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Articles 121 - 150 of 203
Full-Text Articles in Microbiology
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
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 …
The Role Of Cd4 T Cells In The Pathogenesis Of Murine Aids, Wen Li, William R. Green
The Role Of Cd4 T Cells In The Pathogenesis Of Murine Aids, Wen Li, William R. Green
Dartmouth Scholarship
LP-BM5, a retroviral isolate, induces a disease featuring retrovirus-induced immunodeficiency, designated murine AIDS (MAIDS). Many of the features of the LP-BM5-induced syndrome are shared with human immunodeficiency virus-induced disease. For example, CD4 T cells are critical to the development of MAIDS. In vivo depletion of CD4 T cells before LP-BM5 infection rendered genetically susceptible B6 mice MAIDS resistant. Similarly, MAIDS did not develop in B6.nude mice. However, if reconstituted with CD4 T cells, B6.nude mice develop full-blown MAIDS. Our laboratory has shown that the interaction of B and CD4 T cells that is central to MAIDS pathogenesis requires ligation of …
Talking To Themselves: Autoregulation And Quorum Sensing In Fungi, Deborah A. Hogan
Talking To Themselves: Autoregulation And Quorum Sensing In Fungi, Deborah A. Hogan
Dartmouth Scholarship
Extracellular autoinducing compounds in the supernatants of microbial cultures were first recognized for their roles in the induction of genetic competence in gram-positive bacteria and in the regulation of light production in marine vibrios. In 1994, this form of population-level regulation in microbes was dubbed “quorum sensing” since it enabled bacterial cells to chemically measure the density of the surrounding population. Subsequently, many examples of cell density-dependent regulation by extracellular factors have been found in diverse microorganisms. The widespread incidence of diverse quorum-sensing systems strongly suggests that regulation in accordance with cell density is important for the success of microbes …
Conservation Of The Pho Regulon In Pseudomonas Fluorescens Pf0-1, Russell D. Monds, Peter D. Newell, Julia A. Schwartzman, George A. O'Toole
Conservation Of The Pho Regulon In Pseudomonas Fluorescens Pf0-1, Russell D. Monds, Peter D. Newell, Julia A. Schwartzman, George A. O'Toole
Dartmouth Scholarship
The Pho regulon integrates the sensing of environmental inorganic phosphate (Pi) availability with coregulation of gene expression, mediating an adaptive response to Pi limitation. Many aspects of the Pho regulon have been addressed in studies of Escherichia coli; however, it is unclear how transferable this knowledge is to other bacterial systems. Here, we report work to discern the conservation of the Pho regulon in Pseudomonas fluorescens Pf0-1. We demonstrate by mutational studies that PhoB/PhoR and the Pst system have conserved functions in the regulation of Pi-induced phosphatase activities, as well as expression of other Pi-regulated genes. A genetic screen was …
Bacteriophage Migration Via Nematode Vectors: Host-Parasite-Consumer Interactions In Laboratory Microcosms, John J. Dennehy, Nicholas A. Friedenberg, Yul W. Yang, Paul E. Turner
Bacteriophage Migration Via Nematode Vectors: Host-Parasite-Consumer Interactions In Laboratory Microcosms, John J. Dennehy, Nicholas A. Friedenberg, Yul W. Yang, Paul E. Turner
Dartmouth Scholarship
Pathogens vectored by nematodes pose serious agricultural, economic, and health threats; however, little is known of the ecological and evolutionary aspects of pathogen transmission by nematodes. Here we describe a novel model system with two trophic levels, bacteriophages and nematodes, each of which competes for bacteria. We demonstrate for the first time that nematodes are capable of transmitting phages between spatially distinct patches of bacteria. This model system has considerable advantages, including the ease of maintenance and manipulation at the laboratory bench, the ability to observe many generations in short periods, and the capacity to freeze evolved strains for later …
Keeping Their Options Open: Acute Versus Persistent Infections, S. Furukawa, S. L. Kuchma, G. A. O'Toole
Keeping Their Options Open: Acute Versus Persistent Infections, S. Furukawa, S. L. Kuchma, G. A. O'Toole
Dartmouth Scholarship
No abstract provided.
