Transcriptomic Analysis of \u3ci\u3eSalmonella enterica\u3c/i\u3e Newport Adaptive Response to Oxidative Antimicrobials

Agricultural water systems are a known potential source for contamination of fresh produce with foodborne pathogens including Listeria monocytogenes, pathogenic Escherichia coli, as well as Salmonella enterica strains. To reduce this associated risk, antimicrobials such as hypochlorite compounds and peroxyracetic acid (PAA) have been utilized as in-line irrigation disinfectants and as post-harvest treatments. These treatments are considered effective and commonly used; however, pathogenic microbes like Salmonella have the ability to adapt to many environmental conditions. Adaptive response to environmental factors like oxidative conditions, contributes to transfer, attachment, and resilience of Salmonella on surfaces. Through RNA sequencing and analysis of the obtained transcriptome, adaptive response of Salmonella to hypochlorite and PAA can be quantitatively evaluated. Within this study, an outbreak clinical isolate strain of Salmonella enterica Newport was utilized in simulating contaminated agricultural water exposed to hypochlorite and PAA treatment. Treatments consisted of two samples, each repeated in duplicate of 10 and 20 ppm hypochlorite and PAA each, with an aqueous control. Following exposure, cells were isolated for RNA extraction and sequencing to analyze mRNA for relative differential gene expression levels. The dynamic range of cellular adaptive response to chemical stressors was seen in the overall number of differentially expressed genes. They numbered from 11 genes to 316, 1,719, and 2,010 genes respectively for 10 ppm hypochlorite, 20 ppm hypochlorite, 10 ppm PAA, and 20 ppm PAA treatments, respectively. These transcripts ranged in cellular function from biosynthesis to degradation, energy generation, and non-metabolically linked functions. Notably, exposure to oxidative antimicrobials resulted in genes associated with key virulence, attachment, and gene transfer factors showing positive degrees of differential expression. These findings stress the importance of monitoring disinfectant application, and the potential for increasing the efficacy of treatments through further elucidation of microbial adaptive response

Copy Number Variation Shapes Genome Diversity in Arabidopsis Over Immediate Family Generational Scales

Arabidopsis thaliana is the model plant and is grown worldwide by plant biologists seeking to dissect the molecular underpinning of plant growth and development. Gene copy number variation (CNV) is a common form of genome natural diversity that is currently poorly studied in plants and may have broad implications for model organism research, evolutionary biology, and crop science. Herein, comparative genomic hybridization (CGH) was used to identify and interrogate regions of gene CNV across the A. thaliana genome. A common temperature condition used for growth of A. thaliana in our laboratory and many around the globe is 22 °C. The current study sought to test whether A. thaliana, grown under different temperature (16 and 28 °C) and stress regimes (salicylic acid spray) for five generations, selecting for fecundity at each generation, displayed any differences in CNV relative to a plant lineage growing under normal conditions. Three siblings from each alternative temperature or stress lineage were also compared with the reference genome (22 °C) by CGH to determine repetitive and nonrepetitive CNVs. Findings document exceptional rates of CNV in the genome of A. thaliana over immediate family generational scales. A propensity for duplication and nonrepetitive CNVs was documented in 28 °C CGH, which was correlated with the greatest plant stress and infers a potential CNV–environmental interaction. A broad diversity of gene species were observed within CNVs, but transposable elements and biotic stress response genes were notably overrepresented as a proportion of total genes and genes initiating CNVs. Results support a model whereby segmental CNV and the genes encoded within these regions contribute to adaptive capacity of plants through natural genome variation

More Than 1,001 Problems with Protein Domain Databases: Transmembrane Regions, Signal Peptides and the Issue of Sequence Homology

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false-positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users

Draft genome sequence of Chromatium okenii isolated from the stratified alpine Lake Cadagno

Blooms of purple sulfur bacteria (PSB) are important drivers of the global sulfur cycling oxidizing reduced sulfur in intertidal flats and stagnant water bodies. Since the discovery of PSB Chromatium okenii in 1838, it has been found that this species is characteristic of for stratified, sulfidic Environments worldwide and its autotrophic metabolism has been studied in depth since. We describe here the first high-quality draft genome of a large-celled, phototrophic, γ-proteobacteria of the genus Chromatium isolated from the stratified alpine Lake Cadagno, C. okenii strain LaCa. Long read technology was used to assemble the 3.78 Mb genome that encodes 3,016 protein-coding genes and 67 RNA genes. Our findings are discussed from an ecological perspective related to Lake Cadagno. Moreover, findings of previous studies on the phototrophic and the proposed chemoautotrophic metabolism of C. okenii were confirmed on a genomic level. We additionally compared the C. okenii genome with other genomes of sequenced, phototrophic sulfur bacteria from the same environment. We found that biological functions involved in chemotaxis, movement and S-layer-proteins were enriched in strain LaCa. We describe these features as possible adaptions of strain LaCa to rapidly changing environmental conditions within the chemocline and the protection against phage infection during blooms. The high quality draft genome of C. okenii strain LaCa thereby provides a basis for future functional research on bioconvection and phage infection dynamics of blooming PSB

