Microbiology Commons^™

Coordinated Regulation By Agra, Sara, And Sarr To Control Agr Expression In Staphylococcus Aureus, Dindo Reyes, Diego O. Andrey, Antoinette Monod, William L. Kelley, Gongyi Zhang, Ambrose L. Cheung

Dartmouth Scholarship

The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that …

Type Ii Toxoplasma Gondii Ku80 Knockout Strains Enable Functional Analysis Of Genes Required For Cyst Development And Latent Infection, Barbara A. Fox, Alejandra Falla, Leah M. Rommereim, Tadakimi Tomita

Dartmouth Scholarship

Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057 …

Systematic Analysis Of Diguanylate Cyclases That Promote Biofilm Formation By Pseudomonas Fluorescens Pf0-1, Peter D. Newell, Shiro Yoshioka, Kelli L. Hvorecny, Russell D. Monds, George A. O'Toole

Dartmouth Scholarship

Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for …

Non-Identity-Mediated Crispr-Bacteriophage Interaction Mediated Via The Csy And Cas3 Proteins, Kyle C. Cady, George A. O'Toole

Dartmouth Scholarship

Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins …

Cellulose- And Xylan-Degrading Thermophilic Anaerobic Bacteria From Biocompost, M. V. Sizova, J. A. Izquierdo, N. S. Panikov, L. R. Lynd

Dartmouth Scholarship

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S …

H-Ns Binding And Repression Of The Ctx Promoter In Vibrio Cholerae, Emily A. Stonehouse, Robin R. Hulbert, Melinda B. Nye, Karen Skorupski, Ronald K. Taylor

Dartmouth Scholarship

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at …

The Armadillo Repeat Protein Pf16 Is Essential For Flagellar Structure And Function In Plasmodium Male Gametes, Ursula Straschil, Arthur M. Talman, David J. P. Ferguson, Karen A. Bunting, Zhengyao Xu, Elizabeth Bailes, Robert E. Sinden, Anthony A. Holder, Elizabeth F. Smith

Dartmouth Scholarship

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that …

Constraint-Based Model Of Shewanella Oneidensis Mr-1 Metabolism: A Tool For Data Analysis And Hypothesis Generation, Grigoriy E. Pinchuk, Eric A. Hill, Oleg V. Geydebrekht, Jessica De Ingeniis, Xiaolin Zhang, Andrei Osterman, James H. Scott

Dartmouth Scholarship

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based …

Natural Competence In Thermoanaerobacter And Thermoanaerobacterium Species, A Joe Shaw, David A. Hogsett, Lee R. Lynd

Dartmouth Scholarship

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant.

In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence- mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to …

Functional Characterization Of Mat1-1-Specific Mating-Type Genes In The Homothallic Ascomycete Sordaria Macrospora Provides New Insights Into Essential And Nonessential Sexual Regulators, V. Klix, M. Nowrousian, C. Ringelberg, J. J. Loros

Dartmouth Scholarship

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed …

Diversity Of Bacteria And Glycosyl Hydrolase Family 48 Genes In Cellulolytic Consortia Enriched From Thermophilic Biocompost, Javier A. Izquierdo, Maria V. Sizova, Lee R. Lynd

Dartmouth Scholarship

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured …

Farnesol Induces Hydrogen Peroxide Resistance In Candida Albicans Yeast By Inhibiting The Ras-Cyclic Amp Signaling Pathway, Aurélie Deveau, Amy E. Piispanen, Angelyca A. Jackson, Deborah A. Hogan

Dartmouth Scholarship

Farnesol, a Candida albicans cell-cell signaling molecule that participates in the control of morphology, has an additional role in protection of the fungus against oxidative stress. In this report, we show that although farnesol induces the accumulation of intracellular reactive oxygen species (ROS), ROS generation is not necessary for the induction of catalase (Cat1)-mediated oxidative-stress resistance. Two antioxidants, α-tocopherol and, to a lesser extent, ascorbic acid effectively reduced intracellular ROS generation by farnesol but did not alter farnesol-induced oxidative-stress resistance. Farnesol inhibits the Ras1-adenylate cyclase (Cyr1) signaling pathway to achieve its effects on morphology under hypha-inducing conditions, and we demonstrate …

Microbial Nad Metabolism: Lessons From Comparative Genomics, Francesca Gazzaniga, Rebecca Stebbins, Sheila Z. Chang, Mark A. Mcpeek, Charles Brenner

Dartmouth Scholarship

NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life …

Characterization Of Two Outer Membrane Proteins, Flgo And Flgp, That Influence Vibrio Cholerae Motility, Raquel M. Martinez, Madushini N. Dharmasena, Thomas J. Kirn, Ronald K. Taylor