Transposon Disruption Of The Complex I Nadh Oxidoreductase Gene (Snod) In Staphylococcus Aureus Is Associated With Reduced Susceptibility To The Microbicidal Activity Of Thrombin-Induced Platelet Microbicidal Protein 1, Arnold S. Bayer, Peter Mcnamara, Michael R. Yeaman, Natalie Lucindo, Tiffanny Jones, Ambrose L. Cheung
Transposon Disruption Of The Complex I Nadh Oxidoreductase Gene (Snod) In Staphylococcus Aureus Is Associated With Reduced Susceptibility To The Microbicidal Activity Of Thrombin-Induced Platelet Microbicidal Protein 1, Arnold S. Bayer, Peter Mcnamara, Michael R. Yeaman, Natalie Lucindo, Tiffanny Jones, Ambrose L. Cheung
Dartmouth Scholarship
The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1(r)) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1(r) in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene …
Rhamnolipids Modulate Swarming Motility Patterns Of Pseudomonas Aeruginosa, Nicky C. Caiazza, Robert M. Q. Shanks, G. A. O'Toole
Rhamnolipids Modulate Swarming Motility Patterns Of Pseudomonas Aeruginosa, Nicky C. Caiazza, Robert M. Q. Shanks, G. A. O'Toole
Dartmouth Scholarship
Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and …
Role Of Spontaneous Current Oscillations During High-Efficiency Electrotransformation Of Thermophilic Anaerobes, Michael V. Tyurin, Charles R. Sullivan, Lee R. Lynd
Role Of Spontaneous Current Oscillations During High-Efficiency Electrotransformation Of Thermophilic Anaerobes, Michael V. Tyurin, Charles R. Sullivan, Lee R. Lynd
Dartmouth Scholarship
Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes Clostridium thermocellum ATCC 27405, C. thermocellum DSM 1313, and Thermoanaerobacterium saccharolyticum YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both C. thermocellum strains only at field strengths of >12 kV/cm, and ET was only observed above the same threshold. Similarly, for T. saccharolyticum, oscillations were …
Sara Positively Controls Bap-Dependent Biofilm Formation In Staphylococcus Aureus, María P. Trotonda, Adhar C. Manna, Ambrose L. Cheung, Iñigo Lasa, José R. Penadés
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 …
Susceptibility Of Biofilms To Bdellovibrio Bacteriovorus Attack, Daniel Kadouri, George A. O'Toole
Susceptibility Of Biofilms To Bdellovibrio Bacteriovorus Attack, Daniel Kadouri, George A. O'Toole
Dartmouth Scholarship
Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that …
Cellulose Utilization By Clostridium Thermocellum: Bioenergetics And Hydrolysis Product Assimilation, Yi-Heng P. Zhang, Lee R. Lynd
Cellulose Utilization By Clostridium Thermocellum: Bioenergetics And Hydrolysis Product Assimilation, Yi-Heng P. Zhang, Lee R. Lynd
Dartmouth Scholarship
The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated …
Integrated Recombinant Protein Expression And Purification Platform Based On Ralstonia Eutropha, Gavin C. Barnard, Jesse D. Mccool, David W. Wood, Tillman U. Gerngross
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
Requirements For Vibrio Cholerae Hapr Binding And Transcriptional Repression At The Hapr Promoter Are Distinct From Those At The Apha Promoter, Wei Lin, Gabriela Kovacikova, Karen Skorupski
Requirements For Vibrio Cholerae Hapr Binding And Transcriptional Repression At The Hapr Promoter Are Distinct From Those At The Apha Promoter, Wei Lin, Gabriela Kovacikova, Karen Skorupski
Dartmouth Scholarship
Virulence gene expression in certain strains of Vibrio cholerae is regulated in response to cell density by a quorum-sensing cascade that influences the levels of the LuxR homolog HapR through small regulatory RNAs that control the stability of its message. At high cell density, HapR represses the expression of the gene encoding the virulence gene activator AphA by binding to a site between −85 and −58 in the aphA promoter. We show here that a second binding site for HapR lies within the hapR promoter from which it functions to repress its own transcription. This site, as determined by gel …
Sara Is An Essential Positive Regulator Of Staphylococcus Epidermidis Biofilm Development, Maria A. Tormo, Miguel Marti, Jaione Valle, Adhar C. Manna
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, …
Identification Of A Tcpc-Tcpq Outer Membrane Complex Involved In The Biogenesis Of The Toxin-Coregulated Pilus Of Vibrio Cholerae, Niranjan Bose, Ronald K. Taylor
Identification Of A Tcpc-Tcpq Outer Membrane Complex Involved In The Biogenesis Of The Toxin-Coregulated Pilus Of Vibrio Cholerae, Niranjan Bose, Ronald K. Taylor
Dartmouth Scholarship
The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation …
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
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 …
Electrotransformation Of Clostridium Thermocellum, Michael V. Tyurin, Sunil G. Desai, Lee R. Lynd
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.
