In clinical settings it is often important to know not just the identity of a microorganism, but also the danger posed by that particular strain. For instance, Escherichia coli can range from being a harmless commensal to being a very dangerous enterohemorrhagic (EHEC) strain. Determining pathogenic phenotypes can be both time consuming and expensive. Here we propose a simple, rapid and inexpensive method of predicting pathogenic phenotypes on the basis of the presence or absence of short homologous DNA segments in an isolate.

Our method compares completely sequenced genomes without the necessity of genome alignments in order to identify the presence or absence of the segments to produce an automatic alignment of the binary string that describes each genome.

Analysis of the segment alignment allows identification of those segments whose presence strongly predicts a phenotype. Clinical application of the method requires nothing more that PCR amplification of each of the set of predictive segments.

Here we apply the method to identifying EHEC strains of E. coli and to distinguishing E. coli from Shigella. We show in silico that with as few as 8 predictive sequences, if even three of those predictive sequences are amplified the probability of being EHEC or Shigella is >0.99. The method is thus very robust to the occasional amplification failure for spurious reasons.

Experimentally, we apply the method to screening a set of 98 isolates to distinguishing E. coli from Shigella, and EHEC from non-EHEC E. coli strains and show that all isolates correctly identified.

The molecular processes that govern speciation are not completely understood, especially within marine ecosystems. Insight on the topic of speciation can be provided by focusing on adaptive radiations, because of the rapid amount of lineages that have arisen within a short natural historical time frame. Rockfishes (genus: Sebastes) are known as a system for adaptive radiations within marine systems and have been considered an ancient species flock. This dissertation concentrates on marine rockfishes from the subgenus (Pteropodus) and closely related congeners to make inferences about speciation and adaptation within marine systems. Brain, kidney, testes, ovaries, and spleen tissues were selected from multiple rockfish species to extract total RNA in order to sequence the transcriptome with the use of Sanger and next generation sequencing techniques. These tissues types are intended to give us a broad spectrum of the rockfish transcriptome and were utilized to identify genes subject to positive selection. Pair-wise and multiple species comparisons were conducted to identify orthologous sequence pairs between/among species and estimated nonsynonymous (Ka) and synonymous (Ks) substitutions.

In the first study, a comparative analysis of Expressed Sequence Tags (ESTs) from Sebastes caurinus and S. rastrelliger was conducted to identify candidate genes under positive selection within the subgenus Pteropodus. Genes with elevated Ka/Ks values belonged to the following functional categories: immune function, metabolism, longevity, and reproductive behavior, indicating that adaptive divergence at immunological, physiological, and reproductive loci may be important in the diversification of this group of fishes. In the second study, ESTs from S. goodei and S. saxicola, a pair of related congeners to Pteropodus, were utilized for identifying positive selection with the use of testes and ovary tissue types. Gonadal tissues were selected from these two species to elucidate patterns of adaptation and speciation because of their recognition to contribute to reproductive barriers between species. Sequence divergence was estimated within the untranslated regions (UTRs) between these two species and was compared with the rate of divergence within coding regions of these genes to gain a clear depiction of neutral substitutional mutation rates. Orthologous gene pairs between the two species were identified and tested for positive selection. In addition, a candidate gene approach from the zona pellucida (ZP) family was selected to determine whether these genes are under positive selection. In the third study of this dissertation, a multi-transcriptomic comparison was conducted, in which three Pteropodus species were analyzed with the addition of S. rastrelliger and S. caurinus datasets to identify a pattern of positive selection within this subgenus. Brain tissue was used to gain the most diverse set of transcripts from the rockfish genome. Genes under positive selection belonged to a variety of gene functions that included sensory perception, growth, and metabolism. In addition we identified 10 sequences under positive selection that were part of the phosphatidylinositol signaling system pathway, although genes under positive selection had a broad range of gene functions and genes identified under positive selection across all five Pteropodus species were not identified in the first study.

These collections of studies are intended to further advance the field of evolutionary biology by providing support of which functional genes are important for adaptation and speciation. Currently, there is no rockfish genome available, in which by sequencing the transcriptome this provides a foundation for future genomic projects to gain a better understanding about the genus. This dissertation was developed to determine whether traditional gene categories, which are known to be under positive selection such as immune function, reproduction, and apoptosis, also play an important role in marine speciation. With transcriptomic data from multiple species within Sebastes, we obtained a suite of candidate genes under positive selection, which can be used to assess whether these genes are responsible for the radiation and how adaptation and speciation occurred across the entire genus of Sebastes.

