Tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Aminoglycosides, fluoroquinolones, and tetracyclines can be used to treat tularemia; however, there is a high incidence of relapse and treatment failures when using these drugs. Furthermore, there is no tularemia vaccine licensed for use in the United States. Therefore, new antibiotics that target F. tularensis are being investigated. A novel family of resazurin-based antibiotics called resazomycins exhibit antimicrobial activity against F. tularensis and other Gram-negative pathogens including Neisseria gonorrhoeae. The mode of action of resazomycins has yet to be determined. To elucidate potential targets of resazurin (Rz), we screened for spontaneous Rz-resistant (Rzr) F. tularensis LVS mutants. Through the screen, 93% of all Rzr mutants sequenced contained mutations within the coding regions of FTL_0421, FTL_0895, and FTL_1504. In addition, 100% of all Rzr mutants sequenced contained a mutation approximately 50bp upstream of the gene FTL_0445. To understand the effect the mutation has on transcription of FTL_0445, RNA was isolated from wild-type LVS and an Rzr mutant (Rzr1). Quantitative reverse transcription PCR revealed FTL_0445 was upregulated in Rzr1 compared to wild-type LVS. To determine the role of FTL_0445 in Rz resistance, a FTL_0445 null deletion mutant is being generated in Rzr1 using standard molecular genetic techniques. Upon completion, the Rz sensitivity of the deletion mutant will be assessed using agar dilution assays. Understanding the role of FTL_0445 in Rz susceptibility would facilitate further development of these compounds as potential treatments for tularemia and gonorrhea.

Resistance to antibiotic treatments coupled with the decline in antibiotic discovery has resulted in a steady increase in deaths caused by once “curable” bacterial infections. Developing new drugs is crucial to prevent more loss of life in the future. We discovered the compound resazurin exhibits antimicrobial activity against gram-negative bacteria including Francisella tularensis (Ft), however, certain strains of Ft have developed resistance to resazurin. Understanding how Ft develops resistance to resazurin will help with defining the mechanism by which resazurin elicits its antimicrobial effect. Whole genome sequencing of resazurin-resistant (Rzr) Ft LVS mutants revealed four mutations found in 93% of the isolates sequenced. Three mutations were within the coding regions of FTL_0421, FTL_0895, and FTL_1504 and the other mutation was 50 bp upstream of FTL_0445, likely disrupting expression of this gene. The focus of my project was to explore the role of FTL_0895 in resazurin susceptibility. To confirm this gene plays a role in the reduced susceptibility of the Rzr strains to resazurin, we cloned the wild-type copy of FTL_0895 into the Francisella vector pABST which contains the robust groE promotor of Ft. The resulting plasmid will be electroporated into one of the Rzr mutants and we will test the susceptibility of the complemented strain to resazurin, using time kill and agar dilution assays. If the susceptibility of the complemented strain to resazurin is restored, then it can be determined that FTL_0895 is a potential target of resazurin.

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean (Glycine max) that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from P. sojae and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and GmMYB29A2, up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1, GmMYB29A1, and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing GmMYB29A1 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing GmMYB29A2 in Williams 82, a soybean variety that encodes the resistance gene Rps1k, rendered it compatible with race 1 P. sojae, whereas overexpressing GmMYB29A2 rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus, GmMYB29A2 is essential for accumulation of glyceollin I and expression of Phytophthora resistance.

Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases in Ruminococcaceae . Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation. Clinical Perspectives The microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO. Our results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice. These results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery.

Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42–1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42–1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42–1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42–1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42–1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42–1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42–1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.

Coenzyme A (CoA) is a cofactor that is central to energy metabolism and CoA synthesis is controlled by the enzyme pantothenate kinase (PanK). A transgenic mouse strain expressing human PANK2 was derived to determine the physiological impact of PANK overexpression and elevated CoA levels. The Tg(PANK2) mice expressed high levels of the transgene in skeletal muscle and heart; however, CoA was substantially elevated only in skeletal muscle, possibly associated with the comparatively low endogenous levels of acetyl-CoA, a potent feedback inhibitor of PANK2. Tg(PANK2) mice were smaller, had less skeletal muscle mass and displayed significantly impaired exercise tolerance and grip strength. Skeletal myofibers were characterized by centralized nuclei and aberrant mitochondria. Both the content of fully assembled complex I of the electron transport chain and ATP levels were reduced, while markers of oxidative stress were elevated in Tg(PANK2) skeletal muscle. These abnormalities were not detected in the Tg(PANK2) heart muscle, with the exception of spotty loss of cristae organization in the mitochondria. The data demonstrate that excessively high CoA may be detrimental to skeletal muscle function.

