Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity

ABSTRACT TraR of Agrobacterium tumefaciens is a member of the LuxR family of quorum-sensing transcription factors and regulates genes required for conjugation and vegetative replication of the tumor-inducing (Ti) plasmid in the presence of the autoinducer 3-oxooctanoyl-homoserine lactone (OOHL). In the absence of OOHL, TraR is rapidly destroyed by proteolysis, suggesting that this ligand is required for TraR folding. To date, no TraR variant has been found that is active in the absence of OOHL. In this study, we conducted whole-cell and plasmid mutagenesis experiments to search for constitutive mutations of traR and identified two constitutive alleles. Surprisingly, neither contained a point mutation within the traR gene, but rather, both encoded fusion proteins between TraR and the N-terminal domain of an aminoglycoside N-acetyltransferase, encoded by a plasmid-borne antibiotic resistance gene present in the original strain. Data from Western immunoblot assays, pulse-chase assays, and immunoprecipitation assays show that these fusion proteins are far more stable to proteolysis than native apo-TraR. We also constructed a library of traR alleles encoding random amino-terminal fusions and selected for constitutive TraR activity. Five independent fusion proteins were identified by this approach. These fusion proteins accumulated to far higher levels than wild-type TraR in the absence of OOHL. One of these fusions was overexpressed in Escherichia coli and showed detectable tra box binding in the absence of OOHL. These data suggest that the native amino terminus of TraR may signal proteolysis and that fusing it to other proteins might sequester it from intracellular proteases.

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N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms22147565 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7565

Author(s):

Kyungho Woo ◽

Dong Ho Kim ◽

Man Hwan Oh ◽

Ho Sung Park ◽

Chul Hee Choi

Keyword(s):

Bone Marrow ◽

Quorum Sensing ◽

Deletion Mutant ◽

Transmembrane Protein ◽

Cell Communication ◽

Homoserine Lactone ◽

Host Responses ◽

Wild Type ◽

Mutant Strains

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

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Quorum Sensing: a Transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

Infection and Immunity ◽

10.1128/iai.72.11.6589-6596.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6589-6596 ◽

Cited By ~ 57

Author(s):

Ricky L. Ulrich ◽

David DeShazer ◽

Harry B. Hines ◽

Jeffrey A. Jeddeloh

Keyword(s):

Quorum Sensing ◽

Virulence Factor ◽

Regulatory System ◽

In Silico Analysis ◽

Homoserine Lactone ◽

Burkholderia Mallei ◽

Wild Type ◽

Transcriptional Regulatory ◽

A Cell

ABSTRACT Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues. Using mass spectrometry, we showed that wild-type B. mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the B. mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 104 CFU (10 50% lethal doses [LD50s]). For the B. mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B. mallei and each QS mutant. An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B. mallei (<13 CFU). These findings demonstrate that B. mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B. mallei in vivo.

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A role for rhamnolipid in biofilm dispersion

Biofilms ◽

10.1017/s147905050400119x ◽

2004 ◽

Vol 1 (2) ◽

pp. 91-99 ◽

Cited By ~ 48

Author(s):

S. R. Schooling ◽

U. K. Charaf ◽

D. G. Allison ◽

P. Gilbert

Keyword(s):

Surface Tension ◽

Quorum Sensing ◽

Biofilm Formation ◽

Culture Medium ◽

High Cell Density ◽

Culture Media ◽

Homoserine Lactone ◽

Wild Type ◽

In The Wild ◽

Biofilm Dispersion

Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P. aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N-butyryl-L-homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.

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Transgenic Plants Producing the Bacterial Pheromone N-Acyl-Homoserine Lactone Exhibit Enhanced Resistance to the Bacterial Phytopathogen Erwinia carotovora

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.9.1035 ◽

2001 ◽

Vol 14 (9) ◽

pp. 1035-1042 ◽

Cited By ~ 108

Author(s):

Andres Mäe ◽

Marcos Montesano ◽

Viia Koiv ◽

E. Tapio Palva

Keyword(s):

Quorum Sensing ◽

Transgenic Tobacco ◽

Plant Cell Wall ◽

Ectopic Expression ◽

Erwinia Carotovora ◽

Homoserine Lactone ◽

Defense Responses ◽

Dependent Manner ◽

Wild Type ◽

Enhanced Resistance

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

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Quorum-Sensing Systems in the Plant Growth-Promoting Bacterium Paraburkholderia phytofirmans PsJN Exhibit Cross-Regulation and Are Involved in Biofilm Formation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0008-r ◽

