Intragenic suppression of a mutation: Characterization of an autoinducer-independent LuxR

Poellinger, Kristi A.; Lee, Jean Pyo; Parales, J.V.; Greenberg, E. Peter

doi:10.1016/0378-1097(95)00145-u

Cited by 11 publications

(12 citation statements)

References 3 publications

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“…Those in the N-terminal domain are defective in signal binding and thus transcriptional activation. Those with substitutions in the C-terminal domain are capable of signal binding but are nevertheless defective in transcriptional activation (21,31). We sought to determine whether signal binding mutant LuxR proteins were capable of repressing p35LB10 lacZ transcription.…”

Section: Resultsmentioning

confidence: 99%

Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor

Egland

Greenberg

2000

J Bacteriol

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The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with 70 RNA polymerase it can activate transcription of the lux operon. Both the truncated LuxR and RNA polymerase are required for binding to lux regulatory DNA in vitro. We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus ؊35 and ؊10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiple lux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to lux boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native lux operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.Acyl-homoserine lactone (acyl-HSL)-dependent quorum sensing is common to a number of different genera and species of gram-negative bacteria. This type of cell density-dependent control of gene expression was first discovered in the marine luminescent bacterium Vibrio fischeri. The V. fischeri system remains one of the best-studied examples of quorum sensing. Quorum sensing in V. fischeri involves the interaction of the signal molecule N-3-(oxohexanoyl)homoserine lactone (3-oxo-C 6 -HSL) with LuxR, the transcriptional activator of the luminescence (lux) operon. Over 20 LuxR homologs have been described in the past 10 years (for recent reviews on quorum sensing and the LuxR family of transcription factors, see references 13 to 15 and 24).The V. fischeri lux operon consists of seven genes of which the first is luxI (12, 36). The luxI gene codes for an enzyme required for synthesis of 3-oxo-C 6 -HSL. The luxI gene has a 70 RNA polymerase (RNAP)-dependent promoter with a lux box, a 20-bp inverted repeat, centered at position Ϫ42.5 from the transcriptional start site (11, 34). The lux box and its location with respect to the other promoter elements are critical for LuxR-dependent activation of the lux genes (7, 11).

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Section: Resultsmentioning

confidence: 99%

Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor

Egland

Greenberg

2000

J Bacteriol

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“…The amino-terminal regions of the LuxR-like proteins are involved in HSL binding, and the C-terminal domains of the proteins are responsible for oligomerization and promoter DNA binding. A similar promoter element is bound by each of the LuxR-type proteins (Slock et al 1990;Choi andGreenberg 1991, 1992;Stevens et al 1994;Poellinger et al 1995;Stevens and Greenberg 1997;Stevens et al 1999).…”

Section: Gram-negative Bacteria Use Homoserine Lactones As Wordsmentioning

confidence: 99%

The languages of bacteria

Schauder¹,

Bassler²

2001

Genes Dev.

420

295

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Bacteria communicate with one another using chemical signaling molecules as words. Specifically, they release, detect, and respond to the accumulation of these molecules, which are called autoinducers. Detection of autoinducers allows bacteria to distinguish between low and high cell population density, and to control gene expression in response to changes in cell number. This process, termed quorum sensing, allows a population of bacteria to coordinately control the gene expression of the entire community. Quorum sensing confuses the distinction between prokaryotes and eukaryotes because it allows bacteria to behave as multicellular organisms, and to reap benefits that would be unattainable to them as individuals. Many bacterial behaviors are regulated by quorum sensing, including symbiosis, virulence, antibiotic production, and biofilm formation. Recent studies show that highly specific as well as universal quorum sensing languages exist which enable bacteria to communicate within and between species. Finally, both prokaryotic and eukaryotic mechanisms that interfere with bacterial quorum sensing have evolved. Specifically, the secretion of enzymes that destroy the autoinducers, and the production of autoinducer antagonists, are used by competitor bacteria and susceptible eukaryotic hosts to render quorum sensing bacteria mute and deaf, respectively. Analogous synthetic strategies are now being explored for the development of novel antimicrobial therapies.Bacteria in communities convey their presence to one another by releasing and responding to the accumulation of chemical signaling molecules called autoinducers. This process of intercellular communication, called quorum sensing, was first described in the bioluminescent marine bacterium Vibrio fischeri (Hastings and Nealson 1977;Nealson and Hastings 1979). V. fischeri lives in symbiotic associations with a number of marine animal hosts. In these partnerships, the host uses the light produced by V. fischeri for specific purposes such as attracting prey, avoiding predators, or finding a mate. In exchange for the light it provides, V. fischeri obtains a nutrient-rich environment in which to reside (Ruby 1996;Visick and McFall-Ngai 2000). A luciferase enzyme complex is responsible for light production in V. fischeri. Bioluminescence only occurs when V. fischeri is at high cell number, and this process is controlled by quorum sensing. Specifically, the production and accumulation of, and the response to, a minimum threshold concentration of an acylated homoserine lactone (HSL) autoinducer regulates density-dependent light production in V. fischeri, and enables V. fischeri to emit light only inside the specialized light organ of the host but not when free-living in the ocean. The reason for this is twofold. First, only under the nutrient-rich conditions of the light organ can V. fischeri grow to high population densities, and second, trapping of the diffusible autoinducer molecule in the light organ with the bacterial cells allows it to accumulate to a sufficient ...