Antibiotic resistance is a major public health crisis in the United States and around the world, with at least 23,000 people dying each year in the U.S. from antibiotic resistant infections; it is a major cause of death. Technical advancements to facilitate studies and diagnosis of resistance are an important step for finding solutions to this problem. For this study, we partnered with a local community hospital Mercy Medical Center. Our study focuses on resistant isolates that have been identified as possessing the Extended Spectrum β-Lactamases (ESBLs). We have used a novel and sensitive method for measuring the fitness of these isolates. By using growth rates as a measurement for fitness we have discovered previously unknown relationships between co-expressed resistance genes. We identified that in the presence of ampicillin, isolates that have both CTX-M-15 and TEM-1 genes have a slightly higher mean growth rate than isolates that have the CTX-M-15 gene but not the TEM-1 gene leading us to believe that there may be a synergistic effect between CTX-M15 and TEM-1 for the resistance of ampicillin. Surprisingly we also found that isolates in the presence of cephalosporin showed a decrease in growth rates if they had the TEM-1 resistance gene. This suggests that the presence of TEM-1 has a slightly inhibitory effect on growth in the presence of cephalosporins, which is still not understood at a cellular level. We also found that in the presence of ampicillin-sulbactam (SAM) there was no difference in growth rates for isolates that were TEM-1 (+) with those that are TEM-1 (-). However there was a difference in mean growth rates when comparing CTX-M-15 (+) isolates and CTX-M-15 (-) isolates in the presence of SAM. Indicating that CTX-M-15 is interfering with resistance for SAM when TEM-1 is present. Growth rates are a reliable measure of fitness. They calculate the maximum growth by measuring the optical density of that culture over time. The sensitivity of these assays may lead to novel findings about the nature of antibiotic resistance evolution. Future studies of fitness outcomes using growth rate assays are recommended.

I initiated the collection of Gram-negative clinical isolates of bacteria expressing resistance to extended spectrum β-lactam antibiotics. That collection has proved to be a valuable resource for studying the evolution of antibiotic resistance in the Central Valley. It has been used to study in hospitals, agricultural settings and to study the evolution of specific resistance phenotypes. To answer questions about where the major reservoirs of resistance lie, where resistance evolves, and how we might better identify the sources of new resistance as it enters a population. Firstly, Urinary tract infections (UTIs) are the most common bacterial infection among hospitalized patients at Dignity Health Mercy Medical Center (DHMMC). Secondly, CTX-M and OXA have high frequencies at DHMMC and lastly hospitals can have some effect on but are not a major contributor to the evolution of antibiotic resistance. Overall, we have found that the population dynamics of ESBLs rely on transmission of urinary tract infections and that hospital, agricultural and community settings are all important in the transmission of ESBLs.

Background: Reconstructing the higher relationships of pulmonate gastropods has been difficult. Morphology is problematic due to high homoplasy. Molecular studies have suffered from low taxon sampling. Forty-one complete mitochondrial genomes are available for gastropods, seven of which are pulmonates. Ten new complete mitochondrial genomes of pulmonates are presented here: Salinator rhamphidia (Amphiboloidea); Auriculinella bidentata, Myosotella myosotis, Ovatella vulcani, and Pedipes pedipes (Ellobiidae); Peronia peronii (Onchidiidae); Siphonaria gigas (Siphonariidae); Succinea putris (Stylommatophora); Trimusculus reticulatus (Trimusculidae); and Rhopalocaulis grandidieri (Veronicellidae). Also, 94 new pulmonate-specific primers across the entire mitochondrial genome are provided, which were designed for amplifying genomes through simultaneous, short reactions. Results: The structural features of the 10 new mitochondrial genomes are summarized. All genomes share similar gene orders. Phylogenetic analyses were performed including the 10 new genomes and 16 genomes from Genbank (outgroup, opisthobranch, and other pulmonate species). Bayesian Inference and Maximum Likelihood analyses produced the same topology, based on the concatenated amino acid sequences of the 13 protein-coding genes. The pulmonates are paraphyletic and basal to the opisthobranchs that are monophyletic at the tip of the tree. Siphonaria, traditionally regarded as a basal pulmonate, is nested within opisthobranchs. Pyramidella, traditionally regarded as a basal (non-euthyneuran) heterobranch, is nested within pulmonates. Several hypotheses are rejected, such as the Systellommatophora, Geophila, and Eupulmonata. The Ellobiidae is polyphyletic, but the false limpet Trimusculus reticulatus is closely related to some ellobiids. Conclusions: Despite recent efforts for increasing the taxon sampling in euthyneuran gastropods (opisthobranchs and pulmonates) phylogenies, most deep nodes are still uncertain, mainly due to low support values as well as some incongruence between analyses based on complete mitochondrial genomes and those based on individual genes (18S, 28S, 16S, CO1). Additional genomes are still needed, especially for veronicellids, ellobiids, freshwater pulmonates, taxa for which no genome is currently available (Williamia, Otina, and, Smeagol), as well as basal heterobranchs, closely related to euthyneurans. Step by step, however, new relationships are being unveiled, such as the close relationships between the false limpet Trimusculus reticulatus and ellobiids, the fact that a pyramidelloid is nested within pulmonates, and that Siphonaria is closely related to sacoglossan opisthobranchs.

