scholarly journals The Replicator of the Nopaline-Type Ti Plasmid pTiC58 Is a Member of the repABC Family and Is Influenced by the TraR-Dependent Quorum-Sensing Regulatory System

2000 ◽  
Vol 182 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Pei-Li Li ◽  
Stephen K. Farrand
Keyword(s):  
Quorum Sensing ◽  
Copy Number ◽  
Constitutive Expression ◽  
Plasmid Copy Number ◽  
Ti Plasmid ◽  
Conjugal Transfer ◽  
Plasmid Copy ◽  
Sensing System ◽  
Promoter Recognition ◽  
Quorum Sensing System

ABSTRACT The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034–1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, andrepC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids fromRhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region betweentraI and repA contained two copies of thetra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from thetra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal repplasmid increased five- to sevenfold in strains expressingtraR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.

scholarly journals Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 43-58 ◽  
Author(s):  
Geneviève Girard ◽  
E. Tjeerd van Rij ◽  
Ben J. J. Lugtenberg ◽  
Guido V. Bloemberg
Keyword(s):  
Quorum Sensing ◽  
Constitutive Expression ◽  
Homoserine Lactone ◽  
Pseudomonas Chlororaphis ◽  
Acylhomoserine Lactone ◽  
Sensing System ◽  
New Genes ◽  
Regulatory Cascade ◽  
Quorum Sensing System ◽  
Microarray Analyses

Production of the secondary metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis PCL1391 is crucial for biocontrol activity against the phytopathogen Fusarium oxysporum f. sp. radicis lycopersici on tomato. Regulation of PCN production involves the two-component signalling system GacS/GacA, the quorum-sensing system PhzI/PhzR and the regulator PsrA. This paper reports that a functional rpoS is required for optimal PCN and N-hexanoyl-l-homoserine lactone (C6-HSL) production. Constitutive expression of rpoS is able to complement partially the defect of a psrA mutant for PCN and N-acylhomoserine lactone production. Western blotting shows that rpoS is regulated by gacS. Altogether, these results suggest the existence of a cascade consisting of gacS/gacA upstream of psrA and rpoS, which influence expression of phzI/phzR. Overproduction of phzR complements the effects on PCN and C6-HSL production of all mutations tested in the regulatory cascade, which shows that a functional quorum-sensing system is essential and sufficient for PCN synthesis. In addition, the relative amounts of PCN, phenazine-1-carboxylic acid and C6-HSL produced by rpoS and psrA mutants harbouring a constitutively expressed phzR indicate an even more complex network of interactions, probably involving other genes. Preliminary microarray analyses of the transcriptomics of the rpoS and psrA mutants support the model of regulation described in this study and allow identification of new genes that might be involved in secondary metabolism.

scholarly journals The BlcC (AttM) Lactonase of Agrobacterium tumefaciens Does Not Quench the Quorum-Sensing System That Regulates Ti Plasmid Conjugative Transfer

2008 ◽  
Vol 191 (4) ◽  
pp. 1320-1329 ◽  
Author(s):  
Sharik R. Khan ◽  
Stephen K. Farrand
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Ti Plasmid ◽  
Mutation Induction ◽  
Conjugative Transfer ◽  
Wild Type ◽  
In Planta ◽  
Sensing System ◽  
Quorum Sensing System

ABSTRACT The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

#99: Streptococcus pyogenes Utilizes the Peptide-Based Rgg 2/3 Quorum Sensing System During Oropharyngeal Colonization

2021 ◽  
Vol 10 (Supplement_1) ◽  
pp. S10-S10
Author(s):  
Artemis Gogos ◽  
Michael J Federle
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Pyogenes ◽  
Lymphoid Tissue ◽  
Transcriptional Activator ◽  
Rt Pcr ◽  
Sensing System ◽  
Bacterial Rna ◽  
Quorum Sensing System ◽  
Luciferase Reporters

Abstract Background Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors have been found to contribute to persistent colonization of this niche, and many of these factors are important in mucosal immunity and vaccine development. In this work, we infected mice intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system—a peptide-based signaling system conserved in all sequenced isolates of S. pyogenes. Methods Three-week-old CD1 mice were intranasally infected with ~107 CFU of S. pyogenes strain MGAS315. Calcium alginate throat swabs were used to monitor nasopharyngeal colonization by the bacteria over time. Luciferase reporters used alongside an IVIS camera were able to show quorum sensing activity levels after inoculation into the mouse nose. Bacterial RNA was isolated from the throat of the mice and quantitative RT–PCR was performed on the samples to corroborate the luciferase reporter data. The nasal-associated lymphoid tissue (NALT) was excised and its supernatants were subjected to 32-plex murine cytokine and chemokine analysis (Millipore). Results Deletion of the QS system’s transcriptional activator (Δrgg2) dramatically diminished the percentage of colonized mice. Deletion of the transcriptional repressor (Δrgg3) increased the percentage of colonized mice compared with wild type. Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that activity of the QS system is responsive to conditions of the host nasopharynx. Mice inoculated with QS-dependent luciferase reporters were subjected to in vivo imaging and showed activation within 1 hour. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative RT–PCR subsequently confirmed QS upregulation within 1 hour of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the Δrgg2 bacteria suggests that their rapid elimination fails to elicit the previously characterized response to intranasal inoculation of GAS. Conclusions Deletion of the Rgg2 transcriptional activator of the Rgg 2/3 quorum sensing system eliminates colonization of the murine nasopharynx and changes the transcriptional profile of the bacteria in this niche. An existing small-molecule inhibitor of the Rgg2/3 system was unable to inhibit QS activation in vivo, likely due to the suboptimal achievable doses; however, results of our study indicate inhibition of QS may diminish the oropharyngeal colonization of S. pyogenes and argue for further development.

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