Bacteriology Commons^™

Understanding The Role Of Toxs As A Bile Sensing Component Of The Toxrs Virulence Regulatory System In Vibrio Spp., Minje Kim

Dartmouth College Ph.D Dissertations

Pathogenic Vibrio bacteria, such as V. cholerae and V. parahaemolyticus, colonize in the human small intestine to cause severe gastrointestinal disease. During the infection, these bacteria encounter bile salts, antimicrobial cholesterol metabolites secreted into the intestine. Pathogenic Vibrio species have evolved to utilize bile salts as signals to regulate virulence. The signaling depends in part on ToxRS, a conserved co-component transmembrane transcription regulator. ToxRS consists of the transcription factor ToxR and its membrane-tethered binding partner ToxS. ToxS is required for ToxR stability and full transcriptional activity. Although bile salts are known to influence ToxRS-dependent virulence gene expression, the molecular …

Evolution Of Antibiotic Responses During Chronic P. Aeruginosa Infections In The Human Lung, David Frank Ritz

Dartmouth College Ph.D Dissertations

Typical antibiotic susceptibility testing (AST) of microbial samples is performed in homogeneous cultures in batch environments, which does not account for the highly heterogeneous and dynamic nature of antibiotic responses. The most common mutation found in P. aeruginosa lineages evolved during chronic cystic fibrosis infections in the human lung, a loss of function of repressor MexZ, increases basal levels of multidrug efflux MexXY, but does not increase resistance by traditional minimal inhibitory concentration (MIC) assays. Here, we use single cell microfluidics to show that P. aeruginosa’s response to aminoglycosides is highly heterogeneous, with only a subpopulation of cells surviving exposure. …

Biofilm Architecture Determines Proliferation Of Phages And Plasmids, James B. Winans

Dartmouth College Ph.D Dissertations

This thesis explores how biofilm architecture determines the spread and ecological consequences for the bacteria-dependent genetic elements phages and plasmids. Using Escherichia coli and Vibrio cholerae as model microbes, and the genetic elements T7 phages, λ phages, and conjugative plasmid pED208, we investigate how spatial structure influences phage infection dynamics, plasmid transfer, and interbacterial interactions within biofilms. We show that densely packed biofilms can protect sensitive bacteria from phage exposure and modulate competitive outcomes between obligately lytic and temperate phages. We find that entirely different outcomes can coöccur within microns of each other dependent on biofilm structure. We also demonstrate …

Investigating The Mechanisms Of Microbial Interactions Using A Model Cystic Fibrosis Polymicrobial Community, Bassam El Hafi

Dartmouth College Ph.D Dissertations

Cystic fibrosis (CF) airway microbial communities are complex ecosystems where interspecies interactions influence pathogen survival and disease progression. We leverage a CF-relevant in vitro four-species model composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, alongside a microbiome dataset with over 4,000 respiratory samples from publicly available sequencing data of CF patient samples, to explore the dynamics of microbial interactions in CF. On a broader scale, we performed microbial network analysis to examine how the latest modulator therapy, elexacaftor/tezacaftor/ivacaftor (ETI), might influence CF microbial communities. Our analysis revealed a reduction in node connections and …

Modeling The Cystic Fibrosis Intestinal Environment To Study Microbial Interactions, Kaitlyn Emma Barrack

Dartmouth College Ph.D Dissertations

The microbiome of the cystic fibrosis (CF) intestine is altered significantly in structure and function beginning in early life. Key taxa associated with health, digestion and immune-training are reduced in abundance, including but not limited to Bacteroides, Faecalibacterium, Akkermansia, Ruminococcus and other short-chain fatty acid producers. Conversely, there is an expansion of microbes with pathogenic potential, such as Escherichia, Veillonella, Pseudomonas and Staphylococcus. The shifts in microbiota composition direct community-wide changes with differences in beta diversity by genotype, whereby children with CF (cwCF) exhibit significantly different gut microbial compositions compared to nonCF children. Additionally, shifts in microbiota structure often …