T-Cell Responses To The M3 Immune Evasion Protein Of Murid Gammaherpesvirus 68 Are Partially Protective And Induced With Lytic Antigen Kinetics, Joshua J. Obar, Douglas C. Donovan, Sarah G. Crist, Ondine Silvia, James P. Stewart, Edward J. Usherwood
T-Cell Responses To The M3 Immune Evasion Protein Of Murid Gammaherpesvirus 68 Are Partially Protective And Induced With Lytic Antigen Kinetics, Joshua J. Obar, Douglas C. Donovan, Sarah G. Crist, Ondine Silvia, James P. Stewart, Edward J. Usherwood
Dartmouth Scholarship
DNA vaccination with the M3 gene, encoding an immune evasion molecule expressed during both the acute lytic and persistent phases of murid gammaherpesvirus 68 infection, yielded a significantly lower titer of virus in the lung than controls. The protection seen was dependent on T cells, and we mapped an epitope recognized by CD8 T cells. The immune response to this epitope follows the same kinetics as lytic cycle antigens, despite the fact that this gene is expressed in both lytic and persistent stages of infection. This has important implications for our understanding of T-cell responses to putative latency-associated gammaherpesvirus proteins …
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
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 …
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
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 …
Isolation And Characterization Of A Generalized Transducing Phage For Pseudomonas Aeruginosa Strains Pao1 And Pa14, Jonathan M. Budzik, William A. Rosche, Arne Rietsch, George A. O'Toole
Isolation And Characterization Of A Generalized Transducing Phage For Pseudomonas Aeruginosa Strains Pao1 And Pa14, Jonathan M. Budzik, William A. Rosche, Arne Rietsch, George A. O'Toole
Dartmouth Scholarship
A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between P. aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P. aeruginosa.
A Dominant-Negative Fur Mutation In Bradyrhizobium Japonicum, Heather P. Benson, Kristin Levier, Mary Lou Guerinot
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 …
Cd40-Associated Traf 6 Signaling Is Required For Disease Induction In A Retrovirus-Induced Murine Immunodeficiency, Kathy A. Green, Cory L. Ahonen, W. James Cook, William R. Green
Cd40-Associated Traf 6 Signaling Is Required For Disease Induction In A Retrovirus-Induced Murine Immunodeficiency, Kathy A. Green, Cory L. Ahonen, W. James Cook, William R. Green
Dartmouth Scholarship
LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.
