The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform

J. Thomas Beatty - One of the best experts on this subject based on the ideXlab platform.

  • The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.
    Journal of bacteriology, 2021
    Co-Authors: Reynold G. Farrera-calderon, Christina L Wiesmann, Alexander B Westbye, Andrew S Lang, Purvikalyan Pallegar, J. Thomas Beatty
    Abstract:

    ABSTRACT Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphoTransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the Gene Transfer Agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphoTransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphoTransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphoTransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

  • The CckA-ChpT-CtrA phosphorelay controlling Rhodobacter capsulatus Gene Transfer Agent (RcGTA) production is bi-directional and regulated by cyclic-di-GMP.
    Journal of bacteriology, 2021
    Co-Authors: Reynold G. Farrera-calderon, Christina L Wiesmann, Alexander B Westbye, Andrew S Lang, Purvikalyan Pallegar, J. Thomas Beatty
    Abstract:

    Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, histidine phosphoTransferase ChpT and response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus these proteins are key regulators of the Gene Transfer Agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphoTransfer to ChpT and thence to CtrA to biochemically demonstrate that they form a phosphorelay. The secondary messenger cyclic-di-GMP changed CckA from a kinase to a phosphatase resulting in reversal of the phosphoTransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for lytic release of RcGTA. The binding of cyclic-di-GMP to the wild type and mutant CckA proteins was evaluated directly using a pull-down assay based on biotinylated cyclic-di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work biochemically demonstrates that CckA, ChpT and CtrA also form a functional phosphorelay in this latter group, and that the direction of phosphoTransfer is reversed by cyclic-di-GMP. It is important to improve the understanding of more representatives of this pathway to obtain a deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

  • Structure and mechanism of DNA delivery of a Gene Transfer Agent
    Nature communications, 2020
    Co-Authors: Pavol Bárdy, J. Thomas Beatty, Tibor Füzik, Dominik Hrebík, Roman Pantůček, Pavel Plevka
    Abstract:

    Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called Gene Transfer Agents (GTAs) that mediate lateral Gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

  • The Rhodobacter capsulatus Gene Transfer Agent is induced by nutrient depletion and the RNAP omega subunit.
    Microbiology (Reading England), 2017
    Co-Authors: Alexander B Westbye, Zoe R. O’neill, Tegan Schellenberg-beaver, J. Thomas Beatty
    Abstract:

    Small bacteriophage-like particles called Gene Transfer Agents (GTAs) that mediate DNA Transfer between cells are produced by a variety of prokaryotes. The model GTA, produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), is controlled by several cellular regulators, and production is induced upon entry into the stationary phase. We report that RcGTA production and Gene Transfer are stimulated by nutrient depletion. Cells depleted of organic carbon or blocked for amino acid biosynthesis increased RcGTA production and release from cells. Furthermore, cells lacking the sole RelA-SpoT homologue produced decreased levels of RcGTA, and the RNA polymerase omega (ω) subunit was required for appreciable production of RcGTA.

  • Guaranteeing a captive audience: coordinated regulation of Gene Transfer Agent (GTA) production and recipient capability by cellular regulators.
    Current opinion in microbiology, 2017
    Co-Authors: Alexander B Westbye, J. Thomas Beatty, Andrew S Lang
    Abstract:

    Gene Transfer Agents (GTAs) are bacteriophage-like particles produced by many prokaryotes. Several members of the Alphaproteobacteria produce a class of Genetically-related GTAs that is best studied in Rhodobacter capsulatus. DNA Transfer by the R. capsulatus GTA (RcGTA) combines aspects of both transduction and natural transformation, as recipient cells require a natural transformation-like system to incorporate donated DNA. The Genes involved in RcGTA production and recipient capability are located at multiple loci in the bacterial genome; however, a conserved phosphorelay containing the response regulator CtrA and a quorum sensing system regulate both RcGTA production and recipient capability. This review highlights recent discoveries in RcGTA biology, and focuses on the co-regulation of Genes involved in RcGTA production and recipient capability.

Andrew S Lang - One of the best experts on this subject based on the ideXlab platform.

  • The CckA-ChpT-CtrA phosphorelay controlling Rhodobacter capsulatus Gene Transfer Agent (RcGTA) production is bi-directional and regulated by cyclic-di-GMP.
    Journal of bacteriology, 2021
    Co-Authors: Reynold G. Farrera-calderon, Christina L Wiesmann, Alexander B Westbye, Andrew S Lang, Purvikalyan Pallegar, J. Thomas Beatty
    Abstract:

    Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, histidine phosphoTransferase ChpT and response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus these proteins are key regulators of the Gene Transfer Agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphoTransfer to ChpT and thence to CtrA to biochemically demonstrate that they form a phosphorelay. The secondary messenger cyclic-di-GMP changed CckA from a kinase to a phosphatase resulting in reversal of the phosphoTransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for lytic release of RcGTA. The binding of cyclic-di-GMP to the wild type and mutant CckA proteins was evaluated directly using a pull-down assay based on biotinylated cyclic-di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work biochemically demonstrates that CckA, ChpT and CtrA also form a functional phosphorelay in this latter group, and that the direction of phosphoTransfer is reversed by cyclic-di-GMP. It is important to improve the understanding of more representatives of this pathway to obtain a deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

  • The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.
    Journal of bacteriology, 2021
    Co-Authors: Reynold G. Farrera-calderon, Christina L Wiesmann, Alexander B Westbye, Andrew S Lang, Purvikalyan Pallegar, J. Thomas Beatty
    Abstract:

    ABSTRACT Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphoTransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the Gene Transfer Agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphoTransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphoTransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphoTransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

  • induction of rhodobacter capsulatus Gene Transfer Agent Gene expression is a bistable stochastic process repressed by an extracellular calcium binding rtx protein homologue
    Journal of Bacteriology, 2019
    Co-Authors: Hao Ding, Marc P Grull, Martin E. Mulligan, Andrew S Lang, Thomas J Beatty
    Abstract:

    ABSTRACT Bacteriophage-like Gene Transfer Agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phyloGenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in IMPORTANCE GTAs catalyze horizontal Gene Transfer (HGT), which is important for genomic evolution because the majority of Genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of Gene Transfer and thereby specify the tempo of evolution. This work describes a new type of Genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.

  • Guaranteeing a captive audience: coordinated regulation of Gene Transfer Agent (GTA) production and recipient capability by cellular regulators.
    Current opinion in microbiology, 2017
    Co-Authors: Alexander B Westbye, J. Thomas Beatty, Andrew S Lang
    Abstract:

    Gene Transfer Agents (GTAs) are bacteriophage-like particles produced by many prokaryotes. Several members of the Alphaproteobacteria produce a class of Genetically-related GTAs that is best studied in Rhodobacter capsulatus. DNA Transfer by the R. capsulatus GTA (RcGTA) combines aspects of both transduction and natural transformation, as recipient cells require a natural transformation-like system to incorporate donated DNA. The Genes involved in RcGTA production and recipient capability are located at multiple loci in the bacterial genome; however, a conserved phosphorelay containing the response regulator CtrA and a quorum sensing system regulate both RcGTA production and recipient capability. This review highlights recent discoveries in RcGTA biology, and focuses on the co-regulation of Genes involved in RcGTA production and recipient capability.

  • Identification of a predicted partner-switching system that affects production of the Gene Transfer Agent RcGTA and stationary phase viability in Rhodobacter capsulatus
    BMC microbiology, 2014
    Co-Authors: Ryan G. Mercer, Andrew S Lang
    Abstract:

    Background: Production of the Gene Transfer Agent RcGTA in the α-proteobacterium Rhodobacter capsulatus is dependent upon the response regulator protein CtrA. Loss of this regulator has widespread effects on transcription in R. capsulatus, including the dysregulation of numerous Genes encoding other predicted regulators. This includes a set of putative components of a partner-switching signaling pathway with sequence homology to the σ-regulating proteins RsbV, RsbW, and RsbY that have been extensively characterized for their role in stress responses in gram-positive bacteria. These R. capsulatus homologues, RbaV, RbaW, and RbaY, have been investigated for their possible role in controlling RcGTA Gene expression. Results: A mutant strain lacking rbaW showed a significant increase in RcGTA Gene expression and production. Mutation of rbaV or rbaY led to a decrease in RcGTA Gene expression and production, and these mutants also showed decreased viability in the stationary pha se and produced unusual colony morphologies.In vitro and in vivo protein interaction assays demonstrated that RbaW and RbaV interact. A combination of Gene disruptions and protein-protein interaction assays were unsuccessful in attempts to identify a cognate σ factor, and the Genetic data support a model where the RbaV protein that is the determinant regulator of RcGTA Gene expression in this system. Conclusions: These findings provide new information about RcGTA regulation by a putative partner-switching system and further illustrate the integration of RcGTA production into R. capsulatus physiology.

Thomas J Beatty - One of the best experts on this subject based on the ideXlab platform.

  • induction of rhodobacter capsulatus Gene Transfer Agent Gene expression is a bistable stochastic process repressed by an extracellular calcium binding rtx protein homologue
    Journal of Bacteriology, 2019
    Co-Authors: Hao Ding, Marc P Grull, Martin E. Mulligan, Andrew S Lang, Thomas J Beatty
    Abstract:

    ABSTRACT Bacteriophage-like Gene Transfer Agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phyloGenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in IMPORTANCE GTAs catalyze horizontal Gene Transfer (HGT), which is important for genomic evolution because the majority of Genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of Gene Transfer and thereby specify the tempo of evolution. This work describes a new type of Genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.

  • the protease clpxp and the pas domain protein divl regulate ctra and Gene Transfer Agent production in rhodobacter capsulatus
    Applied and Environmental Microbiology, 2018
    Co-Authors: Alexander B Westbye, Christina L Wiesmann, Lukas Kater, Hao Ding, Thomas J Beatty
    Abstract:

    ABSTRACT Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal Gene Transfer vector, called the Gene Transfer Agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus. We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Rhodobacterales. IMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal Gene Transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales. We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

  • the Gene Transfer Agent rcgta contains head spikes needed for binding to the rhodobacter capsulatus polysaccharide cell capsule
    Journal of Molecular Biology, 2016
    Co-Authors: Alexander B Westbye, Kevin Kuchinski, Thomas J Beatty
    Abstract:

    Abstract Viruses and bacteriophages recognize cell surface proteins using receptor-binding proteins. In most tailed bacteriophages, receptor-binding proteins are located on the bacteriophage tail. The Gene Transfer Agent of Rhodobacter capsulatus , RcGTA, morphologically resembles a tailed bacteriophage and binds to a capsular polysaccharide covering R . capsulatus cells. Here, we report that the RcGTA capsid (head) is decorated by spikes that are needed for binding to the capsule. The triangular spikes measured ~ 12 nm and appeared to be attached at the capsid vertices. Head spike production required the putative carbohydrate-binding protein ghsB ( rcc01080 ) previously thought to encode a side tail fiber protein. We found that ghsB is likely co-transcribed with ghsA ( rcc01079 ) and that ghsA/ghsB is regulated by the CckA-ChpT-CtrA phosphorelay homologues and a quorum-sensing system. GhsA and GhsB were found to be CckA-dependent RcGTA maturation factors, as GhsA- and GhsB-deficient particles were found to have altered native-gel electrophoresis migration. Additionally, we provide electron microscopy images showing that RcGTA contains side tail fibers and a baseplate-like structure near the tip of the tail, which are independent of ghsB .

  • homologues of Genetic transformation dna import Genes are required for rhodobacter capsulatus Gene Transfer Agent recipient capability regulated by the response regulator ctra
    Journal of Bacteriology, 2015
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Jeanette A. Johnson, Thomas J Beatty
    Abstract:

    Gene Transfer Agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and Transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three Genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated Gene acquisition. This paper gives Genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and Genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for Gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated Gene Transfer combines key aspects of two bacterial horizontal Gene Transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCE Horizontal Gene Transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be Transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key Genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.

  • rhodobacter capsulatus dpra is essential for reca mediated Gene Transfer Agent rcgta recipient capability regulated by quorum sensing and the ctra response regulator
    Molecular Microbiology, 2014
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Thomas J Beatty
    Abstract:

    Gene Transfer Agents (GTAs) are Genetic exchange elements that resemble small DNA bacteriophages that Transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated Gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated Gene acquisition, and that dprA expression is induced by gtaI-dependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.

Cedric A. Brimacombe - One of the best experts on this subject based on the ideXlab platform.

  • The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA
    Journal of bacteriology, 2016
    Co-Authors: Kevin Kuchinski, Alexander B Westbye, Hao Ding, Cedric A. Brimacombe, J. Thomas Beatty
    Abstract:

    ABSTRACT The Gene Transfer Agent of Rhodobacter capsulatus (RcGTA) is a Genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation ( 5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. IMPORTANCE This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like Genetic exchange carried out by the R. capsulatus Gene Transfer Agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne Genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA Gene expression differs radically.