Biochemical And Molecular Analysis Of Anr-Dependent Microoxic Fitness Factors In Pseudomonas Aeruginosa, Stacie E. Balsam

Dartmouth College Ph.D Dissertations

Pseudomonas aeruginosa is a prominent opportunistic pathogen that is often found in microoxic environments like sites of infection and colony biofilms. P. aeruginosa can grow at oxygen (O2) at concentrations as little as 3 μM and also grows anaerobically by generating energy using nitrate as an alternate electron acceptor. An important way P. aeruginosa adapts to low O2 is through Anr, a transcription factor that upregulates many genes important for microoxic and anoxic fitness.

Specifically, Anr regulates mhr which encodes a hemerythrin protein (Mhr) that binds O2 with affinities relevant to microoxia and is also necessary for full fitness of …

Fleq And Gac/Rsm-Mediated Regulation Of Biofilm Formation And Swarming Motility In Pseudomonas Fluorescens, Alexander Benito Pastora

Dartmouth College Ph.D Dissertations

Pseudomonas fluorescens is a gram-negative Gammaproteobacterium that survives and persists in diverse environments. This survivability is dependent on the bacterium’s ability to form a biofilm. Within Pseudomonas fluorescens, biofilm formation is a complex and dynamic process involving multiple protein- and polysaccharide-based components which each individually serve a specific purpose. Of these components, the repeats-in-toxin (RTX) adhesins LapA and MapA are critical for biofilm formation. Specifically, LapA is important for initial establishment of a biofilm and MapA is important in forming a fully mature biofilm. Deployment of the RTX adhesins during biofilm formation is a highly coordinated process involving multiple …

Social Interactions And Spatial Structure Drive Community Assembly Of Bacterial Biofilm, Matthew C. Bond

Dartmouth College Ph.D Dissertations

Despite the critical nature which microbial communities play in the natural world and human civilization, the breadth of understanding remains shallow. Challenged by scale, high variability between environments, and extensive diversity, microbial ecologists strive to understand connections between a community’s structure and function, as well as the ecological and evolutionary mechanisms underlying observed natural patterning of communities. Biofilms are the predominate mode of growth for microbial communities—characterized by cellular attachment to a surface via a self-produced matrix and heterogeneous structure, often resulting in a primary growth front along the biofilm surface due to differential access to bulk nutrients (cite). This …

Causes And Consequences Of Lasr Mutant Selection In Pseudomonas Aeruginosa Populations, Dallas L. Mould

Dartmouth College Ph.D Dissertations

Change is the only constant in life, and these changes, though random in nature, can have consequences. Quorum sensing is heterogeneous in phenotype and prone to negative selection. In P. aeruginosa, the regulator LasR is frequently non-functional in phylogenetically diverse isolates. Through repeated experimental evolution and mathematical modeling, we show that differences in growth enable lasR mutant evolutionary success and this requires a system enabling metabolic choices, known as carbon catabolite repression (or catabolite repression). The differences in catabolite repression between wild type and lasR mutants enable altered metabolite preferences, and the resulting differences in metabolic states enable intraspecies …

Both Adhe And A Separate Nadph-Dependent Alcohol Dehydrogenase Gene, Adha, Are Necessary For High Ethanol Production In Thermoanaerobacterium Saccharolyticum, Tianyong Zheng, Daniel G. Olson, Sean J. Murphy, Xiongjun Shao, Liang Tian, Lee Lynd

Dartmouth Scholarship

Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at ∼90% theoretical yield and titer of 70 g/L. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenases except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked ADH activity (acetaldehyde-reducing) by …

The Vibrio Cholerae Minor Pilin Tcpb Initiates Assembly And Retraction Of The Toxin-Coregulated Pilus, Dixon Ng, Tony Harn, Tuba Altindal, Subramania Kolappan, Jarrad Marles, Rajan Lala, Ingrid Spielman, Yang Gao, Caitlyn Hauke, Gabriela Kovacikova

