tibet Plateau Marmot strain through biochemical identification test; it had F1,P1,Pgm and Pst four virulence factors; the mouse virulence test showed that the absolute lethal dose(LD100) was 1000 Y.pestis,and the median lethal dose(LD50) was 10 Y.pestis; DFR genotyping analysis showed that the genotype was type 5.Conclusions The strain separated from the death case is Qinghai-Tibet Plateau Marmot strain.Traditional epidemiological investigation combined with molecular epidemiological techniques has an important practical significance for timely detection of outbreaks,and trace the source of infection.

Objective To explore the plague epidemical trend of nearly a 10 years data in Qinghai province to provide basis for making the prevention and control measures. Method The regional distribution and time distribution of animal and human plague, monitoring and plague foci of survey data in Qinghai from 2001 to 2010 were analyzed with Excel software 2003. Results In Qinghai province, a total of 167 strains of Yersinia pestis were isolated from infected animals and insects in 10 years. Yersinia pestis was mainly distributed in Wulan,Delinha, Geermu, and Tianjun, along the Qinghai-Xizang railway. Human plague was occurred every year from 2001 to 2010 except 2002, 2007, 2008, and 2010. In the 10 years, there were 37 plague cases and 16 of these cases died, the mortality was 43.24％. The plague cases were mainly distributed in Nangqian, Qumalai, Chenduo,Zhiduo, Xinghai, Tongde, Tianjun, Wulan and Qilian. And these cases were found mostly in the period from May to October, especially in the period from August to October. Major clinical type of the plague cases was lung-type (62.16％,23/37). Conclusions The plague epidemic situation in Qinghai province is still severe, animal plague occurred year after year, and human plague outbreaks occasionally. Monitoring and early warning in the key areas should be strengthened, and the comprehensive measures of plague prevention and control should be carried out to reduce the incidence and prevalence of plague.

The subunit vaccine SV1 (20 mu g F1 + 10 mu g rV270) has been identified as the optimal formulation in mice, which provided a good protection against plague in mice, guinea pigs and rabbits. To compare SV1 and SV2 (200 mu g F1 + 100 mu g rV270) with live attenuated vaccine EV76, antibody responses, protective efficacy, cytokines (IFN-gamma, TNF-alpha, IL-2, IL-4, IL-10 and IL-12) production, CD4/CD8 ratio and CD69+ T-cell activation marker were determined in sera of the immunized Chinese-origin rhesus macaques, Macaca mulatta. The immunized animals with SV1 or SV2 developed higher anti-rV270 IgG titre, while those immunized with EV76 elicited a negligible IgG to V antigen, indicating that subunit vaccine (SV) had an advantage over EV76 in terms of the indispensable role of anti-V antibody against Yersinia pestis. There was no significant antibody titre difference between SV1 and SV2, suggesting that the immune response may have been saturated at the dose level of SV1. There were no statistical changes for CD4/CD8 ratios, IL-4 and CD69 levels between the three-vaccine immunized groups. However, a significant higher level of IL-12 was observed in the EV76 immunized animals, indicating that EV76 had an advantage over SV in respect of cellular immunity. Complete protection was observed for the immunized animals with SV and EV76, revealing that SV has a similar protective efficacy with EV76 against 6 x 106 CFU of Y. pestis challenge by subcutaneous route in Chinese-origin rhesus macaques.

OBJECTIVETo study the biological characteristics of Yersinia pestis and to develop prevention and control program on plague in Sanjiangyuan areas, Qinghai province.METHODSTo identify the biologic types and molecular biological features of Y.pestis isolated in Sanjiangyuan area from 1954-2007.RESULTSAmong the 411 strains of Y. pestis, 12 strains belonged to the microtus type Y. pestis with denitrification (-) and donkey-hide gelatin carbohydrate (-) and glycerine (+). 399 strains belonged to classic type Y. pestis with denitrification (+) and donkey-hide gelatin carbohydrate (+) and glycerine (+). 411 Y. pestis strains had factor F I and Pst I. Among them, VW+ strains of Y. pestis accounted for 95.13% (391/411), VW-accounted for 4.87% (20/411), Pgm(+) accounted for 80.78% (332/ 411), Pgm(+/-) accounted for 9% (37/411) and Pgm(-) accounted for 10.22% (42/411) respectively. 96.82% (213/220) of the Y. pestis strains showed strong virulence to laboratory mice while 3.18% (7/220) of the strains carried medium virulence. 90.02% of the tested Y. pestis (370/411) strains had 6 x10(6), 45 x 10(6), 65 x 10(6) plasmids. 8 types of genome were found among 80 strains of Y. pestis, with 6 of them resembling ZHOU Dongsheng' s classification. Two new genome types were found.CONCLUSIONThe Y. pestis in the Sanjiangyuan area had the characteristics of plague pathogen, identified in Qinghai-Tibet plateau. It is estimated that human beings are highly susceptible to the disease which spread fast, causing serious signs and symptoms with high death rate.

Objective To study the features of Yersinia pestis(Y.pestis)in areas along Qinghai-Tibet Railroad in Qinghai Province.Methods To identify the biologic types and the molecular biological feathers of Y.pestis isolated from areas along Qinghai-Tibet Railroad in Qinghai from 2001-2006.Results All the tested Y.pestis was biologically of classical type and ecologically of Qinghai-Tibet plateau type.The Y.pestis had high virulence.The Y.pestis of 65×106 plasmids was distributed in the Tanggula area,the Y.pestis of 52×106plasmids,in Tianjun and Delingha areas.The Y.pestis srains carried 52 × 106 plasmids.except the two containing 65 X 106 plasmids in Wulan County.The genetic type of Y.pestis in Tanggula was type 5 and that in Zongwulong of Delingha,Saishike,Keke,Tongpu of Wulan was type 8 except 2 strains of Y.pestis isolated from woodchuck and the patients in Dananwan of Tongpu,Wulan County were type 15.Conclusion The Y.pestis in the area along Qinghai-Tibet Railroad in Qinghai belongs to Qinghai-Tibet plateau type with high virulence.