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 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.

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 Identification of Genes of VSH-1, a Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae

2005 ◽  
Vol 187 (17) ◽  
pp. 5885-5892 ◽  
Author(s):  
Eric G. Matson ◽  
M. Greg Thompson ◽  
Samuel B. Humphrey ◽  
Richard L. Zuerner ◽  
Thad B. Stanton
Keyword(s):  
Amino Acid ◽  
Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Bacterial Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bacterial Genome ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Brachyspira Hyodysenteriae ◽  
Gene Transfer Agent

ABSTRACT VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A λ clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

scholarly journals The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA

2016 ◽  
Vol 198 (7) ◽  
pp. 1137-1148 ◽  
Author(s):  
Kevin S. Kuchinski ◽  
Cedric A. Brimacombe ◽  
Alexander B. Westbye ◽  
Hao Ding ◽  
J. Thomas Beatty
Keyword(s):  
Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Natural Transformation ◽  
Regulatory Gene ◽  
Evolutionary Relationship ◽  
Sos Response ◽  
Genetic Exchange ◽  
Master Regulator ◽  
Content Type ◽  
Gene Transfer Agent

ABSTRACTThe gene transfer agent ofRhodobacter 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 (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surroundingR. capsulatuscells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of thelexAgene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in theR. capsulatusgenome sequence identified a number of putative binding sites located 5′ of typical SOS response coding sequences and also 5′ of the RcGTA regulatory genecckA, which encodes a hybrid histidine kinase homolog. Expression ofcckAwas increased >5-fold in thelexAmutant, and alexA cckAdouble mutant was found to have the same phenotype as a ΔcckAsingle 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 thelexAmutant is largely due to the overexpression ofcckA.IMPORTANCEThis work describes an unusual phenotype of alexAmutant of the alphaproteobacteriumRhodobacter capsulatusin respect to the phage transduction-like genetic exchange carried out by theR. capsulatusgene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, thislexAmutation 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.

scholarly journals Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA

2015 ◽  
Vol 197 (16) ◽  
pp. 2653-2663 ◽  
Author(s):  
Cedric A. Brimacombe ◽  
Hao Ding ◽  
Jeanette A. Johnson ◽  
J. Thomas Beatty
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Natural Transformation ◽  
Response Regulator ◽  
Content Type ◽  
Gene Acquisition ◽  
Metabolic Properties ◽  
Cell Genome ◽  
Key Aspects

ABSTRACTGene 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 ofRhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria,comEC,comF, andcomM, 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.IMPORTANCEHorizontal 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 theR. capsulatusRcGTA 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.

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 Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Ville Hoikkala ◽  
Janne Ravantti ◽  
César Díez-Villaseñor ◽  
Marja Tiirola ◽  
Rachel A. Conrad ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Flavobacterium Columnare ◽  
Content Type ◽  
Immune Systems ◽  
Dna Cleaving ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Rna Targeting ◽  
Microbial Strains ◽  
Target Rna

ABSTRACT CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system “borrows” acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.

Sign in / Sign up

Export Citation Format

Share Document