The evolution of antibiotic resistance among bacteria threatens our continued ability to treat infectious diseases. The need for sustainable strategies to cure bacterial infections has never been greater. So far, all attempts to restore susceptibility after resistance arises have been unsuccessful, including restrictions on prescribing antibiotics (Andersson DI et al.2011) and antibiotic cycling (Andersson DI et al. 2005, Bergstrom CT et al. 2004). Part of the problem may be that those efforts have implemented different classes of unrelated antibiotics, and relied on removal of resistance by random loss of resistance genes from bacterial populations (drift). Moreover, antibiotic resistance results from genetic changes therefore it is a problem that needs to be investigated under the scope of evolution of the causal genetic determinants. TEM β-lactamases enzymatic action is the most prevalent antibiotic resistance mechanism in many clinical populations of bacteria. Nevertheless, the systematic study and analysis of evolutionary traits of TEM β-lactamases are still recent. Here, we show that alternating structurally similar antibiotics can restore susceptibility to antibiotics after resistance has evolved. We found that the resistance phenotypes conferred by variant alleles of the resistance gene encoding the TEM β-lactamase (blaTEM) varied greatly among 15 different β-lactam antibiotics. We captured those differences by characterizing complete adaptive landscapes for the resistance alleles blaTEM-50 and blaTEM-85, each of which differs from its ancestor blaTEM-1 by four mutations. We identified pathways through those landscapes where selection for increased resistance moved in a repeating cycle among a limited set of alleles as antibiotics were alternated. Furthermore, in this study, we investigate the selection process of each variant allele of the gene blaTEM-85 and blaTEM-50 by the 15 different antibiotics we employed in this investigation. Finally, we investigate fitness landscapes

as a central concept in analyzing evolution, in particular for drug resistance mutations in bacteria. We show that the fitness landscapes associated with antibiotic resistance are not compatible with any of the classical models; additive, uncorrelated and block fitness landscapes. It is frequently stated that virtually nothing is known about fitness landscapes in nature. We demonstrate that available records of antimicrobial drug mutations can reveal interesting properties of fitness landscapes in general. We apply the methods to analyze the TEM family of β-lactamases associated with antibiotic resistance. Laboratory results agree with our observations. We suggest that fitness landscapes analysis might provide the necessary tools for finding relations between recombination strategies.

Our results showed that susceptibility to antibiotics can be sustainably renewed by cycling structurally similar antibiotics. We anticipate that these results may provide a conceptual framework for managing antibiotic resistance. This approach may also guide sustainable cycling of the drugs used to treat malaria and HIV.

Our results also showed that among the three categories of β-lactam antibiotics employed in this study, cephalosporins, specifically cefepime have selected for different evolutionary trajectories on the evolution of blaTEM-1 to blaTEM-85. Also our results demonstrate that blaTEM-1 evolved from a specialist gene coding for β-lactamases capable of hydrolyzing only penicillins to blaTEM-50 a generalist gene coding for β-lactamases capable of hydrolyzing a large range of cephlosporins. We anticipate that the knowledge of how β-lactams impact each blaTEM variant allele can aid on the choice of antibiotics to be prescribed on cycling drugs regimes in order to keep antibiotic resistance evolution in a cyclic fashion.

The diauxic shift is a change in metabolism in Saccharomyces cerevisiae whereby glucose consumption fuels glycolytic fermentation but then shifts to respiration by ethanol import upon glucose exhaustion. In comparison, cancer cells have increased aerobic glycolysis and lactic acid excretion compared to noncancerous cells. Cancer cells undergo glycolysis upon glucose induction, much like yeast cells do. One protein that regulates the diauxic shift is the glucose response transcription factor, GCR1. After a high growth state and as glucose becomes limited, Gcr1 is deactivated as the cell switches to respiration. By studying the mechanisms and metabolism of the diauxic shift, much can be learned about diseases in other organisms that have deregulated glycolytic metabolism, such as cancer in humans.

