Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the importance of the QS regulation in the bacteria-yeast interactions.

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Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii

Genomics ◽

10.1016/j.ygeno.2021.11.017 ◽

2021 ◽

Author(s):

Elisa Violeta Bertini ◽

Mariela Analía Torres ◽

Thibaut Léger ◽

Camille Garcia ◽

Kar-Wai Hong ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

Meyerozyma Guilliermondii

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The Oligopeptide Transport System Is Essential for the Development of Natural Competence in Streptococcus thermophilus Strain LMD-9

Journal of Bacteriology ◽

10.1128/jb.00257-09 ◽

2009 ◽

Vol 191 (14) ◽

pp. 4647-4655 ◽

Cited By ~ 97

Author(s):

Rozenn Gardan ◽

Colette Besset ◽

Alain Guillot ◽

Christophe Gitton ◽

Véronique Monnet

Keyword(s):

Quorum Sensing ◽

Streptococcus Thermophilus ◽

Defined Medium ◽

Mass Spectrometry Analysis ◽

Transport Systems ◽

Label Free ◽

Spectrometry Analysis ◽

Proteomic Approach ◽

Liquid Chromatography Tandem Mass ◽

Oligopeptide Transport

ABSTRACT In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

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Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility

Microbiology ◽

10.1099/mic.0.039032-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2670-2681 ◽

Cited By ~ 42

Author(s):

Amelia D. Tomlinson ◽

Bronwyn Ramey-Hartung ◽

Travis W. Day ◽

Peter M. Merritt ◽

Clay Fuqua

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Sinorhizobium Meliloti ◽

Plant Tissues ◽

Flagellar Motility ◽

Regulatory Pathway ◽

Flow Conditions ◽

Two Component System ◽

Form Complex ◽

Abiotic Surfaces

The ubiquitous plant pathogen Agrobacterium tumefaciens attaches efficiently to plant tissues and abiotic surfaces and can form complex biofilms. A genetic screen for mutants unable to form biofilms on PVC identified disruptions in a homologue of the exoR gene. ExoR is a predicted periplasmic protein, originally identified in Sinorhizobium meliloti, but widely conserved among alphaproteobacteria. Disruptions in the A. tumefaciens exoR gene result in severely compromised attachment to abiotic surfaces under static and flow conditions, and to plant tissues. These mutants are hypermucoid due to elevated production of the exopolysaccharide succinoglycan, via derepression of the exo genes that direct succinoglycan synthesis. In addition, exoR mutants have lost flagellar motility, do not synthesize detectable flagellin and are diminished in flagellar gene expression. The attachment deficiency is, however, complex and not solely attributable to succinoglycan overproduction or motility disruption. A. tumefaciens ExoR can function independently of the ChvG–ChvI two component system, implicated in ExoR-dependent regulation in S. meliloti. Mutations that suppress the exoR motility defect suggest a branched regulatory pathway controlling succinoglycan synthesis, motility and biofilm formation.

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Transcriptome-Based Analysis of the Pantoea stewartii Quorum-Sensing Regulon and Identification of EsaR Direct Targets

Applied and Environmental Microbiology ◽

10.1128/aem.01489-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5790-5800 ◽

Cited By ~ 22

Author(s):

Revathy Ramachandran ◽

Alison Kernell Burke ◽

Guy Cormier ◽

Roderick V. Jensen ◽

Ann M. Stevens

Keyword(s):

Quorum Sensing ◽

Cell Envelope ◽

Regulatory Protein ◽

Homoserine Lactone ◽

Sequencing Analysis ◽

Mobility Shift ◽

Content Type ◽

Proteomic Approach ◽

Stewart's Wilt ◽

Cell Densities

ABSTRACTPantoeastewartiisubsp.stewartiiis a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression ofesaRitself as well as the genes for the capsular synthesis operon transcription regulator,rcsA, and a 2,5-diketogluconate reductase,dkgA. It simultaneously directly activates expression of genes for a putative small RNA,esaS, the glycerol utilization operon,glpFKX, and another transcriptional regulator,lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficientP. stewartiistrains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon ofP. stewartiiorchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response.

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Co-Dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955

10.1101/2020.12.15.422598 ◽

2020 ◽

Author(s):

Ian S Barton ◽

Justin L Eagan ◽

Priscila A Nieves-Otero ◽

Ian P Reynolds ◽

Thomas G Patt ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

Homoserine Lactone ◽

Ti Plasmid ◽

Conjugative Transfer ◽

Gene Promoters ◽

Transcription Activator ◽

Conjugation System ◽

Direct Production ◽

Sensing Systems

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologues of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR preferentially biased towards its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

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Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts Agrobacterium tumefaciens from Phenotypically Female to Male

Journal of Bacteriology ◽

10.1128/jb.01608-08 ◽

2009 ◽

Vol 191 (10) ◽

pp. 3375-3383 ◽

Cited By ~ 10

Author(s):

Hongbaek Cho ◽

Uelinton M. Pinto ◽

Stephen C. Winans

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

Host Cell ◽

Ti Plasmid ◽

Conjugative Plasmids ◽

Acylhomoserine Lactone ◽

Ti Plasmids ◽

Quorum Sensing Signals

ABSTRACT Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.

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Rapid Screening of Quorum-Sensing Signal N-Acyl Homoserine Lactones by an In Vitro Cell-Free Assay

Applied and Environmental Microbiology ◽

10.1128/aem.02869-07 ◽

2008 ◽

Vol 74 (12) ◽

pp. 3667-3671 ◽

Cited By ~ 54

Author(s):

Tomohiro Kawaguchi ◽

Yung Pin Chen ◽

R. Sean Norman ◽

Alan W. Decho

Keyword(s):

Agrobacterium Tumefaciens ◽

Quorum Sensing ◽

High Sensitivity ◽

Homoserine Lactone ◽

Assay System ◽

Rapid Screening ◽

Whole Cell ◽

Bacterial Quorum ◽

Whole Cell Biosensor

ABSTRACT A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of β-galactosidase upon addition of exogenous AHLs. We have shown that β-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (−80°C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.

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