Role of the C-terminal domain of the alpha subunit of RNA polymerase in LuxR-dependent transcriptional activation of the lux operon during quorum sensing

Abstract

During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [N-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the lux operon promoter at a position centered at -42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the alpha-subunit C-terminal domain (alphaCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effects of 70 alanine substitution variants of the alpha subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRDeltaN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDeltaN, and 14 alanine substitutions in the alphaCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these 14 alpha variants were also involved in the mechanisms of both LuxR- and LuxRDeltaN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in alpha that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in alpha play roles in DNA recognition and residues 290 and 314 play roles in alpha-LuxR interactions at the lux operon promoter during quorum sensing.

FIG. 1.

(A) Cartoon model of the proposed interactions between RNAP and LuxR at the lux operon promoter. See the text for details. (B) Nucleotide sequence of the lux operon promoter. The positions of various features of the region are highlighted as follows: dashed arrows, binding sites for primers AMS4 and LuxR2A; divergent solid arrows, lux box; boldface letters, extended −10 and transcription start site; the Shine-Delgarno (SD) sequence and initiation codon are underlined, and a putative proximal UP element half site is in boldface in a different font with the two bases that differ from the consensus underlined.

FIG. 2.

Effects of alanine substitutions in α on LuxRΔN-dependent (A) and LuxR-dependent (B) cellular luciferase levels in recombinant E. coli. The value for each variant form of α represents the average of two independent experiments with individual luciferase assays performed in quadruplicate. The error bars represent the range of each experiment from the mean. Luciferase activities from strains containing either of the two wild-type (WT) controls, pHTf1α or pREIIα, were set to 100% for each experiment. The solid bars highlight variants producing <50% wild-type levels of luciferase activity, the hatched bar highlights a variant producing >200% wild-type levels of luciferase activity, the open bars are used at positions where the variants exhibited luciferase levels within twofold of the wild-type, and the shaded bars are used to represent positions were alanine is already present in wild-type α.

FIG. 3.

LuxRΔN-dependent in vitro transcription from the luxI promoter generated by wild-type or variant RNAPs. Representative results for RNAPs in set 1 (A) and set 2 (B) are shown. Transcripts produced by wild-type RNAP (set 1, 30 nM, or set 2, 54 nM) in the absence (−) and presence (+) of LuxRΔN (10 μM) are shown in lanes − and +, respectively. Lane Δ235 illustrates the results when a truncated form of RpoA missing the C-terminal 94 amino acid residues was used in the assays and serves as an additional control. The remaining lanes are labeled with the residue numbers indicating the positions of the alanine substitutions in α present in RNAP. The open arrows point to the lux mRNA products, and the solid arrows indicate the RNA-1 transcripts, which serve as an internal control for LuxRΔN-independent activity and permit normalization of the activity of the variant RNAPs under investigation. The identities of the extra transcripts produced from the DNA template in some lanes are unknown. (C) Graphical representation of the relative average value of the luxI transcript in comparison to the wild-type (WT; 100%) from two independently run experiments. The error bars represent the range of each experiment from the mean. The solid bars highlight variants (or negative controls) with <50% wild-type levels, the shaded bars highlight variants with <100% but >50% wild-type levels, the hatched bars highlight variants with >200% wild-type levels, and the open bars are used at positions where alanine substitutions in α had >100% but <200% wild-type levels.

FIG. 4.

Autoradiograms of gel mobility shift assays. The numbers above each lane refer to the concentrations of α or α variant used in the assays. The results shown are representative of two independent trials. F, free DNA; C, α-DNA complexes; CR, LuxRΔN-α-DNA complexes. (A) Interactions between purified wild-type RNAP α and a 228-bp PCR fragment containing a strong UP element from the rrnB P1 promoter amplified from pRLG4238 (UP element) or a 132-bp PCR fragment with the luxI promoter region amplified from pAHF100 (luxI promoter). The last two lanes show interactions when 10 μM LuxRΔN is added. (B) Interactions between the L290A α variant and the UP element or the luxI promoter. Control reactions containing no α (−) or 8 μM wild-type α (+) are also shown.

FIG. 5.

Space-filling model of the αCTD based on the atomic coordinates of Jeon et al. (24) showing the positions of some of the residues identified as important for LuxR-dependent (A) and LuxRΔN-dependent (B) activation of the lux operon. (A) Amino acid residues producing >2-fold effects on LuxR-dependent luciferase levels in vivo when changed to alanine are highlighted. The residues in red (262, 265, 295, and 296) are hypothesized to be involved in DNA recognition, and the residues in blue (290 and 314) are hypothesized to be involved in protein-protein interactions. (B) Amino acid residues producing >2-fold effect on either LuxRΔN-dependent luciferase levels in vivo or transcription rates in vitro are highlighted. The residues in red (262, 265 and 296) and blue (290 and 314) were found to be important in both LuxR- and LuxRΔN-dependent activation, while the residues in violet are unique to the LuxRΔN-dependent mechanism.