First, to model metabolism and to be able to characterize the phenotype of a biological system based off of transcriptomic, proteomic, or genomic datasets, condition-specific metabolic modeling software was developed. The Algorithm for Simplified Metabolic ANalysIs by Altering Networks and Deducing flux Estimates for VIsuaLization (TASMANIAN DEVIL) is comprised of four independently functional modules: gene activity determination, genome-scale metabolic model importation and simplification to reduce network complexity, robust heuristic model building and metabolic flux prediction using steady-state flux balance analysis, and flux visualization from a reference network topology. Publically available transcriptomic and measured flux datasets for yeast were used to validate the software and assumptions used for simplifying networks. TASMANIAN DEVIL is easily installable and can be utilized by researchers in many fields.

After TASMANIAN DEVIL was developed, it was used to character the metabolism of GCR1 mutant transcriptomic studies before and after the diauxic shift. It was modeled that prior to the diauxic shift, there is a high rate of metabolic flux through glycolysis, the pentose phosphate pathway, and biosynthesis, in part generated by anaplerosis. Upon ethanol import, there is a decrease in these pathways and an increase in the citric acid cycle, oxidative phosphorylation, and arginine metabolism. The modeling predicted that the upregulation of the glycine cleavage complex (GCV1, GCV2, and GCV3 in the mitochondria) by GCR1 creates an efficient way to generate one carbon building blocks for biosynthesis for adenine production along with other purines. Gcr1 is deactivated by inositol pyrophosphorylation from 5-diphosphoinositol pentakisphosphate (5PP-IP5), which is the addition of a phosphate group to a prephosphorylated serine residue through a nonenzymatic cleavage. The process is highly endergonic, requiring a near physiological level Michaelis constant for ATP. This predicts that GCR1 is therefore a regulator of its own deactivation, and it becomes downregulated after a previous high growth state.

Finally, it was investigated how pyrophosphorylation may be conserved in humans to regulate homeostasis. A conserved multi-domain was identified over the residues where pyrophosphorylation occurs on GCR1, which may enable pyrophosphorylation to take place. In humans, several inositol polyphosphatases contain this domain near their phosphatases, perhaps indicating a positive feedback loop for the continued formation of inositol pyrophosphates. GCR1 and its transcriptional partners were also found to be cyclically expressed. The levels of 5PP-IP5 are periodically modulated with the cell cycle in mammals, influencing the activities of the proteins it pyrophosphorylates and binds to, thus governing cell cycle checkpoints. It has been shown that 5PP-IP5 regulates p53-mediated apoptosis upon DNA damage and that it inactivates AKT to downregulate glycolytic metabolism. In cancer cells, there are several ways that the inositol pyrophosphate pathway can become deregulated. However, focusing on reactivating these mechanisms of regulation may provide an effective way to target cancer cells.

Antibiotic resistant infections are a public health threat. According to the CDC, the incidence of antibiotic resistant infections has increased 50% in the last five years1. Urinary tract infections (UTIs) are the most commonly occurring bacterial infection and resistance among them is highly prevalent. Lack of mandatory reporting of these infections, and a vast array of treatment options, place UTIs at the frontiers of newly evolved resistance. To investigate this important human health threat, I performed three studies. First, I performed a surveillance study of antibiotic resistance trends in uropathogenic E. coli (UPEC) at Dignity Health Mercy Medical Center (DHMMC) in Merced, and compared them to resistance trends found nationwide. I found higher incidence of resistance at DHMMC than nationwide. Second, to identify causes for this elevated resistance, I investigated potential environmental sources of antibiotic resistant infections at DHMMC. In doing so, I developed a rapid PCR screen for identifying UPEC using a suite of six genetic markers significantly associated with UPEC. Using this screen, I found evidence of UPEC in all environmental sites surveyed, most associated with livestock for retail meat. Third, to identify the fitness effects of antibiotic resistance genes upon bacteria being treated with antibiotics, and to further infer the effects of antibiotic consumption on the selection of the antibiotic resistance genes, I created a customizable plasmid vector construct library. This library contains all possible combinations of the four most identified resistance genes at DHMMC, and it can be used to evaluate their fitness, relative to one another, in the presence and absence of different antibiotics. In summary, I characterized the antibiotic resistance trends at DHMMC, identified potential sources of the resistance, and developed an experimental tool to evaluate the ongoing selection from antibiotic consumption resulting in the resistance trends at DHMMC.