Dartmouth Scholarship

Vibrio cholerae is highly motile by the action of a single polar flagellum. The loss of motility reduces the infectivity of V. cholerae, demonstrating that motility is an important virulence factor. FlrC is the sigma-54-dependent positive regulator of flagellar genes. Recently, the genes VC2206 (flgP) and VC2207 (flgO) were identified as being regulated by FlrC via a microarray analysis of an flrC mutant (D. C. Morris, F. Peng, J. R. Barker, and K. E. Klose, J. Bacteriol. 190:231-239, 2008). FlgP is reported to be an outer membrane lipoprotein required for motility that functions as a colonization factor. The study reported …

Long-Distance Delivery Of Bacterial Virulence Factors By Pseudomonas Aeruginosa Outer Membrane Vesicles, Jennifer M. Bomberger, Daniel P. Maceachran, Bonita A. Coutermarsh, Siying Ye, George A. O'Toole, Bruce A. Stanton, Frederick M. Ausubel

Dartmouth Scholarship

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma …

Regulation Of The Mazef Toxin-Antitoxin Module In Staphylococcus Aureus And Its Impact On Sigb Expression, Niles P. Donegan, Ambrose L. Cheung

Dartmouth Scholarship

In Staphylococcus aureus, the sigB operon codes for the alternative sigma factor σ^Band its regulators that enable the bacteria to rapidly respond to environmental stresses via redirection of transcriptional priorities. However, a full model of σ^Bregulation in S. aureus has not yet emerged. Earlier data has suggested that mazEF, a toxin-antitoxin (TA) module immediately upstream of the sigB operon, was transcribed with the sigB operon. Here we demonstrate that the promoter P_mazE upstream of mazEF is essential for full σ^B activity and that instead of utilizing autorepression typical of TA systems, sigB …

Lapd Is A Bis-(3′,5′)-Cyclic Dimeric Gmp-Binding Protein That Regulates Surface Attachment By Pseudomonas Fluorescens Pf0–1, Peter D. Newell, Russell D. Monds, George A. O'Toole

Dartmouth Scholarship

The second messenger cyclic dimeric GMP (c-di-GMP) regulates surface attachment and biofilm formation by many bacteria. For Pseudomonas fluorescens Pf0-1, c-di-GMP impacts the secretion and localization of the adhesin LapA, which is absolutely required for stable surface attachment and biofilm formation by this bacterium. In this study we characterize LapD, a unique c-di-GMP effector protein that controls biofilm formation by communicating intracellular c-di-GMP levels to the membrane-localized attachment machinery via its periplasmic domain. LapD contains degenerate and enzymatically inactive diguanylate cyclase and c-di-GMP phosphodiesterase (EAL) domains and binds to c-di-GMP through a degenerate EAL domain. We present evidence that LapD …

Overexpression Of Mazfsa In Staphylococcus Aureus Induces Bacteriostasis By Selectively Targeting Mrnas For Cleavage, Zhibiao Fu, Sandeep Tamber, Guido Memmi, Niles P. Donegan, Ambrose L. Cheung

Dartmouth Scholarship

The role of chromosomally encoded toxin-antitoxin (TA) loci in bacterial physiology has been under debate, with the toxin proposed as either an inducer of bacteriostasis or a mediator of programmed cell death (PCD). We report here that ectopic expression of MazFSa, a toxin of the TA module from Staphylococcus aureus, led to a rapid decrease in CFU counts but most cells remained viable as determined by differential Syto 9 and propidium iodide staining after MazFSa induction. This finding suggested that the toxin MazFSa induced cell stasis rather than cell death. We also showed that MazFSa selectively cleaves cellular mRNAs in …

Identification Of Two Gene Clusters And A Transcriptional Regulator Required For Pseudomonas Aeruginosa Glycine Betaine Catabolism, Matthew J. Wargo, Benjamin S. Szwergold, Deborah A. Hogan

Dartmouth Scholarship

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-(13)C-labeled choline …

Metabolic Engineering Of A Thermophilic Bacterium To Produce Ethanol At High Yield, A. Joe Shaw, Kara K. Podkaminer, Sunil G. Desai, John S. Bardsley, Stephen R. Rogers, Philip G. Thorne, David A. Hogsett, Lee R. Lynd

Dartmouth Scholarship

We report engineering Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield. Knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in a strain able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type. Ethanol formation in the engineered strain (ALK2) utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations …

The Bile Response Repressor Brer Regulates Expression Of The Vibrio Cholerae Breab Efflux System Operon, Francisca A. Cerda-Maira, Carol S. Ringelberg, Ronald K. Taylor

