Browsing by Author "Sharma, Parul"

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    Genomic delineation and description of species and within-species lineages in the genus Pantoea
    Crosby, Katherine C.; Rojas, Mariah; Sharma, Parul; Johnson, Marcela A.; Mazloom, Reza; Kvitko, Brian H.; Smits, Theo HM M.; Venter, Stephanus N.; Coutinho, Teresa A.; Heath, Lenwood S.; Palmer, Marike; Vinatzer, Boris A. (Frontiers, 2023-11-09)
    As the name of the genus Pantoea (“of all sorts and sources”) suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.
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    Ice nucleation in a Gram-positive bacterium isolated from precipitation depends on a polyketide synthase and non-ribosomal peptide synthetase
    Failor, Kevin C.; Liu, Haijie; Llontop, Marco E. Mechan; LeBlanc, Sophie; Eckshtain-Levi, Noam; Sharma, Parul; Reed, Austin; Yang, Shu; Tian, Long; Lefevre, Christopher; Menguy, Nicolas; Du, Liangcheng; Monteil, Caroline L.; Vinatzer, Boris A. (2021-10-23)
    Earth's radiation budget and frequency and intensity of precipitation are influenced by aerosols with ice nucleation activity (INA), i.e., particles that catalyze the formation of ice. Some bacteria, fungi, and pollen are among the most efficient ice nucleators but the molecular basis of INA is poorly understood in most of them. Lysinibacillus parviboronicapiens (Lp) was previously identified as the first Gram-positive bacterium with INA. INA of Lp is associated with a secreted, nanometer-sized, non-proteinaceous macromolecule or particle. Here a combination of comparative genomics, transcriptomics, and a mutant screen showed that INA in Lp depends on a type I iterative polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS). Differential filtration in combination with gradient ultracentrifugation revealed that the product of the PKS-NRPS is associated with secreted particles of a density typical of extracellular vesicles and electron microscopy showed that these particles consist in "pearl chain"-like structures not resembling any other known bacterial structures. These findings expand our knowledge of biological INA, may be a model for INA in other organisms for which the molecular basis of INA is unknown, and present another step towards unraveling the role of microbes in atmospheric processes.
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    Meta-analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology
    Sharma, Parul; Johnson, Marcela A.; Mazloom, Reza; Allen, Caitilyn; Heath, Lenwood S.; Lowe-Power, Tiffany M.; Vinatzer, Boris A. (Microbiology Society, 2022-03)
    Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem tissue and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures and physiological traits. To increase our understanding of the evolution, diversity and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4940 genes on average, and a pangenome analysis found that there are 3262 genes in the core genome (similar to 60 % of the mean RSSC genome) with 13 128 genes in the extensive flexible genome. A core genome phylogenetic tree and a whole-genome similarity matrix aligned with the previously named species (R. solanacearum, R. pseudosolanacearum, R. syzygii) and phylotypes (I-IV). These analyses also highlighted a third unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. We also used our genome- based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved, cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome- based identification of newly sequenced RSSC strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service.
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    Strain-level identification of bacterial tomato pathogens directly from metagenomic sequences
    Mechan Llontop, Marco Enrique; Sharma, Parul; Aguilera Flores, Marcela; Yang, Shu; Pollock, Jill; Tian, Long; Huang, Chengjie; Rideout, Steven L.; Heath, Lenwood S.; Li, Song; Vinatzer, Boris A. (Scientific Societies, 2019-12-12)
    Routine strain-level identification of plant pathogens directly from symptomatic tissue could significantly improve plant disease control and prevention. Here we tested the Oxford Nanopore Technologies (ONT) MinIONTM sequencer for metagenomic sequencing of tomato plants either artificially inoculated with a known strain of the bacterial speck pathogen Pseudomonas syringae pv. tomato (Pto), or collected in the field and showing bacterial spot symptoms caused by either one of four Xanthomonas species. After species-level identification using ONT's WIMP software and the third party tools Sourmash and MetaMaps, we used Sourmash and MetaMaps with a custom database of representative genomes of bacterial tomato pathogens to attempt strain-level identification. In parallel, each metagenome was assembled and the longest contigs were used as query with the genome-based microbial identification Web service LINbase. Both the read-based and assembly-based approaches correctly identified Pto strain T1 in the artificially inoculated samples. The pathogen strain in most field samples was identified as a member of Xanthomonas perforans group 2. This result was confirmed by whole genome sequencing of colonies isolated from one of the samples. Although in our case, metagenome-based pathogen identification at the strain-level was achieved, caution still needs to be exerted when interpreting strain-level results because of the challenges inherent to assigning reads to specific strains and the error rate of nanopore sequencing.
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    Strain-level identification of tomato pathogens from metagenomic sequences obtained with the ONT MinION
    Sharma, Parul; Mechan Llontop, Marco E.; Aguilera Flores, Marcela; Li, Song; Vinatzer, Boris A. (Virginia Tech, 2020-03-25)
    Early detection and correct diagnosis of plant diseases is an essential component of sustainable production of food and other plant-derived products. Although molecular technologies are available, many of them are either slow because they depend on culturing the pathogen first, are limited to specific pathogen species and thus cannot detect any newly emerging diseases, or have low resolution. With recent advances in sequencing technologies, it has become possible to sequence the DNA of an entire plant sample, called the metagenome, at relatively low cost and with relatively easy and fast protocols using the Oxford Nanopore Technologies (ONT) MinIONTM device. MinIONTM software What’s in my pot (WIMP) offers read-based taxonomic identification from the metagenome. In this study, we have used the MinIONTM device to sequence laboratory-inoculated tomato plants and field samples of infected tomato plants to establish the efficiency of WIMP in identifying the underlying plant pathogens. The taxonomic classifications, at the species-level, from WIMP were compared with the results from the third party Sourmash and MetaMaps tools. Since species-level identification is not always sufficient, for example, when tracking pathogen dissemination pathways, custom reference libraries were used to attempt strain-level classification with Sourmash and MetaMaps as well as identification with the LINbase Web service based on metagenome-assembled genomes (MAGs). Our study showed that reliable species-level identification is possible with either WIMP, Sourmash, or MetaMaps. There is the potential for strain-level accuracy, however improvements in the error rate of the MinIONTM and availability of appropriate reference databases is necessary.