DNA microarrays encompassing the entire genome of Yersinia pestis were used to characterize global regulatory changes during steady-state vegetative growth occurring after shift from 26 to 37°C in the presence and absence of Ca 2؉ . Transcriptional profiles revealed that 51, 4, and 13 respective genes and open reading frames (ORFs) on pCD, pPCP, and pMT were thermoinduced and that the majority of these genes carried by pCD were downregulated by Ca 2؉ . In contrast, Ca 2؉ had little effect on chromosomal genes and ORFs, of which 235 were thermally upregulated and 274 were thermally downregulated. The primary consequence of these regulatory events is profligate catabolism of numerous metabolites available in the mammalian host.Bubonic plague caused by Yersinia pestis is generally recognized as the most devastating acute infectious disease experienced by mankind. It is therefore of interest that this organism has evolved within the last 10,000 years from Yersinia pseudotuberculosis (1), known to cause chronic enteropathogenic disease. Despite their very close resemblance, plague bacilli have both lost central genes of intermediary metabolism retained in its predecessor and acquired unique genes by lateral transfer (7). For example, even though early studies showed that Y. pestis possesses functional Embden-Meyerhof (28) and Entner-Doudoroff (20) pathways plus a complete tricarboxylic acid (TCA) cycle (13, 27), the species-specific absence of detectable glucose 6-phosphate dehydrogenase (Zwf) prevents use of hexose via the pentose-phosphate pathway (21). Similarly, loss of aspartase (AspA) activity in Y. pestis but not Y. pseudotuberculosis prevents complete catabolism of L-glutamic acid, which undergoes conversion and excretion as L-aspartate (12). In addition, Y. pestis possesses additional species-specific mutations that cause nutritional requirements at 26°C, prevent utilization of potential metabolites, and eliminate host cell invasins and adhesins (7); these events are now characterized by genomic sequencing (11, 23). The nature of nutritional requirements at 37°C is more complex and, as noted below, dependent upon plasmid profile, the presence or absence of Ca 2ϩ , Na ϩ , dicarboxylic amino acids, and regulatory functions addressed in this report.Established functions unique to Y. pestis are encoded by species-specific ϳ10-kb pPCP and ϳ100-kb pMT. The former encodes plasminogen activator (Pla) required for tissue invasion from dermal sites infected by fleabite whereas the structural genes for anti-phagocytic capsular fraction 1 (Caf1) and murine toxin (MT), required for survival in the flea, reside on pMT (7,25). Plague bacilli and the enteropathogenic yersiniae share a ϳ70-kb plasmid (pCD in Y. pestis) encoding a type III protein secretion system (TTSS) that delivers cytotoxins termed Yops to the cytosol of professional and nonprofessional phagocytes (8) and excretes soluble LcrV (V antigen), which inhibits generation of proinflammatory cytokines by upregulating interleukin-10 (6). These functions provide th...

A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique N-mer frequency profiles of 635 microbial genomes publicly available as of February 2008. These profiles are used to train a naive Bayes classifier (NBC) that can be used to identify the genome of any fragment. We show that our method is comparable to BLAST for small 25 bp fragments but does not have the ambiguity of BLAST's tied top scores. We demonstrate that this approach is scalable to identify any fragment from hundreds of genomes. It also performs quite well at the strain, species, and genera levels and achieves strain resolution despite classifying ubiquitous genomic fragments (gene and nongene regions). Cross-validation analysis demonstrates that species-accuracy achieves 90% for highly-represented species containing an average of 8 strains. We demonstrate that such a tool can be used on the Sargasso Sea dataset, and our analysis shows that NBC can be further enhanced.

