Restriction map of pesticinogenicity plasmid pYP1 of Yersinia pestis

The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.     

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Adhesion pili in Yersinia pestis

Y. pestis cells cultivated at 37 degrees C are capable of agglutinating red blood cells of some animals, which is due to the appearance of pili. The adhesion pili consist of protein subunits with a molecular weight of the order of 12000 daltons, their isoionic point being at pH 4.7. The reaction of hemagglutination was inhibited by the mixture of ganglyosides, while the preliminary treatment of red blood cells with neuraminidases increased its effectiveness. The pili are supposed to take part in the expression of virulence.     

Yersinia pestis lineages in Mongolia

Background

Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia.

Methodology/Principal Findings

Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches.

Conclusions/Significance

We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.     

Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages

Background

Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections.

Principal Findings

In vitro treatment of Y. pestis culture with sodium tellurite (Na₂TeO₃) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype.

Conclusions

These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

Yersinia pestis L-forms in rodents and ectoparasites

Y. pestis L-forms and bacterial forms persist in the body of great gerbils for 40 days. L-forms are poorly phagocytized and can persist in phagocytes for a long time. In guinea pigs immunized with vaccine EV, Y. pestis antigen could be detected till day 160. An unstable L-form was isolated from Ornithodoros mites 3 years after their experimental infection with Y. pestis. Bacterial forms persist in mites for 1-3 years. For 5 years Y. pestis antigen is regularly detected in a high percentage of mites.     

影响耶尔森氏鼠疫杆菌基因组密码子使用的因素分析

基因组密码子使用的影响因素分析有助于发现影响密码子使用的进化动力学 ,对发现和预测进化的方向和模式有重要的作用。同时 ,分析完整的基因组可以发现特定基因组中密码子的使用模式 ,从而重新设计高效的PCR引物和外源导入基因 ,促进外源基因在特定生物体中的高效率表达。导致瘟疫等外源性感染疾病的耶尔森氏鼠疫杆菌完整基因组序列已经测序公布。为了对鼠疫杆菌的同义密码子使用的进化模式有更加深入的了解 ,详细的研究分析鼠疫杆菌的基因组密码子的使用模式和影响密码子使用的因素。结果发现 ,尽管鼠疫杆菌基因组序列中“G” “C”含量相对较低 (4 7.6 4 % ) ,高水平表达基因的密码子第三位碱基使用胞嘧啶 (C)的频率比表达水平低的基因使用胞嘧啶 (C)有显著的提高 ,表达水平较低的基因在密码子的第三位碱基更趋向使用鸟嘌呤 (G)。在表达水平高低的两组基因中 ,对密码子的第三位碱基使用腺嘌呤 (A)和胸腺嘧啶 (T)总体上趋于随机使用。基因的表达水平与对应分析的第一条向量轴呈高度相关 (R =0 .6 3,P <0 .0 0 0 1)。通过分析比较表达水平高低两组基因的密码子使用模式发现 ,基因的表达水平对于密码子使用有显著的影响。GC skew分析结果显示 ,复制转录阶段的选择对密码子使用有一定的影响。在不同长度     

Aromatic amino acid transport in Yersinia pestis.

The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-Menten-like kinetics with apparent Km values of 6 x 10(-7) to 7.5 x 10(-7) and 2 x 10(-6) M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10(-6) M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 TO8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact alpha amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and tyrosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhydryl reagents or sodium azide. The results on the selective inhibition of aromatic amino acid transport provide additional evidence for multiple transport systems . These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy.     

Consequences of aspartase deficiency in Yersinia pestis.

Growing cells of Yersinia pseudotuberculosis, but not those of closely related Yersinia pestis, rapidly destroyed exogenous L-aspartic and L-glutamic acids, thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were determined at pH 5.5, 7.0, and 8.5. Regardless of pH, Y. pseudotuberculosis destroyed L-glutamic acid, L-glutamine, L-aspartic acid, and L-asparagine at rates greater than those observed for Y. pestis. Although rates of proline degardation were similar, its metabolism by Y. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Similarly, Y. pestis excreted aspartic acid when incubated with L-glutamic acid (pH 8.5) or L-asparagine (pH 5.5, 7.0, and 8.5). Aspartase activity was not detected in extracts of 10 strains of Y. pestis but was present in all 11 isolates of Y. pseudotuberculosis. The latter contained significantly more glutaminase, asparaginase, and L-glutamate-oxalacetate transminase activity than did extracts of Y. pestis; specific activities of L-glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase were similar. The observed differences in dicarboxylic amino acid metabolism are traceable to asparatase deficiency in Y. pestis and may account for the slow doubling time of this organism relative to Y. pseudotuberculosis.     

The evolution of flea-borne transmission in Yersinia pestis

Transmission by fleabite is a recent evolutionary adaptation that distinguishes Yersinia pestis, the agent of plague, from Yersinia pseudotuberculosis and all other enteric bacteria. The very close genetic relationship between Y. pestis and Y. pseudotuberculosis indicates that just a few discrete genetic changes were sufficient to give rise to flea-borne transmission. Y. pestis exhibits a distinct infection phenotype in its flea vector, and a transmissible infection depends on genes that are specifically required in the flea, but not the mammal. Transmission factors identified to date suggest that the rapid evolutionary transition of Y. pestis to flea-borne transmission within the last 1,500 to 20,000 years involved at least three steps: acquisition of the two Y. pestis-specific plasmids by horizontal gene transfer; and recruitment of endogenous chromosomal genes for new functions. Perhaps reflective of the recent adaptation, transmission of Y. pestis by fleas is inefficient, and this likely imposed selective pressure favoring the evolution of increased virulence in this pathogen.     

鼠疫耶尔森氏菌基因表达的环境调节

鼠疫耶尔森氏菌是烈性传染病鼠疫的病原菌,该菌在媒介(跳蚤)和宿主(哺乳动物)之间的循环过程中,基因表达适应环境谱的变化。本介绍鼠疫耶尔森氏菌适应环境信号如不同温度、离子浓度、pH等条件下的基因表达调控研究现状。     

Plasmid content in Yersinia pestis strains of different origin

Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains, 172 (71%) were shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 mDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.