scholarly journals Rhodobacter capsulatus Gene Transfer Agent Transduction Assay

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (4) ◽  
Author(s):  
Molly Leung ◽  
John Beatty
Keyword(s):  
Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Gene Transfer Agent

scholarly journals The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production inRhodobacter capsulatus

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Alexander B. Westbye ◽  
Lukas Kater ◽  
Christina Wiesmann ◽  
Hao Ding ◽  
Calvin K. Yip ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Pas Domain ◽  
Content Type ◽  
Regulatory Systems ◽  
Gene Transfer Agent ◽  
Domain Protein

ABSTRACTSeveral members of theRhodobacterales(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 theRhodobacter capsulatusGTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in theAlphaproteobacteriaand 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 inR. capsulatus. We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels inR. capsulatusand 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 theRhodobacterales.IMPORTANCEMembers of theRhodobacteralesare abundant in ocean and freshwater environments. The conserved GTA produced by manyRhodobacteralesmay 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 inR. capsulatusextends the model of CtrA regulation fromCaulobacter crescentusto a member of theRhodobacterales. 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 of Rhodobacter capsulatus

2010 ◽  
pp. 253-264 ◽  
Author(s):  
Molly M. Leung ◽  
Sarah M. Florizone ◽  
Terumi A. Taylor ◽  
Andrew S. Lang ◽  
J. Thomas Beatty
Keyword(s):  
Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Gene Transfer Agent

scholarly journals Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA

2019 ◽  
Vol 93 (23) ◽  
Author(s):  
D. Sherlock ◽  
J. X. Leong ◽  
P. C. M. Fogg
Keyword(s):  
Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Bacterial Genome ◽  
Structural Basis ◽  
Content Type ◽  
Double Stranded Dna ◽  
Antimicrobial Resistance Genes ◽  
Gene Transfer Agent ◽  
Primary Driver ◽  
Apparent Similarity

ABSTRACT Genetic exchange mediated by viruses of bacteria (bacteriophages) is the primary driver of rapid bacterial evolution. The priority of viruses is usually to propagate themselves. Most bacteriophages use the small terminase protein to identify their own genome and direct its inclusion into phage capsids. Gene transfer agents (GTAs) are descended from bacteriophages, but they instead package fragments of the entire bacterial genome without preference for their own genes. GTAs do not selectively target specific DNA, and no GTA small terminases are known. Here, we identified the small terminase from the model Rhodobacter capsulatus GTA, which then allowed prediction of analogues in other species. We examined the role of the small terminase in GTA production and propose a structural basis for random DNA packaging. IMPORTANCE Random transfer of any and all genes between bacteria could be influential in the spread of virulence or antimicrobial resistance genes. Discovery of the true prevalence of GTAs in sequenced genomes is hampered by their apparent similarity to bacteriophages. Our data allowed the prediction of small terminases in diverse GTA producer species, and defining the characteristics of a “GTA-type” terminase could be an important step toward novel GTA identification. Importantly, the GTA small terminase shares many features with its phage counterpart. We propose that the GTA terminase complex could become a streamlined model system to answer fundamental questions about double-stranded DNA (dsDNA) packaging by viruses that have not been forthcoming to date.

scholarly journals The CckA-ChpT-CtrA phosphorelay controlling Rhodobacter capsulatus gene transfer agent (RcGTA) production is bi-directional and regulated by cyclic-di-GMP

2020 ◽  
Author(s):  
Reynold G. Farrera-Calderon ◽  
Purvikalyan Pallegar ◽  
Alexander B. Westbye ◽  
Christina Wiesmann ◽  
Andrew S. Lang ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Phosphatase Activity ◽  
Regulatory Networks ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Evolutionary Significance ◽  
Secondary Messenger ◽  
Gene Transfer Agent

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.

scholarly journals Long-Chain Acyl-Homoserine Lactone Quorum-Sensing Regulation of Rhodobacter capsulatus Gene Transfer Agent Production

2002 ◽  
Vol 184 (23) ◽  
pp. 6515-6521 ◽  
Author(s):  
Amy L. Schaefer ◽  
Terumi A. Taylor ◽  
J. Thomas Beatty ◽  
E. P. Greenberg
Keyword(s):  
Gene Transfer ◽  
Quorum Sensing ◽  
Rhodobacter Capsulatus ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Homoserine Lactones ◽  
Acyl Homoserine Lactones ◽  
Chain Acyl ◽  
Gene Transfer Agent ◽  
Long Chain

ABSTRACT Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone. The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An R. capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named gtaI, resulted in decreased production of the R. capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R. capsulatus.

scholarly journals Regulation of Rhodobacter capsulatus gene transfer agent production

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Ian Duncan ◽  
David Sherlock ◽  
Paul Fogg
Keyword(s):  
Gene Transfer ◽  
Antimicrobial Resistance ◽  
Rhodobacter Capsulatus ◽  
Fluorescent Protein ◽  
Recipient Cell ◽  
Cell Contact ◽  
Structural Genes ◽  
Antimicrobial Resistance Genes ◽  
Gene Transfer Agent ◽  
A Cell

Horizontal gene transfer (HGT) enables the spread of antimicrobial resistance, virulence, metabolic and other genes conferring an advantage to the organism. HGT is enhanced in biofilms because of increased cell-cell contact (conjugation), and eDNA in the biofilm matrix causing development of competence and providing material for transformation. Production of the Rhodobacter capsulatus gene transfer agent (RcGTA), another mechanism of HGT, could also increase in biofilm as high cell density increases the proportion of GTA particles produced that encounter a target cell. RcGTA is a phage-like particle that packages ∼4.5 kb pieces of random DNA from the producing cell’s genome and transfers it to a recipient cell. Five loci comprise the RcGTA genome: a 15kb cluster containing most of the RcGTA structural genes, a cell lysis locus, two structural loci encoding head spikes and tail fibres, and a maturation/regulation locus that includes that master regulator, gafA. I assayed RcGTA production using gene transfer bioassays and biofilm using a 96 well plate assay. I will present data showing that deletion of four GTA-related genes, including gafA itself, all lead to reduced biofilm production. All four gene knock-outs also strongly reduce GTA-mediated gene transfer, suggesting GTA production and biofilm are co-regulated. I will also present work to characterize GTA production in biofilms, for example by monitoring the transfer of fluorescent protein genes through confocal microscopy, and assessing how specific regulators control this process. Biofilms are ubiquitous in the environment so studying the spread of antimicrobial resistance genes by GTAs is important.

scholarly journals The ATP Synthase atpHAGDC(F1) Operon from Rhodobacter capsulatus

1998 ◽  
Vol 180 (2) ◽  
pp. 416-421 ◽  
Author(s):  
Roberto Borghese ◽  
Massimo Crimi ◽  
Luca Fava ◽  
Bruno Andrea Melandri
Keyword(s):  
Gene Transfer ◽  
Atp Synthase ◽  
Promoter Region ◽  
Rhodobacter Capsulatus ◽  
Growth Conditions ◽  
Primer Extension ◽  
Primer Extension Analysis ◽  
Viable Cells ◽  
Gene Transfer Agent ◽  
Extension Analysis

ABSTRACT The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F1 sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carryingatp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.

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