Association Mapping and Genomic Selection for Yield and Agronomic Traits in Soft Winter Wheat

Tools such as genome-wide association study (GWAS) and genomic selection (GS) have expedited the development of crops with improved genetic potential. While GWAS aims to identify significant markers associated with a trait of interest, the goal of GS is to utilize all marker effects to predict the performance of new breeding lines prior to testing. A GWAS for grain yield (GY), yield components, and agronomic traits was conducted using a diverse panel of 239 soft winter wheat (SWW) lines evaluated in eight site-years in Arkansas and Oklahoma. Broad sense heritability of GY (H2=0.48) was moderate compared to other traits including plant height (H2=0.81) and kernel weight (H2=0.77). Markers associated with multiple traits on chromosomes 1A, 2D, 3B, and 4B serve as potential targets for marker assisted breeding to select for GY improvement. Validation of GY-related loci using spring wheat from the International Maize and Wheat Improvement Center (CIMMYT) in Mexico confirmed the effects of three loci in chromosomes 3A, 4B, and 6B. Lines possessing the favorable allele at all three loci (A-C-G allele combination) had the highest mean GY of possible haplotypes. The same population of 239 lines was used in a GS study as a training population (TP) to determine factors that affect the predictability of GY. The TP size had the greatest effect on predictive ability across the measured traits. Adding covariates in the GS model was more advantageous in increasing prediction accuracies under single population cross validations than in forward predictions. Forward validation of the prediction models on two new populations resulted in a maximum accuracy of 0.43 for GY. Genomic selection was “superior” to marker-assisted selection in terms of response to selection and combining phenotypic selection with GS resulted in the highest response. Results from this study can be used to accelerate the process of GY improvement and increase genetic gains in wheat breeding programs

The Development of a Tractable System to Assess \u3ci\u3eCaulobacter\u3c/i\u3e-Plant Interactions

Bacteria play an integral role in regulating plant growth and development. However, many of the mechanisms encompassing bacteria-plant interactions are poorly understood and thus require detailed assessments (see CHAPTER 1). To this end, I coupled bacterial (Caulobacter sp.) and plant model organisms (Arabidopsis) to determine 1) the degree to which select bacteria can enhance the growth and development of plants, and 2) what functions these bacteria possess that enable them to aid plant development. Employing bacterial isolation techniques, monoculture inoculum-based plant growth assays, biochemical assays, comparative genomics, functional genetics, and real-time quantitative PCR (RT-qPCR), I determined that 1) Caulobacter-Arabidopsis interactions vary from mutualistic to parasitic; 2) common biosynthates are not required for many beneficial Caulobacter-Arabidopsis interactions; 3) redox-related genes and bacterial cell curvature facilitate Caulobacter-Arabidopsis interactions, and 4) bacterial concentration and bacterial induced pH reductions contribute to Caulobacter-mediated seed germination inhibition. Collecting and processing soil and root samples from South Carolina and Florida, I uncovered two novel Caulobacter strains that can enhance the biomass of Arabidopsis. To contextualize these findings, I tested the ability of previously obtained stock cultures of Caulobacter strains (collected from both aquatic and soil environments) to also enhance plant growth. As a result, I determined that 1) plant growth enhancement is not a conserved feature in the Caulobacter genus, and 2) isolation source did not correlate with plant-growth-promoting (PGP) factors (i.e., not all soil-derived strains enhanced plant growth and not all aquatic-derived strains failed to enhance plant growth). Using established biochemical tests as proxies for plant-growth-promotion factors, I determined that (among the 11 Caulobacter strains that I assayed) Caulobacter strains do not use these common PGP factors to enhance plant growth. Employing a comparative genomics approach, I determined that each of the PGP Caulobacter strains that I assayed harbors a unique set of genes (cyo operon) with predicted functions in betalain biosynthesis—a ROS scavenging metabolite—in its genome. Since ROS molecules are critical for plant growth and development, I hypothesized that these genes may be involved in the ability of PGP Caulobacter strains to enhance the growth and development of Arabidopsis (see CHAPTER 2). To determine whether the cyo operon genes are necessary for Caulobacter-mediated plant growth enhancement, I disabled the function of one of the subunits (cyoB) using homologous recombination in two different PGP Caulobacter species and assessed the potential of the resultant mutant strains to enhance plant growth relative to their parental strain. As a result, I determined that a functional cyo operon facilitates Caulobacter-mediated growth enhancement of Arabidopsis since the mutant strains were unable to enhance plant growth relative to their parental strains. Interestingly, using RT-qPCR, I determined that one PGP Caulobacter strain expresses the cyoB gene (and additional genes with predicted betalain biosynthesis functions; see CHAPTER 3) significantly more than other strains and subsequently hinders the germination rate of Arabidopsis seeds. I also constructed a flux balance analysis (FBA) to gauge the relative metabolic activity between Caulobacter strains since a large portion (~80%) of variation in seed germination inhibition was explained by the culturing media type (media used for bacterial-seed plating assays). To this end, the FBA and subsequent pH measurements suggested that increased H+ ion excretion likely contributes to Caulobacter-mediated seed germination inhibition, although abundant bacterial growth also contributes to the observed inhibition. Moreover, I hypothesized that bacterial cell shape would facilitate plant growth since previous reports have shown that Caulobacter cell shape impacts niche habitancy, and I showed that Caulobacter cell curvature is required for this bacterium to enhance the growth of Arabidopsis. Therefore, I established a genetic framework to investigate the mechanisms that undergird Caulobacter-Arabidopsis interactions. Taken together, I fused two reliable genetic models (Caulobacter and Arabidopsis) to generate a working model for bacteria-plant interactions. Leveraging the high-quality genomic database for Caulobacter strains, I discovered genetic factors that facilitate the ability of select Caulobacter strains to enhance the growth of Arabidopsis plants

