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 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 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.

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 An Evolutionary Link between Natural Transformation and CRISPR Adaptive Immunity

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Peter Jorth ◽  
Marvin Whiteley
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Bacterial Diversity ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Comparative Genomic ◽  
Content Type ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Adaptive Immune Systems

ABSTRACTNatural transformation by competent bacteria is a primary means of horizontal gene transfer; however, evidence that competence drives bacterial diversity and evolution has remained elusive. To test this theory, we used a retrospective comparative genomic approach to analyze the evolutionary history ofAggregatibacter actinomycetemcomitans, a bacterial species with both competent and noncompetent sister strains. Through comparative genomic analyses, we reveal that competence is evolutionarily linked to genomic diversity and speciation. Competence loss occurs frequently during evolution and is followed by the loss of clustered regularly interspaced short palindromic repeats (CRISPRs), bacterial adaptive immune systems that protect against parasitic DNA. Relative to noncompetent strains, competent bacteria have larger genomes containing multiple rearrangements. In contrast, noncompetent bacterial genomes are extremely stable but paradoxically susceptible to infective DNA elements, which contribute to noncompetent strain genetic diversity. Moreover, incomplete noncompetent strain CRISPR immune systems are enriched for self-targeting elements, which suggests that the CRISPRs have been co-opted for bacterial gene regulation, similar to eukaryotic microRNAs derived from the antiviral RNA interference pathway.IMPORTANCEThe human microbiome is rich with thousands of diverse bacterial species. One mechanism driving this diversity is horizontal gene transfer by natural transformation, whereby naturally competent bacteria take up environmental DNA and incorporate new genes into their genomes. Competence is theorized to accelerate evolution; however, attempts to test this theory have proved difficult. Through genetic analyses of the human periodontal pathogenAggregatibacter actinomycetemcomitans, we have discovered an evolutionary connection between competence systems promoting gene acquisition and CRISPRs (clustered regularly interspaced short palindromic repeats), adaptive immune systems that protect bacteria against genetic parasites. We show that competentA. actinomycetemcomitansstrains have numerous redundant CRISPR immune systems, while noncompetent bacteria have lost their CRISPR immune systems because of inactivating mutations. Together, the evolutionary data linking the evolution of competence and CRISPRs reveals unique mechanisms promoting genetic heterogeneity and the rise of new bacterial species, providing insight into complex mechanisms underlying bacterial diversity in the human body.

scholarly journals Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Léo Hardy ◽  
Pierre-Alexandre Juan ◽  
Bénédicte Coupat-Goutaland ◽  
Xavier Charpentier
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Genetic Basis ◽  
Natural Transformation ◽  
Essential Genes ◽  
Content Type ◽  
Transposon Insertion ◽  
Life Traits ◽  
Transposon Insertion Sequencing

ABSTRACT Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdom horizontal gene transfer events. Transposon insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, which are possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of the difficulty of obtaining a high-saturation transposon insertion library. Indeed, we found that isolates of sequence type 1 (ST1), which includes the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we then used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. IMPORTANCE Legionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. The environmental life traits of L. pneumophila are essential to its ability to accidentally infect humans. A comprehensive identification of their genetic basis could be obtained through the use of transposon insertion sequencing. However, this powerful approach had not been fully implemented in L. pneumophila. Here, we describe the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step toward identifying the genetic basis of the many life traits of this environmental and pathogenic species.

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 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.

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