Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element

The pigmentation (Pgm+) phenotype of Yersinia pestis encompasses a variety of different physiological traits, all of which are missing in Pgm- mutants. We have previously shown that loss of the Pgm+ phenotype is accompanied by the spontaneous deletion of at least 45 kb of chromosomal DNA, referred to as the pgm locus. Using chromosomal walking, we have now mapped the full extent of the pgm locus in Y. pestis strain KIM6+. Our results indicate that the locus spans 102 kb of DNA which is absent in the spontaneous Pgm- mutant, KIM6. Yersinia pseudotuberculosis PB1/0 contains sequences homologous to the entire pgm locus while only part of this region hybridized to Yersinia enterocolitica WA-LOX DNA. Restriction enzyme mapping and hybridization studies revealed the presence of a repetitive element at both ends of the pgm locus and in multiple copies elsewhere in the Y. pestis genome. This element may be responsible for generating the deletion.     

High-Frequency RecA-Dependent and -Independent Mechanisms of Congo Red Binding Mutations in Yersinia pestis

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar. The hmsHFRS genes required for CR binding (Crb(+)) are genetically linked to virulence-associated genes encoding a siderophore uptake system. These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb(+) phenotype. We constructed a recA mutant strain of Y. pestis KIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flanked pgm locus. Two Pgm-associated phenotypes (Crb(+) and pesticin sensitivity [Pst(s)]) were used as markers for the presence of the pgm locus in the RecA(+) KIM10+ and RecA(-) YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 x 10(-3)) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb(+) phenotype was still lost at a high frequency (4.5 x 10(-5)), although the loss of the Pst(s) phenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 x 10(-7)). These RecA-independent Crb(-) mutants were caused by mutations in both the hmsHFRS locus and in a newly identified gene, hmsT. Nonpigmented Yersinia pseudotuberculosis and Escherichia coli strains transformed with both hmsT and hmsHFRS became Crb(+). This study demonstrates that in a laboratory culture, the Crb(+) phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y. pestis in vitro may contribute to the mutation frequencies observed at the hmsHFRS and hmsT loci.     

Independent acquisition and insertion into different chromosomal locations of the same pathogenicity island in Yersinia pestis and Yersinia pseudotuberculosis

We show that Yersinia pestis and pesticin-sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP-HPI) is marked by IS 100 , has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4-like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis . These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis . The two YP-HPI locations and their possession of an integrase gene support a model of site-specific integration of the YP-HPI into these bacteria.     

The role of IS-elements of Yersinia pestis (Lehmann, Neumann) in the emergence of calcium-independent mutations

Calcium independent mutants of two Yersinia pestis strains were studied. Insertions of IS100 element at three different sites of plasmid pCad within calcium dependence region were detected in Y. pestis EV, as well as two extensive deletions covering the whole region. It was shown that IS100 carries no HindIII sites. Novel IS element of Y. pestis designated IS101 was discovered in strain 358, in addition to IS100. It is distinguished by a slightly smaller size, HindIII site presence and high specificity of integration.     

Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore.

We have sequenced a region from the pgm locus of Yersinia pestis KIM6+ that confers sensitivity to the bacteriocin pesticin to certain strains of Escherichia coli and Y. pestis. The Y. pestis sequence is 98% identical to the pesticin receptor from Yersinia enterocolitica and is homologous to other TonB-dependent outer membrane proteins. Y. pestis strains with an in-frame deletion in the pesticin receptor gene (psn) were pesticin resistant and no longer expressed a group of iron-regulated outer membrane proteins, IrpB to IrpD. In addition, this strain as well as a Y. pestis strain with a mutation constructed in the gene (irp2) encoding the 190-kDa iron-regulated protein HMWP2 could not grow at 37 degrees C in a defined, iron-deficient medium. However, the irp2 mutant but not the psn mutant could be cross-fed by supernatants from various Yersinia cultures grown under iron-deficient conditions. An analysis of the proteins synthesized by the irp2 mutant suggests that HMWP2 may be indirectly required for maximal expression of the pesticin receptor. HMWP2 likely participates in synthesis of a siderophore which may induce expression of the receptor for pesticin and the siderophore.     