Dartmouth Scholarship

Enteric pathogens have developed several resistance mechanisms to survive the antimicrobial action of bile. We investigated the transcriptional profile of Vibrio cholerae O1 El Tor strain C6706 under virulence gene-inducing conditions in the presence and absence of bile. Microarray analysis revealed that the expression of 119 genes was affected by bile. The mRNA levels of genes encoding proteins involved in transport were increased in the presence of bile, whereas the mRNA levels of genes encoding proteins involved in pathogenesis and chemotaxis were decreased. This study identified genes encoding transcriptional regulators from the TetR family (vexR and breR) and …

Integration Host Factor Positively Regulates Virulence Gene Expression In Vibrio Cholerae, Emily Stonehouse, Gabriela Kovacikova, Ronald K. Taylor, Karen Skorupski

Dartmouth Scholarship

Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position 162 …

Genome-Wide Transcriptional Profiling Of The Cyclic Amp-Dependent Signaling Pathway During Morphogenic Transitions Of Candida Albicans, Yong-Sun Bahn, Matthew Molenda, Janet F. Staab, Courtney A. Lyman, Laura J. Gordon, Paula Sundstrom

Dartmouth Scholarship

Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. …

Characterization Of Mazfsa, An Endoribonuclease From Staphylococcus Aureus, Zhibiao Fu, Niles P. Donegan, Guido Memmi, Ambrose L. Cheung

Dartmouth Scholarship

The mazEF homologs of Staphylococcus aureus, designated mazEF(sa), have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF(Sa), as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF(Sa) leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF(Sa) is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF(Sa) cleaves single-strand RNA preferentially at the 5' side of the first U or 3' side of the second …

Bifa, A Cyclic-Di-Gmp Phosphodiesterase, Inversely Regulates Biofilm Formation And Swarming Motility By Pseudomonas Aeruginosa Pa14, Sherry L. Kuchma, Kimberly M. Brothers, Judith H. Merritt, Nicole T. Liberati, Frederick M. Ausubel, George A. O'Toole

Dartmouth Scholarship

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner …

Crystal Structure Of The Vibrio Cholerae Quorum-Sensing Regulatory Protein Hapr, Rukman S. De Silva, Gabriela Kovacikova, Wei Lin, Ronald K. Taylor, Karen Skorupski, F. Jon Kull

Dartmouth Scholarship

Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-Å resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain …

Membrane Association And Multimerization Of Tcpt, The Cognate Atpase Ortholog Of The Vibrio Cholerae Toxin-Coregulated-Pilus Biogenesis Apparatus, Shital A. Tripathi, Ronald K. Taylor

Dartmouth Scholarship

The toxin-coregulated pilus (TCP) is one of the major virulence factors of Vibrio cholerae. Biogenesis of this type 4 pilus (Tfp) requires a number of structural components encoded by the tcp operon. TcpT, the cognate putative ATPase, is required for TCP biogenesis and all TCP-mediated functions. We studied the stability and localization of TcpT in cells containing in-frame deletions in each of the tcp genes. TcpT was detectable in each of the biogenesis mutants except the ΔtcpT strain. TcpT was localized to the inner membrane (IM) in a TcpR-dependent manner. TcpR is a predicted bitopic inner membrane protein …

Inverse Regulation Of Biofilm Formation And Swarming Motility By Pseudomonas Aeruginosa Pa14, Nicky C. Caiazza, Judith H. Merritt, Kimberly M. Brothers, George A. O'Toole

Dartmouth Scholarship

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). …

Cd80 And Cd86 Control Antiviral Cd8+ T-Cell Function And Immune Surveillance Of Murine Gammaherpesvirus 68, Shinichiro Fuse, Joshua J. Obar, Sarah Bellfy, Erica K. Leung, Weijun Zhang, Edward J. Usherwood

Dartmouth Scholarship

The interactions between CD80 and CD86 on antigen-presenting cells and CD28 on T cells serve as an important costimulatory signal in the activation of T cells. Although the simplistic two-signal hypothesis has been challenged in recent years by the identification of different costimulators, this classical pathway has been shown to significantly impact antiviral humoral and cellular immune responses. How the CD80/CD86-CD28 pathway affects the control of chronic or latent infections has been less well characterized. In this study, we investigated its role in antiviral immune responses against murine gammaherpesvirus 68 (MHV-68) and immune surveillance using CD80/CD86^−/− mice. In the …

Saccharomyces Cerevisiae-Based Molecular Tool Kit For Manipulation Of Genes From Gram-Negative Bacteria, Robert M. Q. Shanks, Nicky C. Caiazza, Shannon M. Hinsa, Christine M. Toutain, George A. O'Toole

Dartmouth Scholarship

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface …