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Characterization of the ?? subcomplex of Pseudomonas aeruginosa DNA polymerase III.

ABSTRACT: BACKGROUND: DNA polymerase III, the main enzyme responsible for bacterial DNA replication, is composed of three sub-assemblies: the polymerase core, the ?-sliding clamp, and the clamp loader. During replication, single-stranded DNA-binding protein (SSB) coats and protects single-stranded DNA (ssDNA) and also interacts with the ?? heterodimer, a sub-complex of the clamp loader. Whereas the ? subunits of Escherichia coli and Pseudomonas aeruginosa are about 40% homologous, P. aeruginosa ? is twice as large as its E. coli counterpart, and contains additional sequences. It was shown that P. aeruginosa ?? together with SSB increases the activity of its cognate clamp loader 25-fold at low salt. The E. coli clamp loader, however, is insensitive to the addition of its cognate ?? under similar conditions. In order to find out distinguishing properties within P. aeruginosa ?? which account for this higher stimulatory effect, we characterized P. aeruginosa ?? by a detailed structural and functional comparison with its E. coli counterpart. RESULTS: Using small-angle X-ray scattering, analytical ultracentrifugation, and homology-based modeling, we found the N-terminus of P. aeruginosa ? to be unstructured. Under high salt conditions, the affinity of the ?? complexes from both organisms to their cognate SSB was similar. Under low salt conditions, P. aeruginosa ??, contrary to E. coli ??, binds to ssDNA via the N-terminus of ?. Whereas it is also able to bind to double-stranded DNA, the affinity is somewhat reduced. CONCLUSIONS: The binding to DNA, otherwise never reported for any other ? protein, enhances the affinity of P. aeruginosa ?? towards the SSB/ssDNA complex and very likely contributes to the higher stimulatory effect of P. aeruginosa ?? on the clamp loader. We also observed DNA-binding activity for P. putida ??, making this activity most probably a characteristic of the ? proteins from the Pseudomonadaceae.

SUBMITTER: El Houry Mignan S

PROVIDER: S-EPMC3197488 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Characterization of the χψ subcomplex of Pseudomonas aeruginosa DNA polymerase III.

El Houry Mignan Sirine S Witte Gregor G Naue Natalie N Curth Ute U

BMC molecular biology 20110928

<h4>Background</h4>DNA polymerase III, the main enzyme responsible for bacterial DNA replication, is composed of three sub-assemblies: the polymerase core, the β-sliding clamp, and the clamp loader. During replication, single-stranded DNA-binding protein (SSB) coats and protects single-stranded DNA (ssDNA) and also interacts with the χψ heterodimer, a sub-complex of the clamp loader. Whereas the χ subunits of Escherichia coli and Pseudomonas aeruginosa are about 40% homologous, P. aeruginosa ψ i ...[more]

PMID: 21955458

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Project description:Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

Project description:UNLABELLED:Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE:Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.

Dataset Information

Characterization of the ?? subcomplex of Pseudomonas aeruginosa DNA polymerase III.

Publications

Characterization of the χψ subcomplex of Pseudomonas aeruginosa DNA polymerase III.

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