Publications

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Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocoliticabiovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

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The transfer-messenger RNA (tmRNA) and its partner protein SmpB act together in resolving problems arising when translating bacterial ribosomes reach the end of mRNA with no stop codon. Their genes have been found in nearly all bacterial genomes and in some organelles. The tmRNA Website serves tmRNA sequences, alignments and feature annotations, and has recently moved to http://bioinformatics.sandia.gov/tmrna/. New features include software used to find the sequences, an update raising the number of unique tmRNA sequences from 492 to 1716, and a database of SmpB sequences which are served along with the tmRNA sequence from the same organism.

The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

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Genes for the RNA tmRNA and protein SmpB, partners in the trans-translation process that rescues stalled ribosomes, have previously been found in all bacteria and some organelles. We validate recent identification of tmRNA homologs in oomycete mitochondria by finding partner genes from oomycete nuclei that target SmpB to the mitochondrion. Exhaustive search now identifies a small number of complete, often highly derived, bacterial genomes that appear to lack a functional copy of one or the other partner gene (but not both). Three groups with reduced genomes have lost the central loop of SmpB, which is thought to improve alanylation and EF-Tu activation: Carsonella, Hodgkinia and the hemplasmas (hemotropic Mycoplasma). Carsonella has also lost the SmpB C-terminal tail, thought to stimulate the decoding center of the ribosome. Carsonella moreover exhibits gene overlap such that tmRNA maturation should produce a non-stop smpB mRNA, and one isolate exhibits complete degradation of the tmRNA gene yet its smpB shows no evidence for relaxed selective constraint. After loss of the SmpB central loop in the hemoplasmas, a subclade apparently lost tmRNA. At least some of the tmRNA/SmpB-deficient strains appear to further lack the ArfA and ArfB backup systems for ribosome rescue. The most frequent neighbors of smpB are the tmRNA gene, a ratA/rnfH unit, and the gene for RNaseR, a known physical and functional partner of tmRNA-SmpB. The tmRNA Website has moved and been updated, adding an SmpB sequence database (http://bioinformatics.sandia.gov/tmrna).

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Lignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. Our project of understanding microbial lignolysis for renewable platform chemicals aimed to understand microbial and enzymatic lignolysis processes to break down lignin for conversion into commercially viable drop-in fuels. We developed novel lignin analytics to interrogate enzymatic and microbial lignolysis of native polymeric lignin and established a detailed understanding of lignolysis as a function of fungal enzyme, microbes and endophytes. Bioinformatics pipeline was developed for metatranscryptomic analysis of aridland ecosystem for investigating the potential discovery of new lignolysis gene and gene products.

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Key goals towards national biosecurity include methods for analyzing pathogens, predicting their emergence, and developing countermeasures. These goals are served by studying bacterial genes that promote pathogenicity and the pathogenicity islands that mobilize them. Cyberinfrastructure promoting an island database advances this field and enables deeper bioinformatic analysis that may identify novel pathogenicity genes. New automated methods and rich visualizations were developed for identifying pathogenicity islands, based on the principle that islands occur sporadically among closely related strains. The chromosomally-ordered pan-genome organizes all genes from a clade of strains; gaps in this visualization indicate islands, and decorations of the gene matrix facilitate exploration of island gene functions. A %E2%80%9Clearned phyloblocks%E2%80%9D method was developed for automated island identification, that trains on the phylogenetic patterns of islands identified by other methods. Learned phyloblocks better defined termini of previously identified islands in multidrug-resistant Klebsiella pneumoniae ATCC BAA-2146, and found its only antibiotic resistance island.

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Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories. © 2013 Kim et al.

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