  • homologues of Genetic transformation dna import Genes are required for rhodobacter capsulatus Gene Transfer Agent recipient capability regulated by the response regulator ctra
    Journal of Bacteriology, 2015
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Jeanette A. Johnson, Thomas J Beatty
    Abstract:

    Gene Transfer Agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and Transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three Genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated Gene acquisition. This paper gives Genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and Genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for Gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated Gene Transfer combines key aspects of two bacterial horizontal Gene Transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCE Horizontal Gene Transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be Transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key Genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.

  • Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA
    Journal of bacteriology, 2015
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Jeanette A. Johnson, J. Thomas Beatty
    Abstract:

    Gene Transfer Agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and Transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three Genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated Gene acquisition. This paper gives Genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and Genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in

  • rhodobacter capsulatus dpra is essential for reca mediated Gene Transfer Agent rcgta recipient capability regulated by quorum sensing and the ctra response regulator
    Molecular Microbiology, 2014
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Thomas J Beatty
    Abstract:

    Gene Transfer Agents (GTAs) are Genetic exchange elements that resemble small DNA bacteriophages that Transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated Gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated Gene acquisition, and that dprA expression is induced by gtaI-dependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.

  • Rhodobacter capsulatus DprA is essential for RecA‐mediated Gene Transfer Agent (RcGTA) recipient capability regulated by quorum‐sensing and the CtrA response regulator
    Molecular microbiology, 2014
    Co-Authors: Cedric A. Brimacombe, Hao Ding, J. Thomas Beatty
    Abstract:

    Gene Transfer Agents (GTAs) are Genetic exchange elements that resemble small DNA bacteriophages that Transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated Gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated Gene acquisition, and that dprA expression is induced by gtaI-dependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.

Hao Ding - One of the best experts on this subject based on the ideXlab platform.

  • induction of rhodobacter capsulatus Gene Transfer Agent Gene expression is a bistable stochastic process repressed by an extracellular calcium binding rtx protein homologue
    Journal of Bacteriology, 2019
    Co-Authors: Hao Ding, Marc P Grull, Martin E. Mulligan, Andrew S Lang, Thomas J Beatty
    Abstract:

    ABSTRACT Bacteriophage-like Gene Transfer Agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phyloGenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in IMPORTANCE GTAs catalyze horizontal Gene Transfer (HGT), which is important for genomic evolution because the majority of Genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of Gene Transfer and thereby specify the tempo of evolution. This work describes a new type of Genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.

  • the protease clpxp and the pas domain protein divl regulate ctra and Gene Transfer Agent production in rhodobacter capsulatus
    Applied and Environmental Microbiology, 2018
    Co-Authors: Alexander B Westbye, Christina L Wiesmann, Lukas Kater, Hao Ding, Thomas J Beatty
    Abstract:

    ABSTRACT Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal Gene Transfer vector, called the Gene Transfer Agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus. We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Rhodobacterales. IMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal Gene Transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales. We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

  • The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA
    Journal of bacteriology, 2016
    Co-Authors: Kevin Kuchinski, Alexander B Westbye, Hao Ding, Cedric A. Brimacombe, J. Thomas Beatty
    Abstract:

    ABSTRACT The Gene Transfer Agent of Rhodobacter capsulatus (RcGTA) is a Genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation ( 5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. IMPORTANCE This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like Genetic exchange carried out by the R. capsulatus Gene Transfer Agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne Genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA Gene expression differs radically.

  • homologues of Genetic transformation dna import Genes are required for rhodobacter capsulatus Gene Transfer Agent recipient capability regulated by the response regulator ctra
    Journal of Bacteriology, 2015
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Jeanette A. Johnson, Thomas J Beatty
    Abstract:

    Gene Transfer Agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and Transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three Genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated Gene acquisition. This paper gives Genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and Genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for Gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated Gene Transfer combines key aspects of two bacterial horizontal Gene Transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCE Horizontal Gene Transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be Transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key Genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.

  • Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA
    Journal of bacteriology, 2015
    Co-Authors: Cedric A. Brimacombe, Hao Ding, Jeanette A. Johnson, J. Thomas Beatty
    Abstract:

    Gene Transfer Agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and Transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three Genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated Gene acquisition. This paper gives Genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and Genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in