The Virulence Activator Apha Links Quorum Sensing To Pathogenesis And Physiology In Vibrio Cholerae By Repressing The Expression Of A Penicillin Amidase Gene On The Small Chromosome, Gabriela Kovacikova, Wei Lin, Karen Skorupski
The Virulence Activator Apha Links Quorum Sensing To Pathogenesis And Physiology In Vibrio Cholerae By Repressing The Expression Of A Penicillin Amidase Gene On The Small Chromosome, Gabriela Kovacikova, Wei Lin, Karen Skorupski
Dartmouth Scholarship
Activation of the tcpPH promoter on the Vibrio pathogenicity island by AphA and AphB initiates the Vibrio cholerae virulence cascade and is regulated by quorum sensing through the repressive action of HapR on aphA expression. To further understand how the chromosomally encoded AphA protein activates tcpPH expression, site-directed mutagenesis was used to identify the base pairs critical for AphA binding and transcriptional activation. This analysis revealed a region of partial dyad symmetry, TATGCA-N6-TNCNNA, that is important for both of these activities. Searching the V. cholerae genome for this binding site permitted the identification of a second one upstream of a …
Crystal Structure Of The Sars Protein From Staphylococcus Aureus, Ronggui Li, Adhar C. Manna, Shaodong Dai, Ambrose L. Cheung, Gongyi Zhang
Crystal Structure Of The Sars Protein From Staphylococcus Aureus, Ronggui Li, Adhar C. Manna, Shaodong Dai, Ambrose L. Cheung, Gongyi Zhang
Dartmouth Scholarship
The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). One of these determinants, protein A (spa), is activated by sarS, which encodes a 250-residue DNA-binding protein. Genetic analysis indicated that the agr locus likely mediates spa repression by suppressing the transcription of sarS. Contrary to SarA and SarR, which require homodimer formation for proper function, SarS is unusual within the SarA protein family in that it contains two homologous halves, with each half sharing sequence similarity to SarA and SarR. Here we report the 2.2 Å …
Alpha-Toxin Is Required For Biofilm Formation By Staphylococcus Aureus, Nicky C. Caiazza, George A. O'Toole
Alpha-Toxin Is Required For Biofilm Formation By Staphylococcus Aureus, Nicky C. Caiazza, George A. O'Toole
Dartmouth Scholarship
Staphylococcus aureus is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the hla gene, plays an integral role in biofilm formation. The hla mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.
Rhamnolipid Surfactant Production Affects Biofilm Architecture In Pseudomonas Aeruginosa Pao1, Mary E. Davey, Nicky C. Caiazza, George A. O'Toole
Rhamnolipid Surfactant Production Affects Biofilm Architecture In Pseudomonas Aeruginosa Pao1, Mary E. Davey, Nicky C. Caiazza, George A. O'Toole
Dartmouth Scholarship
In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen Pseudomonas aeruginosa in the maintenance of biofilm architecture. …
The Cd154/Cd40 Interaction Required For Retrovirus-Induced Murine Immunodeficiency Syndrome Is Not Mediated By Upregulation Of The Cd80/Cd86 Costimulatory Molecules, Kathy A. Green, W. James Cook, Arlene H. Sharpe, William R. Green
The Cd154/Cd40 Interaction Required For Retrovirus-Induced Murine Immunodeficiency Syndrome Is Not Mediated By Upregulation Of The Cd80/Cd86 Costimulatory Molecules, Kathy A. Green, W. James Cook, Arlene H. Sharpe, William R. Green
Dartmouth Scholarship
C57BL/6 (B6) mice infected with LP-BM5 retroviruses develop disease, including an immunodeficiency similar to AIDS. This disease, murine AIDS (MAIDS), is inhibited by in vivo anti-CD154 monoclonal antibody treatment. The similar levels of insusceptibility of CD40−/− and CD154−/− B6 mice indicate that CD154/CD40 molecular interactions are required for MAIDS. CD4+ T and B cells, respectively, provide the CD154 and CD40 expression needed for MAIDS induction. Here, the required CD154/CD40 interaction is shown to be independent of CD80 and CD86 expression: CD80/CD86−/− B6 mice develop MAIDS after LP-BM5 infection.
Mechanism Of Toxt-Dependent Transcriptional Activation At The Vibrio Cholerae Tcpa Promoter, Robin R. Hulbert, Ronald K. Taylor
Mechanism Of Toxt-Dependent Transcriptional Activation At The Vibrio Cholerae Tcpa Promoter, Robin R. Hulbert, Ronald K. Taylor
Dartmouth Scholarship
The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae. ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others. In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA. Deletion analysis showed that a region from −95 to +2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to −63. A collection of point mutations generated by error-prone PCR revealed two small regions required …