We propose an efficient framework for genetic subtyping of SARS-CoV-2, the novel coronavirus that causes the COVID-19 pandemic. Efficient viral subtyping enables visualization and modeling of the geographic distribution and temporal dynamics of disease spread. Subtyping thereby advances the development of effective containment strategies and, potentially, therapeutic and vaccine strategies. However, identifying viral subtypes in real-time is challenging: SARS-CoV-2 is a novel virus, and the pandemic is rapidly expanding. Viral subtypes may be difficult to detect due to rapid evolution; founder effects are more significant than selection pressure; and the clustering threshold for subtyping is not standardized. We propose to identify mutational signatures of available SARS-CoV-2 sequences using a population-based approach: an entropy measure followed by frequency analysis. These signatures, Informative Subtype Markers (ISMs), define a compact set of nucleotide sites that characterize the most variable (and thus most informative) positions in the viral genomes sequenced from different individuals. Through ISM compression, we find that certain distant nucleotide variants covary, including non-coding and ORF1ab sites covarying with the D614G spike protein mutation which has become increasingly prevalent as the pandemic has spread. ISMs are also useful for downstream analyses, such as spatiotemporal visualization of viral dynamics. By analyzing sequence data available in the GISAID database, we validate the utility of ISM-based subtyping by comparing spatiotemporal analyses using ISMs to epidemiological studies of viral transmission in Asia, Europe, and the United States. In addition, we show the relationship of ISMs to phylogenetic reconstructions of SARS-CoV-2 evolution, and therefore, ISMs can play an important complementary role to phylogenetic tree-based analysis, such as is done in the Nextstrain project. The developed pipeline dynamically generates ISMs for newly added SARS-CoV-2 sequences and updates the visualization of pandemic spatiotemporal dynamics, and is available on Github at https:// github.com/EESI/ISM (Jupyter notebook), https://github.com/EESI/ncov_ism (command line tool) and via an interactive website at https://covid19-ism.coe.drexel.edu/.

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. Yersinia pestis, the causative agent of bubonic plague, is a recently evolved clone of Yersinia pseudotuberculosis serotype O:1b (1, 29). Strains of Y. pestis are divided into three biovars on the basis of their abilities to ferment glycerol and to reduce nitrate. These phenotypic differences have proven useful in distinguishing strains thought to be responsible for the three plague pandemics (7). Isolates of the biovar antiqua (able to ferment glycerol and reduce nitrate) are believed to remain as holdovers from the first pandemic that started with the Justinian plague of the 6th century. Strains of the biovar medievalis (glycerol positive and nitrate negative) evidently caused the second pandemic of Europe (Black Death), which was initiated during the 14th century, and strains of the biovar orientalis (glycerol negative and nitrate positive) are responsible for the third plague pandemic of modern times (15,22).Several molecular methods that generate fingerprinting patterns of Y. pestis DNA have been successfully used for genotyping strains of this microorganism, such as pulse-field gel electrophoresis and ribotyping (15,16,18,25) as well as newly described variable-number tandem repeat analysis (2). Genotyping of Y. pestis was also accomplished by restriction fragment length polymorphism (RFLP) detected on Southern blots using probes originating from IS100, which is found at least singly on each of the three plasmids of the species and as multiple copies within the chromosome (24). This insertion sequence (IS) element was sequenced (11, 23) and then used by Filippov et al. (9) in conjunction with the novel IS285 to obtain fingerprint patterns establishing phylogenetic relationships. Further IS100-and IS285-based RFLP analysis became the method of choice used by many researchers for extensive analysis of Y. pestis collections (1,5,14,20). Finally, a third insertion sequence (IS1541) was discovered that disrupts inv, which encodes invasin (28). This element is now known to be present in many copies in the Y. pestis genome and has also been used to obtain RFLP fingerprinting profiles (21, 28).Many insertions of IS1541 in the genome of Y. pestis strain 6/69 M biovar orientalis were characterized and then tested to see if the insert flanked the same genes in other strains of Y. pestis (21). The authors employed five unrelated strains (four of biovar orientalis and one of medievalis) of different ribotypes and IS1541 hybridization patterns that were isol...

Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts considerably less pathway throughput than observed in experimental in vitro assays. This finding, in combination with the effects of pathway cooperativity on model throughput, supports the hypothesis of cooperation during abasic site repair and between the apurinic/apyrimidinic (AP) endonuclease, Ape1, and the 8-oxoguanine DNA glycosylase, Ogg1. The quantitative model also predicts that for 8-oxoguanine and hydrolytic AP site damage, short-patch Polbeta-mediated BER dominates, with minimal switching to the long-patch subpathway. Sensitivity analysis of the model indicates that the Polbeta-catalyzed reactions have the most control over pathway throughput, although other BER reactions contribute to pathway efficiency as well. The studies within represent a first step in a developing effort to create a predictive model for BER cellular capacity.