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

Abstract

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).

Inhibition of the Agrobacterium tumefaciens TraR Quorum-sensing Regulator

“…A region in the amino-terminal half of the protein has conserved residues known to be required for 3-oxohexanoyl-homoserine lactone binding by LuxR (14). The carboxyl-terminal region of the protein contains a helix-turn-helix motif (position 193-211) that has been implicated in DNA binding for both TraR and LuxR (15,16). There is genetic and biochemical evidence that interaction with 3-oxo-C8-HSL results in the formation of stable TraR dimers that can bind tra boxes and activate transcription (17,18).…”

Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter

“…It is important that synthetic biologists learn to investigate the reasons for failed designs in the interest of basic research and the purpose of redesign. In this case, we discovered that the transcription factor LuxR and the Plux promoter do not function as previously reported 14,15 . This result contributes to basic research that complements our successful demonstration of time-delayed bacterial growth, which is a contribution to applied research.…”

“…Perhaps the lower average and larger error bars is due to stochastic binding of 3OC6 and the resulting inconsistent forward vs. backward transcription from the same promoter. The literature indicates that LuxR binds Plux only after 3OC6 binds to LuxR 14,15,[24][25][26] . Our results show that the BioBrick part containing Plux supports backwards transcription when LuxR is present in the cell and 3OC6 is absent.…”

“…Our results indicated that the Plux promoter is bidirectional and is induced "backwards" by LuxR in the absence of 3OC6. The backwards transcription in the absence of 3OC6 had not been documented in the literature 14,15,[24][25][26] . In the V. fischeri genome, the Plux promoter points in the direction of the LuxI gene and away from the LuxR gene.…”

“…One of the promoters is the Plux promoter, a well-documented and widely used synthetic biology part from the LuxR quorum sensing mechanism. One of the first quorum sensing systems discovered, the luxR operon was isolated from V. fischeri, a marine bacterium living symbiotically within the squid Euprymna scolopes 14 . According to the literature 14,15 the transcriptional activator protein LuxR must first bind to its ligand, the chemical autoinducer 3-oxo-C6-homoserine lactone (3OC6), before LuxR can bind to, and subsequently activate, the promoter Plux.…”