Genetic analysis of biochemical differences of Yersinia pestis strains

Literature data and results of our experimental studies on genetic base of biochemical differentiation of Yersinia pestis strains of various subspecies and biovars are summarized in the review. Data on variability of genes coding biochemical features (sugar and alcohol fermentation, nitrate reduction), the differential development of which are the base of existing phenotypic schemes of Y. pestis strains classification, are presented. Variability of these genes was shown to have possible use for the development of genetic classification of Y. pestis strains of various subspecies and biovars.     

Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD)

A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed.     

Complete genome sequence of Yersinia pestis strains Antiqua and Nepal516: evidence of gene reduction in an emerging pathogen

Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the "classical" antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. We compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans.     

Genotyping of Yersinia pestis strains on the basis of variation of ribosomal rRNA biosynthesis genes

Analysis of restriction fragment length polymorphism of rRNA genes of Yersinia pestis and Y. pseudotuberculosis strains, circulating in Russian Federation and abroad revealed the effectiveness of ribotyping for differentiation between these microorganisms, as well as for differentiation between different Y. pestis biovars and main and nonmain subspecies of this agent. Use of this method was shown to be promising as a component for the complex molecular typing system of Y. pestis. Variant ribotypes of main and non-main subspecies of Y. pestis strains are presented.     

Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus

Yersinia pestis has been historically divided into three biovars: antiqua, mediaevalis, and orientalis. On the basis of this study, strains from Microtus-related plague foci are proposed to constitute a new biovar, microtus. Based on the ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative). A 93-bp in-frame deletion in glpD gene results in the glycerol-negative characteristic of biovar orientalis strains. Two kinds of point mutations in the napA gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively. A 122-bp frameshift deletion in the araC gene may lead to the arabinose-negative phenotype of biovar microtus strains. Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar. Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of Y. pestis and plague pathogenesis. Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine.     

IS1OO周围序列多态性分析技术的建立及其在鼠疫耶尔森氏菌分型中的应用

建立鼠疫耶尔森氏菌IS1000周围序列多态性（ISCP）分析技术,并探讨其在鼠疫耶尔森氏菌分型中的应用,根据鼠疫杆菌CO92株IS100的基因序列在其两端设计两条向外延伸的引物进行PCR扩增,电泳,获得的指纹图用RAPD,PHYLIP和Treeview软件分析,建立的ISCP分析技术稳定,可靠,利用该技术分析17个生态型的271株鼠疫耶尔森氏菌,扩增结果表明,指纹图有一定的差异,经RAPD,PHYLIP和Treeview分析可分为3个类型,IS100在鼠疫耶尔森氏菌染色体中虽然分布较广,但其周围序列变异较小,在遗传上较稳定,可作为鼠疫耶尔森氏菌的基因标志,研究该菌的分型与进化。     

Homology with a repeated Yersinia pestis DNA sequence IS100 correlates with pesticin sensitivity in Yersinia pseudotuberculosis.

We have identified IS100 sequences in a specific subset of Yersinia pseudotuberculosis isolates that were also sensitive to the Y. pestis-produced bacteriocin, pesticin. In contrast, Y. pseudotuberculosis strains which did not contain IS100 sequences were not sensitive to pesticin. We propose that IS100 serves as a molecular marker that identifies a subset of Y. pseudotuberculosis isolates that have a particularly close evolutionary and/or ecological relationship with Y. pestis.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.     

Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression.