Background: The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. Results: A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. Conclusions: A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, largescale, multi-institutional reverse genetic pipelines to improve crop traits.

Reverse transcription-PCR (RT-PCR) is a core technique for detecting and quantifying alternative pre-mRNA splicing. RT-PCR is multistep process involving RNA isolation, reverse transcription, and PCR that is often performed using radiolabeled primers. As a result RT-PCR analysis of alternative splicing is a laborious technique that quickly becomes prohibitively expensive when applied to large numbers of samples. Here, we describe an RT-PCR approach for detecting alternative splicing in multi-well plates that can be applied to effortlessly quantify exon inclusion levels in large number of samples. The procedures outlined here can also be automated on standard liquid handling equipment to produce medium throughput assay capable of handling thousands of samples per day.

Completion of genome sequences for the diploid Setaria italica reveals features of C4 photosynthesis that could enable improvement of the polyploid biofuel crop switchgrass (Panicum virgatum). The genetic basis of biotechnologically relevant traits, including drought tolerance, photosynthetic efficiency and flowering control, is also highlighted. We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ∼400-Mb assembly covers ∼80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).

Background: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues.Results: PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues.Conclusions: The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical eight helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 muM(-1) s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (K(d) or P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 muM(-1) s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 muM(-1) s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

The success of viral and non-viral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm toward the nucleus and cross the nuclear envelope into the nucleus, the site of transcription in the cell. While the dense latticework of the cytoskeleton impedes free diffusion of large macromolecules, including DNA, transfections do work, and consequently there must be mechanisms by which DNA moves toward the nucleus. We and others have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. We have identified a set of proteins that interact with the DNA to form protein-DNA complexes that mediate this trafficking and that into the non-dividing nucleus. While studying the effects of mechanical strain on the transfection process, we discovered that cyclic stretch causes large scale reorganization of the microtubule cytoskeleton and induction of a pool of stabilized microtubules that are posttranslationally acetylated and which lead to increased gene delivery to the nucleus. Using a variety of approaches, we have found that by increasing the levels of acetylated microtubules we can increase gene transfer, not at the level of transcriptional activation, but rather at the level of cytoplasmic trafficking toward the nucleus. Taken together, these results suggest that modulation of the microtubule network and the formation of protein-DNA complexes can increase the efficiency of gene transfer.

One of the top-selling medicinal products worldwide is Hypericum perforatum (St. John's Wort). Despite its cosmopolitan distribution and utilization, little is known regarding the relationship of the bioactive compounds in H. perforatum to the plants from which they are purportedly derived. In this study, amplified fragment length polymorphism (AFLP) analysis of 56 Hypericum accessions, representing 11 species, was conducted to gain a better understanding of diversity within Hypericum species, especially within cultivated accessions of H. perforatum, and to establish a molecular methodology that will provide breeders and regulators with a simple, affordable, and accurate tool with which to identify purported H. perforatum material. Utilizing four primer combinations, a total of 298 polymorphic markers were generated, of which 17 were present in all H. perforatum accessions and 2 were specific to only H. perforatum. This study demonstrates that AFLP can be utilized not only to determine the relationships of closely related Hypericum accessions, but as a tool to authenticate material in herbal remedies through the use of genetic fingerprinting.

Studying gene expression in polyploids is complicated by genomewide gene duplication and the problem of distinguishing transcript pools derived from each of the two homeologous genomes such as the A- and D-genomes of allotetraploid Gossypium. Short oligonucleotide probes designed to specifically target several hundred homeologous gene pairs of Gossypium were printed on custom NimbleGen microarrays. These results demonstrate that relative expression levels of homeologous genes may be measured by microarrays and that deviation from equal expression levels of homeologous loci may be common in the allotetraploid nucleus of Gossypium.

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.