Dartmouth Scholarship

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent …

Presence Of The Cyanotoxin Microcystin In Arctic Lakes Of Southwestern Greenland, Jessica Trout-Haney, Zachary Wood, Kathryn Cottingham

Dartmouth Scholarship

Cyanobacteria and their toxins have received significant attention in eutrophic temperate and tropical systems where conspicuous blooms of certain planktonic taxa release toxins into fresh water, threatening its potability and safe use for recreation. Although toxigenic cyanobacteria are not confined to high nutrient environments, bloom-forming species, or planktonic taxa, these other situations are studied les often studied. For example, toxin production in picoplankton and benthic cyanobacteria—the predominant photoautotrophs found in polar lakes—is poorly understood. We quantified the occurrence of microcystin (MC, a hepatotoxic cyanotoxin) across 18 Arctic lakes in southwestern Greenland. All of the focal lakes contained detectable levels of …

The Pseudomonas Aeruginosa Efflux Pump Mexghi-Opmd Transports A Natural Phenazine That Controls Gene Expression And Biofilm Development, Hassan Sakhtah, Leslie Koyama, Yihan Zhang, Diana K. Morales, Blanche Fields, Alexa Price-Whelan, Deborah Hogan

Dartmouth Scholarship

Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We …

Cyclic Di-Gmp-Mediated Repression Of Swarming Motility By Pseudomonas Aeruginosa Pa14 Requires The Motab Stator, S. L. Kuchma, N. J. Delalez, L. M. Filkins, E. A. Snavely, J. P. Armitage, G. A. O'Toole

Dartmouth Scholarship

The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, …

Deletion Of Nfnab In Thermoanaerobacterium Saccharolyticum And Its Effect On Metabolism, Jonathan Lo, Tianyong Zheng, Daniel G. Olson, Natalie Ruppertsberger, Shital Tripathi, Adam Guss, Lee Lynd

Dartmouth Scholarship

NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP⁺. The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H₂ formation but …

Cofactor Specificity Of The Bifunctional Alcohol And Aldehyde Dehydrogenase (Adhe) In Wild-Type And Mutant Clostridium Thermocellum And Thermoanaerobacterium Saccharolyticum, Tianyong Zheng, Daniel G. Olson, Liang Tian, Yannick J. Bomble, Michael Himmel, Jonathan Lo, Shuen Hon, A. Joe Shaw, Johannes P. Van Dijken, Lee Lynd

Dartmouth Scholarship

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. …

The Bifunctional Alcohol And Aldehyde Dehydrogenase Gene, Adhe, Is Necessary For Ethanol Production In Clostridium Thermocellum And Thermoanaerobacterium Saccharolyticum, Jonathan Lo, Tianyong Zheng, Shuen Hon, Daniel G. Olson, Lee Lynd

Dartmouth Scholarship

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol …

Structural Features Of The Pseudomonas Fluorescens Biofilm Adhesin Lapa Required For Lapg-Dependent Cleavage, Biofilm Formation, And Cell Surface Localization, Chelsea D. Boyd, T. Jarrod Smith, Sofiane El-Kirat-Chatel, Peter D. Newell, Yves F. Dufrêne, George A. O'Toole

Dartmouth Scholarship

The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for …

Role Of The Cipa Scaffoldin Protein In Cellulose Solubilization, As Determined By Targeted Gene Deletion And Complementation In Clostridium Thermocellum, Daniel G. Olson, Richard J. Giannone, Robert L. Hettich, Lee R. Lynd

Dartmouth Scholarship

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In …

Secretion Of Tcpf By The Vibrio Cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires An N-Terminal Determinant, Christina J. Megli, Ronald K. Taylor

Dartmouth Scholarship

Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81–92, 2003). This phenomenon is not unique to V. cholerae; secreted …

Characterization Of Brer Interaction With The Bile Response Promoters Breab And Brer In Vibrio Cholerae, Francisca A. Cerda-Maira, Gabriela Kovacikova, Brooke A. Jude, Karen Skorupski, Ronald Taylor

