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2013 Research Showcase
GAMB Abstracts
Abstract Title : Mechanics of Phage201phi2-1 Infection with the Tubulin Protein PhuZ
Abstract : PhuZ is a distant tubulin relative produced by the Pseudomonas chlororaphis bacteriophage 201phi2-1. Previously we demonstrated that PhuZ assembles into a dynamic cytoskeletal spindle that positions a nucleoid of phage DNA in the center of the host cell, but the mechanistic details of this process are poorly understood. Here we use a combination of proteomics, site directed mutagenesis, and cell biology to further characterize PhuZâ??s role during infection. We used mass spectrometry to identify phage proteins expressed during infection, and we found that PhuZ production begins within 20 minutes of phage infection and continues through 60 minutes. This suggests that the PhuZ spindle is needed very early in the infection process, when the phage DNA is just beginning to replicate. We show that the replicating phage DNA is moved to the center of the cell early in the infection process, which is consistent with an early role for the PhuZ spindle in the lytic cycle. Antibiotics that are predicted to block phage replication have no effect on positioning, suggesting that replication and positioning are uncoupled processes. We also generated a number of mutations in the C-terminal tail of PhuZ that have turned out to be essential to the proteinâ??s function during infection. Our results allow us to propose a more detailed model for how PhuZ polymerization drives positioning of phage DNA and contributes to formation of a viral replication factory, which resembles that of the eukaryotic herpes viruses.
Abstract Title : Genomic Organization and Structural Components of Cluster C1 Phage, QBert
Abstract : Phages provide a vast amount of genomic data which has yet to be fully explored. Research in bacteriophages may lead to numerous applications, varying from phage therapy to development of genetic systems used for manipulating pathogenic bacteria like Mycobacterium tuberculosis. The mycobacteriophage QBert, isolated from a soil sample, infects a bacterial species similar to TB, Mycobacterium smegmatis. QBert?s genome was sequenced and annotated. Tandem mass spectrometry was also used to verify the phage proteins expressed during lytic growth. Furthermore, bioinformatics tools such as Phamerator and BLAST were used to analyze QBert?s genome, which was 153 kb long. Genes encoding phage structural components, such as capsid and tail proteins, were found throughout the genome, as well as a cluster of genes involved in DNA replication of the genome. Comparisons between QBert?s genome and those of other phages in the C1 cluster of mycobacteriophage revealed many highly conserved regions; however, we also found many instances of additions and substitutions in the genome that were indicative of genetic recombination.
Abstract Title : Genes Associated With Lysogeny in the "Lytic" Mycobacteriophage Qbert
Abstract : Bacteriophage can be classified as lytic or temperate. While lytic phage complete their life cycle immediately after infection, temperate phage can form lysogens that incorporate their DNA into the bacterial genome where they can lay dormant for many generations. Some lysogenic phage encode proteins that provide an evolutionary advantage to the host cell. Here we analyze the genome of the mycobacteriophage Qbert to identify its potential to be lysogenic. While experiments with bacteriophage Qbert suggested that it was a lytic phage, a closer inspection of the bacteriophage?s genome revealed multiple genes consistent with lysogeny. The functions of these genes include integrase, iron-permease, beta-lactamase, and lipoprotein/invasion. Our results suggest that Qbert has the potential to be both lytic and lysogenic.
Abstract Title : Using Mass Spectrometry to Identify Novel Bacteriophage
Abstract : A collection of schools are working together to assemble an extensive collection of bacteriophage for the possible use in the research and development of new treatments for Mycobacterium tuberculosis. As the amount of participants have increased over the past five years, the collection of phage has grown exponentially, making it more challenging to identify unique phage to add to the collection. The challenge in identifying novel phage for genomic annotation lies in the fact that conventional methods such as sequencing are not time- and cost-efficient. Here we tested tandem mass spectrometry to see if it could rapidly identify novel phage from a collection of mycobacteriophage isolated from local soil samples. Twenty eight phage lysates were isolated by our class during the fall of 2012 and subjected to mass spectrometry. We found we were able to determine the identify of all 28 phage and group them into five known clusters, or families. Mass spectrometry is a relatively fast and easy method of classifying phage and identifying their close relatives. This allows researchers to examine evolutionary relationships and propose future experiments.
