Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome

Identification of an Attenuated Substrain of Francisella tularensis SCHU S4 by Phenotypic and Genotypic Analyses

Pneumonic tularemia is a highly debilitating and potentially fatal disease caused by inhalation of Francisella tularensis. Most of our current understanding of its pathogenesis is based on the highly virulent F. tularensis subsp. tularensis strain SCHU S4. However, multiple sources of SCHU S4 have been maintained and propagated independently over the years, potentially generating genetic variants with altered virulence. In this study, the virulence of four SCHU S4 stocks (NR-10492, NR-28534, NR-643 from BEI Resources and FTS-635 from Battelle Memorial Institute) along with another virulent subsp. tularensis strain, MA00-2987, were assessed in parallel. In the Fischer 344 rat model of pneumonic tularemia, NR-643 and FTS-635 were found to be highly attenuated compared to NR-10492, NR-28534, and MA00-2987. In the NZW rabbit model of pneumonic tularemia, NR-643 caused morbidity but not mortality even at a dose equivalent to 500x the LD50 for NR-10492. Genetic analyses revealed that NR-10492 and NR-28534 were identical to each other, and nearly identical to the reference SCHU S4 sequence. NR-643 and FTS-635 were identical to each other but were found to have nine regions of difference in the genomic sequence when compared to the published reference SCHU S4 sequence. Given the genetic differences and decreased virulence, NR-643/FTS-635 should be clearly designated as a separate SCHU S4 substrain and no longer utilized in efficacy studies to evaluate potential vaccines and therapeutics against tularemia

OpaR Controls a Network of Downstream Transcription Factors in <i>Vibrio parahaemolyticus</i> BB22OP

<div><p><i>Vibrio parahaemolyticus</i> is an emerging world-wide human pathogen that is associated with food-borne gastroenteritis when raw or undercooked seafood is consumed. Expression of virulence factors in this organism is modulated by the phenomenon known as quorum sensing, which permits differential gene regulation at low versus high cell density. The master regulator of quorum sensing in <i>V</i>. <i>parahaemolyticus </i>is OpaR. OpaR not only controls virulence factor gene expression, but also the colony and cellular morphology associated with growth on a surface and biofilm formation. Whole transcriptome Next Generation sequencing (RNA-Seq) was utilized to determine the OpaR regulon by comparing strains BB22OP (<i>opaR</i>⁺, LM5312) and BB22TR (∆<i>opaR1</i>, LM5674). This work, using the published <i>V</i>. <i>parahaemolyticus</i> BB22OP genome sequence, confirms and expands upon a previous microarray analysis for these two strains that used an Affymetrix GeneChip designed from the closely related <i>V</i>. <i>parahaemolyticus</i> RIMD2210633 genome sequence. Overall there was excellent correlation between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control were identified by both methods and further confirmed by quantitative reverse transcription PCR (qRT-PCR) analysis. Nine of these transcription factors were demonstrated to be direct OpaR targets via <i>in vitro</i> electrophoretic mobility shift assays with purified hexahistidine-tagged OpaR. Identification of the direct and indirect targets of OpaR, including small RNAs, will enable the construction of a network map of regulatory interactions important for the switch between the nonpathogenic and pathogenic states.</p></div

Model of OpaR regulon highlighting downstream transcription factors.

<p>Downstream targets of OpaR include 11 transcription factors differentially regulated by OpaR four-fold or more. The genes controlling nine of the transcription factors are directly controlled by OpaR, as indicated by the solid lines. VPA0606 (AraC family protein) and VP2710 (CsgD/VpsT family protein) appear to be indirectly regulated as indicated by the dashed lines. Blue indicates genes about which some experimental data with relation to hierarchical control has been obtained in <i>V</i>. <i>parahaemolyticus</i>.</p

Putative sRNAs and 5’-untranslated leaders regulated four-fold or more by OpaR.

<p>^asRNAs identified with sRNAPredict2</p><p>^b Not found in either database</p><p>^c sRNAs identified with BSRD</p><p>^d Due to the qRT-PCR size and primer requirements, sRNA expression was not further validated. Only the Srr was confirmed via qRT-PCR since it was not part of one of the existing sRNA databases.</p><p>^e The approximate genomic location of Srr is 1354895–1355085 bp and Candidate 12 is 1776278–1776403 bp. Due to the lack of strand specific data, the location of the other sRNAs was not determined, although all sRNAs were observed in the RNA-Seq data.</p><p>^fRNA-Seq data is fold change of the <i>opaR</i>⁺ strain gene expression divided by the ∆<i>opaR1</i> strain gene expression. Error for the ratios of normalized gene expression levels were conservatively estimated using the standard deviation ratios across the majority of genes with less than 4-fold change. The standard deviation for chromosome 1 is 1.53 and the error for chromosome 2 is 1.59.</p><p>Putative sRNAs and 5’-untranslated leaders regulated four-fold or more by OpaR.</p

Organization of select gene loci of <i>V</i>. <i>parahaemolyticus</i> in the OpaR regulon.

<p>Cartoon gene maps, not to scale, are provided for the promoter regions and gene organization near OpaR-regulated transcription factors identified through the RNA-Seq data analysis. Dark green arrows represent genes of transcription factors activated by OpaR, dark red arrows indicate genes of repressed transcription factors and the lighter arrows illustrate other genes in the operons of interest. The diamonds represent predicted OpaR-binding sites from PATSER data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121863#pone.0121863.t002" target="_blank">Table 2</a> for specific locations and scores).</p

EMSA analysis of putative OpaR direct targets.

<p>Each panel is labeled with the promoter being analyzed. The black arrow indicates unbound DNA and the grey arrow indicates the DNA shift due to bound OpaR, with additional complexes with higher mobility present at some promoters. The concentration of FAM-labeled DNA probe in all lanes is 10 nM. The lanes within each panel (left to right) consist of the following: DNA probe with 0 nM, 25 nM, 50 nM, 100 nM OpaR, 200 nM or 400 nM OpaR. The sample in the far right lane contains DNA probe plus 100 nM of OpaR with 100 nM unlabeled DNA for competition. Gene names are: VPBB_0491(<i>cpsR)</i>, VPBB_0645 (<i>crl</i> family<i>)</i>, VPBB_2530 (<i>cspD/vpsT</i> family), VPBB_1307 <i>(fhlA</i> family<i>)</i>, VPBB_1322 <i>(asnC</i> family<i>)</i>, VPBB_1558 <i>(exsA)</i>, VPBB_2619 <i>(aphA)</i>, VPBB_A0554 (<i>araC</i> family), VPBB_A0869 <i>(ars</i> family<i>)</i>, VPBB_A1319 <i>(cpsQ</i> family<i>)</i>, and VPBB_A1405 (<i>lafK)</i>.</p

Validation of Transcription Factors Highly Regulated by OpaR via qRT-PCR.

<p>^aFor the microarray data, the standard deviation of the normalized expression in four biological replicates of the ∆<i>opaR1</i> strain was compared to three biological replicates of <i>opaR</i>^<i>+</i> strains.</p><p>^b RNA-Seq data is fold change of the <i>opaR</i>^<i>+</i> strain gene expression divided by the ∆<i>opaR1</i> strain gene expression. Error for the ratios of normalized gene expression levels were conservatively estimated using the standard deviation ratios across the majority of genes with less than 4-fold change. The standard deviation for chromosome 1 is 1.53 and the error for chromosome 2 is 1.59.</p><p>^c The error in fold changes for each gene was estimated by computing the standard deviation for four replicates of the qRT-PCR measurements.</p><p>Validation of Transcription Factors Highly Regulated by OpaR via qRT-PCR.</p