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.

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 A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression.

1991 ◽  
Vol 11 (4) ◽  
pp. 1901-1911 ◽  
Author(s):  
J R Schultz ◽  
T Tansey ◽  
L Gremke ◽  
R V Storti
Keyword(s):  
Gene Expression ◽  
Flight Muscle ◽  
Primer Extension ◽  
Primer Extension Analysis ◽  
P Element ◽  
Indirect Flight Muscle ◽  
Mrna Levels ◽  
Enhancer Element ◽  
Mutant Phenotypes ◽  
Extension Analysis

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.

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.

Primer Extension Analysis of mRNA

2003 ◽  
pp. 195-199
Author(s):  
Maggie Walmsley ◽  
Mark Leonard ◽  
Roger Patient
Keyword(s):  
Primer Extension ◽  
Primer Extension Analysis ◽  
Extension Analysis
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