2017 ◽

Vol 30 (7) ◽

pp. 557-565 ◽

Cited By ~ 16

Author(s):

Ana Zúñiga ◽

Raúl A. Donoso ◽

Daniela Ruiz ◽

Gonzalo A. Ruz ◽

Bernardo González

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Homoserine Lactone ◽

Wild Type ◽

Transcript Levels ◽

Sensing Systems ◽

Plant Growth Promoting ◽

Growth Promoting

Quorum-sensing systems play important roles in host colonization and host establishment of Burkholderiales species. Beneficial Paraburkholderia species share a conserved quorum-sensing (QS) system, designated BraI/R, that controls different phenotypes. In this context, the plant growth-promoting bacterium Paraburkholderia phytofirmans PsJN possesses two different homoserine lactone QS systems BpI.1/R.1 and BpI.2/R.2 (BraI/R-like QS system). The BpI.1/R.1 QS system was previously reported to be important to colonize and produce beneficial effects in Arabidopsis thaliana plants. Here, we analyzed the temporal variations of the QS gene transcript levels in the wild-type strain colonizing plant roots. The gene expression patterns showed relevant differences in both QS systems compared with the wild-type strain in the unplanted control treatment. The gene expression data were used to reconstruct a regulatory network model of QS systems in P. phytofirmans PsJN, using a Boolean network model. Also, we examined the phenotypic traits and transcript levels of genes involved in QS systems, using P. phytofirmans mutants in homoserine lactone synthases genes. We observed that the BpI.1/R.1 QS system regulates biofilm formation production in strain PsJN and this phenotype was associated with the lower expression of a specific extracytoplasmic function sigma factor ecf26.1 gene (implicated in biofilm formation) in the bpI.1 mutant strain.

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Mutation of luxS Affects Biofilm Formation in Streptococcus mutans

Infection and Immunity ◽

10.1128/iai.71.4.1972-1979.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 1972-1979 ◽

Cited By ~ 185

Author(s):

Justin Merritt ◽

Fengxia Qi ◽

Steven D. Goodman ◽

Maxwell H. Anderson ◽

Wenyuan Shi

Keyword(s):

Quorum Sensing ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Microscopic Analysis ◽

Homoserine Lactone ◽

Good Evidence ◽

Genome Project ◽

Bacterial Biofilm ◽

Wild Type

ABSTRACT Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html ). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

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Role of Quorum Sensing and Antimicrobial Component Production by Serratia plymuthica in Formation of Biofilms, Including Mixed Biofilms with Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01708-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7294-7300 ◽

Cited By ~ 40

Author(s):

Pieter Moons ◽

Rob Van Houdt ◽

Abram Aertsen ◽

Kristof Vanoirbeek ◽

Yves Engelborghs ◽

...

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Broth Culture ◽

Confocal Laser ◽

Wild Type ◽

Serratia Plymuthica ◽

E Coli

ABSTRACT We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

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AinS Quorum Sensing Regulates the Vibrio fischeri Acetate Switch

Journal of Bacteriology ◽

10.1128/jb.00148-08 ◽

2008 ◽

Vol 190 (17) ◽

pp. 5915-5923 ◽

Cited By ~ 58

Author(s):

Sarah V. Studer ◽

Mark J. Mandel ◽

Edward G. Ruby

Keyword(s):