Abstract Title : Genetic studies of a novel antisense transcript involved in phycobilisome degradation in Anabaena sp. strain PCC 7120
Abstract : The response to nutrient stress is a major area of study in cyanobacteria. Better understanding of nutrient stress-response in cyanobacteria may lead to more efficient and productive industrial applications for production of nutraceuticals and biofuel. When subjected to fixed nitrogen starvation, the cyanobacterium Anabaena sp. strain PCC 7120 (hereafter, Anabaena) forms terminally differentiated nitrogen-fixing cells called heterocysts. The response to nitrogen starvation in Anabaena includes degradation of phycobilisomes (PBS), the light-harvesting complexes of cyanobacterial photosynthesis. The small protein NblA triggers PBS degradation in heterocysts. Previous directional deep sequencing of Anabaena's transcriptome during removal of combined nitrogen from the environment identified a novel antisense RNA transcript that runs the length of and is complementary to the nblA open reading frame. The antisense RNA (hereafter, as_nblA RNA) is expressed before nitrogen deprivation, and the sense transcript of nblA increases after removal of combined nitrogen. The regulatory role of this antisense RNA is especially interesting because it is an example of an antisense transcript that is completely turned off specifically in heterocysts. The as_nblA promoter driving green fluorescent protein expression shows high expression in vegetative cells in nitrogen deplete media. We assembled a double recombinant gene inactivation plasmid for the targeted gene inactivation of the putative as_nblA promoter region. We also cloned the sense nblA promoter driving GFP and visualized its expression in Anabaena during nitrogen step-down in vivo. The sense nblA-GFP reporter fusion shows heightened GFP expression in proheterocysts at 24 and 27 hours after the removal of combined nitrogen.
Abstract Title : Effects of Beta-lactams on the Proteome of Daptomycin Susceptible and Nonsusceptible Methicillin-Resistant Staphylococcus aureus (MRSA)
Abstract : Background. Acquired resistance to the lipopeptide antibiotic daptomycin (DAP) in clinical MRSA poses ongoing medical challenges. A promising recent finding is restoration of DAP susceptibility via synergistic effects with beta-lactam antibiotics such as nafcillin (NAF), even in strains resistant to both NAF and DAP. The goal of this study is to use mass spectrometry to characterize the proteomes before and after NAF treatment in vitro of two isogenic clinical bloodstream MRSA strains, D592 (DAP-susceptible) and D712 (DAP-nonsusceptible). The restoration of DAP susceptibility by NAF was previously observed in vitro with these strains, and DAP plus NAF combination therapy was used to eradicate persistent bacteremia. Methods. Log-phase D592 and D712 cultures were treated with NAF (5 mg/L) or the cephalosporin ceftaroline (0.1 or 1 mg/L). Samples were harvested, digested with trypsin, fractionated using high pH reverse phase HPLC and labeled with isobaric iTRAQ tags for relative quantification using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. This allowed a detailed proteomic analysis through the simultaneous identification of the proteins present and their relative quantities before and after NAF exposure. Results. Treatment of MRSA with NAF resulted in marked increases in proteins involved in cell wall stress and antibiotic resistance, including VraR (8 to 10 fold), SarA (4 to 5 fold), penicillin-binding protein 2a (PBP2a) (22 to 30 fold) and beta-lactamase (24 to 33 fold), in good agreement with previous transcriptional profiling results. We also observed significant decreases in the key virulence factors Protein A and fibronectin binding protein (2 to 9 fold). Ceftaroline at 1 mg/L exerted similar effects. We are currently performing additional experiments to confirm and extend these results. Conclusions. Beta-lactam antibiotics conferred some expected and some unexpected changes to the proteome of MRSA. The decreased level of key virulence factors has potential consequences on bacterial virulence that might explain the successful clinical outcome of DAP plus NAF combination therapy in strains resistant to both drugs.
Abstract Title : Genes Involved in the Lytic Pathway of Mycobacteriophage Qbert
Abstract : Bacteriophage, viruses that infect bacteria, may undergo a lytic pathway to replicate their DNA and produce progeny, resulting in the lysis of the host cell. Some bacteriophages rely heavily on host proteins for replication while others encode their own components and use these to replicate independently of host enzymes. We analyzed the sequenced genome of the mycobacteriophage Qbert, utilizing DNA Master for annotation, as well as the NCBI and Phagesdb BLAST programs to predict the functions of each gene. A substantial number of genes were found to encode proteins involved in DNA replication, structural assembly, and lysis -- characteristics of the lytic pathway. The presence of these genes indicate that Qbert is an efficient and versatile mycobacteriophage.
Abstract Title : Using Next-Generation Mapping Methods to Identify Genes that Mediate Responses to Water Limitation in Plants
Abstract : Plants respond to water limitation in a number of different ways. However, the mechanism(s) by which plants sense the initial osmotic stress signal remain unknown. The Schroeder lab has identified several Arabidopsis mutant lines displaying abnormal osmotic stress-induced calcium responses. Additionally, these mutants exhibit other osmotic stress-related phenotypes. Identification of the precise mutations causing these phenotypes could reveal genes necessary to sense water limitation. The rapid growth of sequencing technology now enables us to use whole-genome sequencing to map mutations. Previously, I have established an analysis pipeline for identification of causative mutations using whole-genome sequencing, which I verified using simulated and previously published data sets. My simulated data allowed me to determine the optimal experimental conditions necessary to yield positive results using this analysis method. Experimental data gathered from candidate calcium response mutants was determined as suboptimal, leading us to begin collection of data from osmotic root skew mutants to more effectively identify a potential osmotic sensor in Arabidopsis.