Ceftolozane/tazobactam (c/t) is a fifth generation β-lactam cephalosporin and β-lactam inhibitor, recently approved in the United States for treatment of complicated Urinary Tract Infections (UTIs) and complicated intra-abdominal infections. While third generation cephalosporins are commonly used for Enterobacteriaceae infection treatment, fourth generation cephalosporins are used less frequently to spare them from rising antibiotic resistance, and fifth generation cephalosporins, such as c/t, are last-resort treatment options. Surveillance for c/t resistance is essential to identifying current resistance patterns and to identify causes of any observed resistance. We performed Kirby-Bauer disk diffusion tests on a collection of Extended Spectrum β-lactamases (ESBL)-producing Enterobacteriaceae collected mainly from urine samples at Dignity Health Mercy Medical Center (DHMMC) in Merced, CA (n = 993), and found low rates of c/t resistance (3.24%). We used a PCR screen to quantify the presence of four common ESBLs (blaTEM, blaOXA, blaSHV, and blaCTX-M) on 852 isolates and assessed their association with c/t resistance. We then screened the genomic sequences of 123 isolates and used PCR to identify the presence of four non-ESBL genes, emrD (gene for efflux pump), ramR (gene for efflux pump repressure), and ompK35 and ompK37 (genes for porins in bacterial outer membrane) in 96 isolates subset. We found that the presence of blaCTX-M, blaSHV independently contributed to c/t resistance. We did not find significant interactions between the non-ESBLs and c/t resistance, nor did we find any significant interactions between having a combination of ESBL and non-ESBL genes with c/t resistance. We then wanted to find the teaching patterns in Science, Technology, Engineering, and Mathematics (STEM) classrooms. We investigated how STEM instructors, who are mainly faculty on the forefront of scientific discoveries, discuss scientific content with their students. We looked at instructional and discourse practices of 35 STEM instructors across variables such as STEM discipline, years of teaching experience, and class size. We found that chemistry instructors used more instructor-centered teaching practices (i.e., lecturing with real world examples) than biology instructors. We also found that teaching faculty use more student-centered teaching practices than lecturers. Additionally, we found that neither years of faculty teaching experience nor class size impacted teaching practices. Investigating resistance patterns to newly available antibiotics is essential for prolonging its efficacy, while studying patterns of effective communication of these scientific findings in undergraduate STEM classrooms are equally important, and both contribute to the betterment of our society.

Antibiotic resistant bacteria generated by agricultural and livestock practices can exchange antibiotic resistance genes (ARG) with soil microbes and generate novel pathogenicity; environmental microbes can then act as reservoirs or vectors of ARG. However, there are many other practices that could be contributing to the generation, spread, and maintenance of antibiotic resistance genes. One industrial agricultural practice that has yet to be investigated for its potential role as a reservoir or vector of antibiotic resistance genes is the use of biopesticides. The use of microorganisms for pest control, frost prevention, and rhizosphere enhancements in agriculture has steadily increased over the last 20 years. Considered safe for consumption, non-toxic, and nonpathogenic, microbial products offer welcome alternatives to traditional chemically synthesized pesticides known to cause damage to human health and the environment. Plasmids bearing antibiotic resistance genes and novel pathogenicity should be considered part of the resistomes when investigating ARG of bacterial biopesticides. The widespread use and application of bacterial biopesticides to crops in high concentrations raises the possibility of unintentional contributions to the movement and generation of antibiotic resistance genes (ARG) in the environment. Are there clinically relevant antibiotic resistance genes present in commercially available bacterial biopesticides? Are the bioinformatically identified antibiotic resistance genes expressed and do they demonstrate resistance to clinically relevant antibiotics? Is there genetic exchange between widely available Bacillus-based bacterial biopesticide products and a bioinformatically informed pathogen? What is the role of plasmids, viruses and other mobile genetic elements in relation to antibiotic resistance genes occurring in commercially available Bacillus-based bacterial biopesticides? This research investigated publicly available genomes sequences for widely used agricultural bacterial biopesticide products in order to identify the presence of antibiotic resistance genes. Commercially available Bacillus-based biopesticides were the basis of antibiotic resistance experiments and whole genome sequencing. I found that biopesticides are reservoirs of clinically relevant antibiotic resistance genes, however, their resistance phenotypes were not matched. Simple contact with carbapenemase positive Klebsiella pneumoniae was enough to change the phenotype from susceptible to resistant for three of the four commercial biopesticide products. A comprehensive look into the viral and integrative and mobile elements showed a likely explanation for the large and diverse suite of genes found in the biopesticides. This work demonstrates the need to include biopesticide use in the cohort of agricultural processes that have been implicated in the generation, spread, and maintenance of ARG in the environment.