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“…Previous studies with recombinant Escherichia coli have identified amino acid point mutations in the C-terminal domain of LuxR that can be placed into two categories: (i) those that affect the ability of LuxR to activate transcription of the lux operon (residues 184,193,195,197,217, and 230) (17,19) and (ii) those that result in a form of LuxR that is capable of autoinducer-independent activation of transcription of the lux operon (residues 164, 221, 223, and 246) (14,18). Deletion mutagenesis analysis of the C-terminal domain of LuxR was also used to identify regions of LuxR critical for its ability to activate transcription of the lux operon (3,4).…”

mentioning

confidence: 99%

“…However, since random mutagenesis of the C-terminal domain of LuxR has previously identified single amino acid substitutions that allow LuxR to activate transcription of the lux operon independent of autoinducer (14,18), all 38 of the alanine-substitution mutants were also tested for this autoinducer-independent phenotype in the luminescence assay. Strains were grown as described above, except in the absence of autoinducer and at 31°C, since the mutation in luxI encoded on pJR551 is a temperaturesensitive mutation that will allow LuxI to synthesize autoinducer below 30°C (14). None of the alanine-substitution mutants was shown to exhibit an autoinducer-independent phenotype (data not shown).…”

mentioning

confidence: 99%

Amino Acid Residues in LuxR Critical for Its Mechanism of Transcriptional Activation during Quorum Sensing in Vibrio fischeri

Trott

Stevens

2001

J Bacteriol

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PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR. This region plays a critical role in the mechanism of LuxRdependent transcriptional activation of the Vibrio fischeri lux operon during quorum sensing. The ability of the variant forms of LuxR to activate transcription of the lux operon was examined by using in vivo assays in recombinant Escherichia coli. Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR. Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type. An in vivo DNA binding-repression assay in recombinant E. coli was subsequently used to measure the ability of the variant forms of LuxR to bind to the lux box, the binding site of LuxR at the lux operon promoter. All eight LuxR variants found to affect cellular luciferase levels were unable to bind to the lux box. An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding. None of the alanine substitutions in LuxR affected activation of transcription of the lux operon without also affecting DNA binding. These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.Vibrio fischeri, a symbiotic bioluminescent bacterium, serves as one of the best-understood model systems for a mechanism of cell density-dependent bacterial gene regulation known as quorum sensing. During quorum sensing in V. fischeri, the chemical signal, 3-oxohexanoyl homoserine lactone (3-oxo-C6-HSL) is synthesized by the bacteria and used to self-sense population levels in a given environment (for recent reviews, see references 8, 9, and 24). As the levels of this autoinducer signal rise, complexes form between it and the N-terminal domain of a 250-amino-acid residue regulatory protein, LuxR. Only when autoinducer has bound to the N-terminal domain of LuxR is the C-terminal domain able to bind to a regulatory region of the lux DNA, known as the lux box, and activate transcription of the luminescence or lux operon (7,10,23). The arrangement of the lux genes in V. fischeri is such that the operon (luxICDABEG) containing the luxI autoinducer synthase gene and the other structural genes necessary for luminescence and luxR are divergently transcribed (reviewed in references 9 and 23). The lux box region, thought to be bound by LuxR in the presence of the V.

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Intragenic suppression of a mutation: Characterization of an autoinducer-independent LuxR

Cited by 11 publications

References 3 publications

Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor

Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor

The languages of bacteria

Amino Acid Residues in LuxR Critical for Its Mechanism of Transcriptional Activation during Quorum Sensing in Vibrio fischeri

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