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Articles 61 - 90 of 105

Full-Text Articles in Microbiology

Role Of Flgt In Anchoring The Flagellum Of Vibrio Cholerae, Raquel M. Martinez, Brooke A. Jude, Thomas J. Kirn, Karen Skorupski, Ronald K. Taylor Apr 2010

Role Of Flgt In Anchoring The Flagellum Of Vibrio Cholerae, Raquel M. Martinez, Brooke A. Jude, Thomas J. Kirn, Karen Skorupski, Ronald K. Taylor

Dartmouth Scholarship

Flagellar motility has long been regarded as an important virulence factor. In Vibrio cholerae, the single polar flagellum is essential for motility as well as for proper attachment and colonization. In this study, we demonstrate that the novel flagellar protein FlgT is involved in anchoring the flagellum to the V. cholerae cell. A screen for novel colonization factors by use of TnphoA mutagenesis identified flgT. An in-frame deletion of flgT established that FlgT is required for attachment, colonization, and motility. Transmission electron microscopy revealed that while the flgT mutant is capable of assembling a phenotypically normal flagellum, …

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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 Mar 2010

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 …

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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 Jan 2010

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 …

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Microbial Nad Metabolism: Lessons From Comparative Genomics, Francesca Gazzaniga, Rebecca Stebbins, Sheila Z. Chang, Mark A. Mcpeek, Charles Brenner Sep 2009

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 …

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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 Sep 2009

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 …

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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 Mar 2009

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 …

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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 Jan 2009

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 …

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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 Oct 2008

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 …

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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 Sep 2008

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 …

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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 Sep 2008

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 …

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Integration Host Factor Positively Regulates Virulence Gene Expression In Vibrio Cholerae, Emily Stonehouse, Gabriela Kovacikova, Ronald K. Taylor, Karen Skorupski Apr 2008

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 …

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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 Dec 2007

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. …

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A Serratia Marcescens Oxyr Homolog Mediates Surface Attachment And Biofilm Formation, Robert M. Q. Shanks, Nicholas A. Stella, Eric J. Kalivoda, Megan R. Doe Aug 2007

A Serratia Marcescens Oxyr Homolog Mediates Surface Attachment And Biofilm Formation, Robert M. Q. Shanks, Nicholas A. Stella, Eric J. Kalivoda, Megan R. Doe

Dartmouth Scholarship

OxyR is a conserved bacterial transcription factor with a regulatory role in oxidative stress response. From a genetic screen for genes that modulate biofilm formation in the opportunistic pathogen Serratia marcescens, mutations in an oxyR homolog and predicted fimbria structural genes were identified. S. marcescens oxyR mutants were severely impaired in biofilm formation, in contrast to the hyperbiofilm phenotype exhibited by oxyR mutants of Escherichia coli and Burkholderia pseudomallei. Further analysis revealed that OxyR plays a role in the primary attachment of cells to a surface. Similar to what is observed in other bacterial species, S. marcescens OxyR …

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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 Jun 2007

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 …

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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 May 2007

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 …

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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 Apr 2007

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 …

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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 Mar 2007

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). …

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Gammaherpesvirus Persistence Alters Key Cd8 T-Cell Memory Characteristics And Enhances Antiviral Protection, Joshua J. Obar, Shinichiro Fuse, Erica K. Leung, Sarah C. Bellfy, Edward J. Usherwood Sep 2006

Gammaherpesvirus Persistence Alters Key Cd8 T-Cell Memory Characteristics And Enhances Antiviral Protection, Joshua J. Obar, Shinichiro Fuse, Erica K. Leung, Sarah C. Bellfy, Edward J. Usherwood

Dartmouth Scholarship

In herpesvirus infections, the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection, we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist, we were able to dissect the changes in the antiviral CD8(+) T-cell response …

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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 Sep 2006

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 …

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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 Jul 2006

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 …

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Sara Positively Controls Bap-Dependent Biofilm Formation In Staphylococcus Aureus, María P. Trotonda, Adhar C. Manna, Ambrose L. Cheung, Iñigo Lasa, José R. Penadés Aug 2005

Sara Positively Controls Bap-Dependent Biofilm Formation In Staphylococcus Aureus, María P. Trotonda, Adhar C. Manna, Ambrose L. Cheung, Iñigo Lasa, José R. Penadés

Dartmouth Scholarship

The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and …

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Integrated Recombinant Protein Expression And Purification Platform Based On Ralstonia Eutropha, Gavin C. Barnard, Jesse D. Mccool, David W. Wood, Tillman U. Gerngross May 2005

