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      The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes

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          Abstract

          AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.

          Author Summary

          The bacterium Pseudomonas aeruginosa causes a variety of human infections and is the leading cause of death in patients with cystic fibrosis. The main reason for the severity of these infections arises from the ability of P. aeruginosa to express virulence factors that protect it from the host immune system. Several of these processes are controlled by a transcription factor called AmrZ, a potential target for anti-microbial therapeutics. AmrZ is unusual in that it has the ability to both activate some genes, such as for alginate biofilm, and repress others, as with flagellum and itself. Here we determine the three dimensional structure of AmrZ bound to DNA containing a repressor sequence. Our structure shows the specific interactions the protein makes with the DNA for binding and repression. It also reveals that both the sequence and shape of the DNA are important for tight association. We next examined the binding of the protein to DNA containing an activator sequence and found that it has different interactions. However, by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence in P. aeruginosa, we show that differences in binding alone do not account for transcriptional regulation.

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          Lung infections associated with cystic fibrosis.

          While originally characterized as a collection of related syndromes, cystic fibrosis (CF) is now recognized as a single disease whose diverse symptoms stem from the wide tissue distribution of the gene product that is defective in CF, the ion channel and regulator, cystic fibrosis transmembrane conductance regulator (CFTR). Defective CFTR protein impacts the function of the pancreas and alters the consistency of mucosal secretions. The latter of these effects probably plays an important role in the defective resistance of CF patients to many pathogens. As the modalities of CF research have changed over the decades from empirical histological studies to include biophysical measurements of CFTR function, the clinical management of this disease has similarly evolved to effectively address the ever-changing spectrum of CF-related infectious diseases. These factors have led to the successful management of many CF-related infections with the notable exception of chronic lung infection with the gram-negative bacterium Pseudomonas aeruginosa. The virulence of P. aeruginosa stems from multiple bacterial attributes, including antibiotic resistance, the ability to utilize quorum-sensing signals to form biofilms, the destructive potential of a multitude of its microbial toxins, and the ability to acquire a mucoid phenotype, which renders this microbe resistant to both the innate and acquired immunologic defenses of the host.
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            The role of DNA shape in protein-DNA recognition

            The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanisms: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analyzing the three dimensional structures of protein-DNA complexes, we show that the binding of arginines to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a striking example of this effect. Minor groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings suggest that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA binding specificity.
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              Gene splicing and mutagenesis by PCR-driven overlap extension.

              Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3' ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3' region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                April 2012
                April 2012
                12 April 2012
                : 8
                : 4
                : e1002648
                Affiliations
                [1 ]Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America
                [2 ]Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America
                [3 ]Departments of Microbiology and Microbial Infection and Immunity, Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio, United States of America
                Yale University School of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: EEP DJW TH. Performed the experiments: EEP EAW BX SDN. Analyzed the data: EEP EAW BX SDN DJW TH. Contributed reagents/materials/analysis tools: EEP EAW BX SDN DJW TH. Wrote the paper: EEP TH.

                Article
                PPATHOGENS-D-11-01495
                10.1371/journal.ppat.1002648
                3325190
                22511872
                ca348535-5209-4f5a-98d2-7e1916ad567d
                Pryor Jr. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 7 July 2011
                : 2 March 2012
                Page count
                Pages: 12
                Categories
                Research Article
                Biology
                Biochemistry
                Nucleic Acids
                DNA
                DNA structure
                Proteins
                Protein Structure
                Biophysics
                Macromolecular Assemblies
                Nucleic Acids
                Microbiology
                Bacterial Pathogens
                Gram Negative
                Microbial Metabolism
                Molecular Cell Biology
                Gene Expression
                DNA transcription

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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