The yopE gene of Yersinia pseudotuberculosis was recently sequenced, and YopE was identified as an indispensable virulence determinant when tested in a mouse model (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). In the study described here, the DNA sequences of the yopE genes of Yersinia pestis EV76 and Yersinia enterocolitica 8081 were determined and compared with that of the Y. pseudotuberculosis gene. Only two codons were found to differ, both leading to amino acid replacements, when the gene from Y. pestis was compared. These two replacements were also present in the gene from Y. enterocolitica; in addition, 18 other codons were found to differ. Thirteen of these substitutions led to amino acid replacements. Downstream of the yopE gene, the plasmid partition locus par was found to be conserved in all three species. In Y. enterocolitica 8081, the sequence homology was interrupted by a putative insertion sequence element inserted between the yopE gene and the par region at a position only 5 base pairs downstream of the yopE stop codon. Upstream of the yopE gene, 620 base pairs were conserved in the three species. This region contained a 130-amino-acid-long open reading frame reading in the opposite direction to the yopE gene and expressed a 14-kilodalton protein in minicells. An insertion mutation in this region constructed in Y. pseudotuberculosis expressed significantly lower amounts of YopE protein in vitro than did the corresponding wild type. The expression level could be restored by transcomplementation. This new locus was designated yerA, for yopE-regulating gene A. The yerA mutant was avirulent when mice were challenged by oral infection.     

Genotyping Yersinia pestis strains from various natural foci

Seven genetic variants of Yersinia pestis were detected by finger-printing of 85 strains of this bacterium from natural foci by means of a BX probe. Variants of Y. pestis strains correlate with certain species of carriers.     

Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.     

Genotyping of Yersinia pestis: variability of locus (CAAA)n in natural strains isolated in territory of former SSSR

Genome polymorphism by the locus (CAAA)n was studied in 69 strains of Yersinia pestis isolated from natural foci of the former Soviet Union. The polymorphism was found to be represented by ten alleles in chromosomes, which could be regarded as evidence of variability of this VNTR-locus (diversity index, DI = 0.86). The value of DI was found to vary substantially: from 0.24 in a group of vole strains from seven isolates from the Transcaucasian highlands to 0.77 in nine strains from the Central Asia desert focus. The allele polymorphism of the variable locus (CAAA)n in natural strains of Y. pestis was suggested to be used as a possible genetic marker of the strain. It was concluded that the oligonucleotide primers used in polymerase chain reaction should be upgraded to the genotyping accuracy.     

Determination of genetic bases of auxotrophy in Yersinia pestis ssp. caucasica strains

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG, aroF, thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.     

The population variability of Yersinia pestis in soil samples from the natural focus of plague

Three Y. pestis strains were found to exist in the experimental soil ecosystem at a temperature of 4 degrees - 8 degrees C for a longer period (10 months, the term of observation) than at room temperature (3.5 months). Y. pestis population structure was characterized by relative stability in strains of the subspecies altaica and heterogeneity in the strain of the main subspecies, manifested by the loss of the pgm locus by vegetative cells and the preservation of pgm+ variants in the latent (uncultivable) form (LF). In the populations of all strains uniformity in calcium dependence, the tendency towards a decrease in the synthesis of factor 1, nutritional requirements in amino acids was observed. An important factor of the preservation of Y. pestis in the soil was LF formation. At room temperature this process quickly resulted in the death of the population. At 4 degrees - 8 degrees C A. pestis altaica avirulent strain could be inoculated onto solid nutrient media for a two-fold longer period (for 4 month) than the strain with selective virulence and for 5.5 months longer than Y. pestis pestis highly virulent strain.     

环介导等温扩增技术检测鼠疫耶尔森菌的敏感性和特异性研究

为观察环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术能否适用于我国不同疫源地鼠疫耶尔森菌所有基因组型的检测,本研究建立了一种基于3a靶序列设计特异性引物快速检测鼠疫耶尔森菌的LAMP方法.选择分离自我国11个鼠疫自然疫源地的65株野生代表性鼠疫耶尔森菌株,同...