Quorum Sensing ◽

Vibrio Fischeri ◽

Growth Yield ◽

Normal Growth ◽

Homoserine Lactone ◽

Acetate Metabolism ◽

Wild Type ◽

Gene Encoding ◽

Sensing Systems ◽

Acetate Utilization

ABSTRACT The marine bacterium Vibrio fischeri uses two acyl-homoserine lactone (acyl-HSL) quorum-sensing systems. The earlier signal, octanoyl-HSL, produced by AinS, is required for normal colonization of the squid Euprymna scolopes and, in culture, is necessary for a normal growth yield. In examining the latter requirement, we found that during growth in a glycerol/tryptone-based medium, wild-type V. fischeri cells initially excrete acetate but, in a metabolic shift termed the acetate switch, they subsequently utilize the acetate, removing it from the medium. In contrast, an ainS mutant strain grown in this medium does not remove the excreted acetate, which accumulates to lethal levels. The acetate switch is characterized by the induction of acs, the gene encoding acetyl coenzyme A (acetyl-CoA) synthetase, leading to uptake of the excreted acetate. Wild-type cells induce an acs transcriptional reporter 25-fold, coincident with the disappearance of the extracellular acetate; in contrast, the ainS mutant did not display significant induction of the acs reporter. Supplementation of the medium of an ainS mutant with octanoyl-HSL restored normal levels of acs induction and acetate uptake. Additional mutant analyses indicated that acs regulation was accomplished through the regulator LitR but was independent of the LuxIR quorum-signaling pathway. Importantly, the acs mutant of V. fischeri has a competitive defect when colonizing the squid, indicating the importance of proper control of acetate metabolism in the light of organ symbiosis. This is the first report of quorum-sensing control of the acetate switch, and it indicates a metabolic connection between acetate utilization and cell density.

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AbaM Regulates Quorum Sensing, Biofilm Formation and Virulence in Acinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00635-20 ◽

2021 ◽

Author(s):

Mario López-Martín ◽

Jean-Frédéric Dubern ◽

Morgan R. Alexander ◽

Paul Williams

Keyword(s):

Quorum Sensing ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Plant Pathogens ◽

Galleria Mellonella ◽

Homoserine Lactone ◽

Infection Model ◽

Wild Type ◽

Surface Motility ◽

Biofilm Development

Acinetobacter baumannii possesses a single divergent luxR/luxI-type quorum sensing (QS) locus named abaR/abaI. This locus also contains a third gene located between abaR and abaI which we term abaM that codes for an uncharacterized member of the RsaM protein family known to regulate N-acylhomoserine lactone (AHL) dependent QS in other β- and γ-proteobacteria. Here we show that disruption of abaM via a T26 insertion in A. baumannii strain AB5075 resulted in increased production of N-(3-hydroxydodecanoyl)-L-homoserine lactone (OHC12) and enhanced surface motility and biofilm formation. In contrast to the wild type and abaI::T26 mutant, the virulence of the abaM::T26 mutant was completely attenuated in a Galleria mellonella infection model. Transcriptomic analysis of the abaM::T26 mutant revealed that AbaM differentially regulates at least 76 genes including the csu pilus operon and the acinetin 505 lipopeptide biosynthetic operon, that are involved in surface adherence, biofilm formation and virulence. A comparison of the wild type, abaM::T26 and abaI::T26 transcriptomes, indicates that AbaM regulates ∼21% of the QS regulon including the csu operon. Moreover, the QS genes (abaI/abaR) were among the most upregulated in the abaM::T26 mutant. A. baumannii lux-based abaM reporter gene fusions revealed that abaM expression is positively regulated by QS but negatively auto-regulated. Overall, the data presented in this work demonstrates that AbaM plays a central role in regulating A. baumannii QS, virulence, surface motility and biofilm formation. Importance Acinetobacter baumanni is a multi-antibiotic resistant pathogen of global healthcare importance. Understanding Acinetobacter virulence gene regulation could aid the development of novel anti-infective strategies. In A. baumannii, the abaR and abaI genes that code for the receptor and synthase components of an N-acylhomoserine (AHL) lactone-dependent quorum sensing system (QS) are separated by abaM. Here we show that although mutation of abaM increased AHL production, surface motility and biofilm development, it resulted in the attenuation of virulence. AbaM was found to control both QS-dependent and QS-independent genes. The significance of this work lies in the identification of AbaM, an RsaM ortholog known to control virulence in plant pathogens, as a modulator of virulence in a human pathogen.

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Leukemic HRX Fusion Proteins Inhibit GADD34-Induced Apoptosis and Associate with the GADD34 and hSNF5/INI1 Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.7050 ◽

1999 ◽

Vol 19 (10) ◽

pp. 7050-7060 ◽

Cited By ~ 107

Author(s):

Haskell T. Adler ◽

Rebecca Chinery ◽

Daniel Y. Wu ◽

Steven J. Kussick ◽

John M. Payne ◽

...

Keyword(s):

Fusion Proteins ◽

Chromosomal Abnormalities ◽

Protein A ◽

Wild Type ◽

Chromosomal Locus ◽

Amino Terminal ◽

Apoptotic Effect ◽

The Difference ◽

Induced Apoptosis ◽

First Time

ABSTRACT One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

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