The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.Author SummaryDimorphic fungal pathogens including Blastomyces are the cause of major fungal diseases in North and South America. The genus Emmonsia includes species infecting small mammals as well as a newly emerging pathogenic species recently reported in HIV-positive patients in South Africa. Here, we synthesize both genome sequencing of four isolates of Blastomyces and two species of Emmonsia as well as deep sequencing of Blastomyces RNA to draw major new insights into the evolution of this group and the pathogen response to infection. We investigate the trajectory of genome evolution of this group, characterizing the phylogenetic relationships of these species, a remarkable genome expansion that formed large isochore-like regions of low GC content in Blastomyces, and variation of gene content, related to host interaction, among the dimorphic fungal pathogens. Using RNA-Seq, we profile the response of Blastomyces to macrophage and mouse pulmonary infection, identifying key pathways and novel virulence factors. The identification of key fungal genes involved in adaptation to the host suggests targets for further study and therapeutic intervention in Blastomyces and related dimorphic fungal pathogens

Development of Functional Markers for Resistance to Leaf Scald in Sugarcane

Leaf scald, caused by Xanthomonas albilineans, is a major sugarcane disease worldwide. The disease is managed primarily with resistant cultivars obtained through classical breeding; however, the erratic symptom expression hinders the reliability and reproducibility of the selection process. The development of molecular markers associated with incompatible/compatible reaction can overcome this limitation. Suppression subtractive hybridization (SSH) and quantitative trait locus (QTL) mapping were the strategies used to find leaf scald resistance-associated genes and molecular markers in sugarcane. SSH results showed that genes involved in signal perception and transduction, and DNA binding, were highly expressed in the resistant clone LCP 85-384 compared to the susceptible clone HoCP 89-846. Also, a higher proportion of overexpressed genes were located in the chloroplast in the resistant clone. Early accumulation and maintenance of high mRNA concentration was hypothesized as the determining factor for leaf scald resistance. A linkage map was constructed using 89 F1 progeny of a cross between the cultivars LCP 85-384 (resistant) and L 99-226 (susceptible) using simple sequence repeat (SSR), leaf scald responsive genes-derived SSR and single nucleotide polymorphic (SNP) markers. Single marker analysis showed that the markers c3-579 (LOD = 3.7189; phenotypic variance explained (PVE = 17.56%), 1x71593 (LOD = 3.0453; PVE = 14.65%) and c1-586b (LOD = 3.013; PVE = 14.48%) were associated with leaf scald resistance. Interval mapping identified 15 QTLs associated with disease resistance that explained 2.5 to 18.6% of the phenotypic variance. Comparative genomic analysis with Sorghum bicolor identified genes previously associated with resistance or tolerance to biotic and abiotic stresses within and flanking the QTLs. The present study resulted in a strong platform for future functional validation of the genes to ascertain their role in leaf scald resistance and marker validation in larger and diverse populations toward development of allele-specific markers for their use in breeding resistant sugarcane varieties