Integrated Recombinant Protein Expression And Purification Platform Based On Ralstonia Eutropha, Gavin C. Barnard, Jesse D. Mccool, David W. Wood, Tillman U. Gerngross

Dartmouth Scholarship

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer poly

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Sara Is An Essential Positive Regulator Of Staphylococcus Epidermidis Biofilm Development, Maria A. Tormo, Miguel Marti, Jaione Valle, Adhar C. Manna Apr 2005

Sara Is An Essential Positive Regulator Of Staphylococcus Epidermidis Biofilm Development, Maria A. Tormo, Miguel Marti, Jaione Valle, Adhar C. Manna

Dartmouth Scholarship

Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, …

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A Three-Component Regulatory System Regulates Biofilm Maturation And Type Iii Secretion In Pseudomonas Aeruginosa, Sherry L. Kuchma, John P. Connolly, George A. O'Toole Feb 2005

A Three-Component Regulatory System Regulates Biofilm Maturation And Type Iii Secretion In Pseudomonas Aeruginosa, Sherry L. Kuchma, John P. Connolly, George A. O'Toole

Dartmouth Scholarship

Biofilms are structured communities found associated with a wide range of surfaces. Here we report the identification of a three-component regulatory system required for biofilm maturation by Pseudomonas aeruginosa strain PA14. A transposon mutation that altered biofilm formation in a 96-well dish assay originally defined this locus, which is comprised of genes for a putative sensor histidine kinase and two response regulators and has been designated sadARS. Nonpolar mutations in any of the sadARS genes result in biofilms with an altered mature structure but do not confer defects in growth or early biofilm formation, swimming, or twitching motility. After …

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Electrotransformation Of Clostridium Thermocellum, Michael V. Tyurin, Sunil G. Desai, Lee R. Lynd Nov 2004

Electrotransformation Of Clostridium Thermocellum, Michael V. Tyurin, Sunil G. Desai, Lee R. Lynd

Dartmouth Scholarship

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 􏰍 0.5) 􏰎 105 transformants per 􏰃g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 􏰍 1.8) 􏰎 104 transformants per 􏰃g of plasmid DNA for strain ATCC 27405 and 􏰁1 􏰎 103 transformants per 􏰃g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was 􏰁50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.

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Identification Of Sarv (Sa2062), A New Transcriptional Regulator, Is Repressed By Sara And Mgra (Sa0641) And Involved In The Regulation Of Autolysis In Staphylococcus Aureus, Adhar C. Manna, Susham S. Ingavale, Marybeth Maloney, Willem Van Wamel, Ambrose L. Cheung Aug 2004

Identification Of Sarv (Sa2062), A New Transcriptional Regulator, Is Repressed By Sara And Mgra (Sa0641) And Involved In The Regulation Of Autolysis In Staphylococcus Aureus, Adhar C. Manna, Susham S. Ingavale, Marybeth Maloney, Willem Van Wamel, Ambrose L. Cheung

Dartmouth Scholarship

The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, sae, arlRS, lytSR, and sarA-like genes. Here we described a novel transcriptional regulator called sarV of the SarA protein family. The transcription of sarV is low or undetectable under in vitro conditions but is significantly augmented in sarA and mgrA (norR or rat) (SA0641) mutants. The sarA and mgrA genes act as repressors of sarV expression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of the …

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Sadb Is Required For The Transition From Reversible To Irreversible Attachment During Biofilm Formation By Pseudomonas Aeruginosa Pa14, Nicky C. Caiazza, George A. O'Toole Jul 2004

Sadb Is Required For The Transition From Reversible To Irreversible Attachment During Biofilm Formation By Pseudomonas Aeruginosa Pa14, Nicky C. Caiazza, George A. O'Toole

Dartmouth Scholarship

Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes …

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Pcr And 16s Rdna Sequencing For Identifying And Comparing Gram-Negative Bacteria, Molly Truax May 2004

Pcr And 16s Rdna Sequencing For Identifying And Comparing Gram-Negative Bacteria, Molly Truax

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

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Caenorhabditis Elegans: A Look At Koch's Postulates And Host-Pathogen Interactions, Nicole Helmrichs May 2004

Caenorhabditis Elegans: A Look At Koch's Postulates And Host-Pathogen Interactions, Nicole Helmrichs

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

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A Dominant-Negative Fur Mutation In Bradyrhizobium Japonicum, Heather P. Benson, Kristin Levier, Mary Lou Guerinot Mar 2004

A Dominant-Negative Fur Mutation In Bradyrhizobium Japonicum, Heather P. Benson, Kristin Levier, Mary Lou Guerinot

Dartmouth Scholarship

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the furgene repress iron uptake systems and starve for iron, whereas fur mutants fail to …

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