Dartmouth Scholarship

The Vibrio cholerae BreR protein is a transcriptional repressor of the breAB efflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol. 190:7441-7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at the breAB promoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in the breAB promoter. However, only one of …

The Crispr/Cas Adaptive Immune System Of Pseudomonas Aeruginosa Mediates Resistance To Naturally Occurring And Engineered Phages, Kyle C. Cady, Joe Bondy-Denomy, Gary E. Heussler, Alan R. Davidson, George A. O'Toole

Dartmouth Scholarship

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer …

Minor Pilins Of The Type Iv Pilus System Participate In The Negative Regulation Of Swarming Motility, S L. Kuchma, E. F. Griffin, G. A. O'Toole

Dartmouth Scholarship

Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming …

Epoxide-Mediated Cifr Repression Of Cif Gene Expression Utilizes Two Binding Sites In Pseudomonas Aeruginosa, Alicia E. Ballok, Christopher D. Bahl, Emily L. Dolben, Allia K. Lindsay, Jessica D. St. Laurent, Deborah Hogan, Dean Madden, George A. O'Toole

Dartmouth Scholarship

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR …

Lapg, Required For Modulating Biofilm Formation By Pseudomonas Fluorescens Pf0-1, Is A Calcium-Dependent Protease, Chelsea D. Boyd, Debashree Chatterjee, Holger Sondermann, George A. O'Toole

Dartmouth Scholarship

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro …

Structural Characterization Of A Conserved, Calcium-Dependent Periplasmic Protease From Legionella Pneumophila, Debashree Chatterjee, Chelsea D. Boyd, George A. O'Toole, Holger Sondermann

Dartmouth Scholarship

The bacterial dinucleotide second messenger c-di-GMP has emerged as a central molecule in regulating bacterial behavior, including motility and biofilm formation. Proteins for the synthesis and degradation of c-di-GMP and effectors for its signal transmission are widely used in the bacterial domain. Previous work established the GGDEF-EAL domain-containing receptor LapD as a central switch in Pseudomonas fluorescens cell adhesion. LapD senses c-di-GMP inside the cytosol and relays this signal to the outside by the differential recruitment of the periplasmic protease LapG. Here we identify the core components of an orthologous system in Legionella pneumophila. Despite only moderate sequence conservation at …

2-Heptyl-4-Quinolone, A Precursor Of The Pseudomonas Quinolone Signal Molecule, Modulates Swarming Motility In Pseudomonas Aeruginosa, Dae-Gon Ha, Judith H. Merritt, Thomas H. Hampton, James T. Hodgkinson, Matej Janecek, David R. Spring, Martin Welch, George A. O'Toole

Dartmouth Scholarship

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors, including biofilm formation and swarming motility. These group behaviors are regulated by both the intracellular signaling molecule c-di-GMP and acylhomoserine lactone quorum-sensing systems. Here, we show that the Pseudomonas quinolone signal (PQS) system also contributes to the regulation of swarming motility. Specifically, our data indicate that 2-heptyl-4-quinolone (HHQ), a precursor of PQS, likely induces the production of the phenazine-1-carboxylic acid (PCA), which in turn acts via an as-yet-unknown downstream mechanism to repress swarming motility. We show that this HHQ- and PCA-dependent swarming repression is apparently independent of changes in global …

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 …

Protection And Attachment Of Vibrio Cholerae Mediated By The Toxin-Coregulated Pilus In The Infant Mouse Model, Shelly J. Krebs, Ronald K. Taylor

Dartmouth Scholarship

Colonization of the human small intestine by Vibrio cholerae is an essential step in pathogenesis that requires the type IV toxin-coregulated pilus (TCP). To date, three functions of TCP have been characterized: it serves as the CTXΦ receptor, secretes the colonization factor TcpF, and functions in microcolony formation by mediating bacterium-bacterium interactions. Although type IV pili in other pathogenic bacteria have been characterized as playing a major role in attachment to epithelial cells, there are very few studies to suggest that TCP acts as an attachment factor. Taking this into consideration, we investigated the function of TCP in attachment to …

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 …