The master transcription factor LuxR precisely controls the expression of more than 600 genes in the V. harveyi
quorum-sensing regulon. LuxR directly regulates approximately one-fifth of these genes (115 genes). Quorum-sensing regulons of similar sizes are known in other vibrios, such as V. vulnificus
, in which the LuxR homolog SmcR binds to 121 promoters (9
). Four of the genes directly controlled by LuxR are predicted to be transcription factors, which likely control second-tier genes in the LuxR regulon.
Our ChIP-seq studies provide a global view of LuxR binding. We identified a significant number of LuxR binding sites in promoters of genes for which we do not observe regulation by LuxR. We propose that LuxR regulates these genes during growth under conditions that are not mimicked by our laboratory experiments. Previous studies of transcription factors in other organisms, such as Drosophila melanogaster
and Candida albicans
, also found that the number of protein binding sites is significantly larger than the number of genes displaying regulation (19
). More than half of the LuxR binding peaks were present within ORFs (671 out of 1,165 LuxR binding peaks), another feature consistent with findings of these earlier studies. For example, LuxR bound within the ORFs of VIBHAR_05222
, the two representative promoters we characterized, although none of these sites is necessary for LuxR regulation of the gene in vivo
. It is noteworthy that among the 75 LuxR-regulated genes with binding peaks within the ORF, 50 of these also harbor additional binding peaks upstream of their start codons. It is therefore possible that binding sites within ORFs paired with binding sites in the promoter are important for LuxR transcriptional regulation under some conditions.
Unlike canonical TetR proteins with stringent consensus motifs, the consensus motif of LuxR-type proteins is degenerate and asymmetric (9
). One-half of the dyad is more strongly conserved than the other half, as observed from 1,165 binding site sequences. However, we discovered how the unusual asymmetric nature of the LuxR consensus motif is generated. As with other TetR-type proteins, LuxR binding sites in repressed promoters contain a palindrome of roughly equal symmetry. In contrast, the LuxR binding sequences present in activated promoters are nonpalindromic, containing only the left half of the repressed promoter site. Thus, the combination of these two distinct sites results in a skewed palindrome in vitro
and in vivo
). We propose that LuxR has evolved the flexibility to tolerate minor changes to the DNA binding sequence at activated and repressed promoters, which could underpin why LuxR proteins have the capability to control the expression of hundreds of quorum-sensing genes. A model showing these ideas is presented in .
FIG 7 Model for LuxR transcriptional control. At activated promoters (e.g., PluxC), LuxR interacts with RNA polymerase (denoted RNAP) and/or other transcription factors to promote transcription. At repressed promoters (e.g., P05222), LuxR binding occludes RNA (more ...)
To study the mechanism that drives LuxR activation and repression activity, we performed a screen to identify LuxR variants that only activate or only repress. None of the resulting LuxR mutants was specifically defective for either activation or repression. Two activation-defective mutants, LuxR L139P and LuxR N142D, were defective only at activating luxC and VIBHAR_p08175. However, LuxR 139P did not exhibit any defects in DNA binding. We predict that the region of LuxR containing amino acids L139 and N142 may be critical for RNA polymerase interaction and thus required for activation of luxC. Several of our LuxR mutants exhibited defects in DNA binding only at specific LuxR binding sites, and each of these mutant LuxR proteins harbored substitutions in the DNA binding domain. This finding suggests that modifications to the amino acids in the conserved HTH motif restrict DNA sequence recognition to specific sequences. Two such LuxR mutants, carrying N55I and T52M/I24V, exhibited the strongest defects in gene regulation, suggesting that these amino acids likely play the most important roles in DNA sequence recognition, at least at the two promoters we examined.
A common theme that we observe is that when repression is considered, LuxR functions similarly to TetR. First, the LuxR consensus motif at repressed promoters is a symmetrical palindrome. Second, LuxR bound a single site at the example repressed promoter P05222
, which is reminiscent of TetR proteins that bind a single operator at a promoter. Third, similar to what we show for LuxR, altering the conserved residues in the DNA binding domain of TetR residues decreases its repression activity (23
). For example, the TetR T40M substitution (compare with LuxR T52M) reduced TetR repression activity 153-fold in vivo
. Finally, we found that the phenotype of a repression-defective mutant, LuxR T52M/I24V, could be suppressed by altering the DNA sequence in P05222
. In an analogous experiment, tet
operator sequence variants also suppress defective repression phenotypes of TetR proteins with substitutions at T40 (23
). Thus, LuxR likely functions similarly to TetR at repressed promoters by binding to a single palindromic operator site via specific interactions with residues in the DNA binding domain. LuxR is not like TetR when one considers activated promoters. At activated promoters, LuxR recognizes a different consensus motif and binds three sites in the example activated promoter PluxC
. Multiple LuxR binding sites commonly exist in each promoter (59% of the LuxR-bound regions contain 2 or more sites; see Fig. S4
in the supplemental material; for example, PluxC
). LuxR may facilitate DNA looping by binding to multiple sites, a mechanism that has been proposed for the LuxR homolog SmcR (24
Our biochemical analyses suggest that the DNA sequence of the LuxR binding site is not the only factor that specifies activation or repression, because LuxR mutants that activated PluxC
, for example, could not activate all LuxR-activated promoters. Likewise, LuxR mutants that repressed P05222
could not repress all LuxR-repressed promoters. In addition, DNA binding affinity alone cannot account for all LuxR transcription activity because LuxR had the weakest affinity for PluxC
site 2, which is the critical binding site in that promoter in terms of regulation. It is also peculiar that LuxR exhibits the strongest affinity for PluxC
site 3 but this site is not required for activation in vivo
. Thus, features such as the DNA sequence, number and relative locations of LuxR binding sites, and productive LuxR interactions with RNA polymerase and other transcription factors likely combine to dictate whether LuxR activates or represses a given promoter and to what extent. For example, we know that the cAMP receptor protein (CRP) and MetR regulate PluxC
), and LuxR may interact with these proteins to activate transcription (). We are currently exploring LuxR interactions with RNA polymerase and other transcription factors at PluxC
as a model to understand the mechanism of LuxR activation.
Our studies of LuxR gene regulation support a role for LuxR as a dual-function global transcriptional regulator. In many ways, our analysis suggests that LuxR is more similar to CRP than to TetR. CRP is an activator and repressor of transcription of >200 genes (26
). CRP binds to a conserved 22-bp operator sequence (32
), and the positioning of the CRP site dictates its mode of action (36
). CRP DNA binding affinity varies between promoters, with the highest affinities corresponding to those sites that are most similar to the consensus site (32
). Finally, activation-defective mutants of CRP have been identified, and they contain substitutions in amino acids that interact with RNA polymerase (40
). While these general parallels suggest that LuxR functions similarly to CRP, there are two striking contrasts: first, there is no known ligand that controls LuxR activity, and second, LuxR repression requires a symmetrical palindrome, whereas LuxR activation requires only a half-site. We are currently determining how the position of LuxR binding sites with respect to RNA polymerase at promoters correlates with activation or repression.
As the master regulator of quorum-sensing gene expression, LuxR controls the timing of expression of hundreds of genes in response to changes in cell density. The concentration of LuxR increases as autoinducers accumulate (7
). This LuxR protein concentration gradient enables LuxR to control promoters via different binding affinities at various cell densities, producing a temporal pattern of gene expression (7
). Absent other modulatory features, promoters containing the highest-affinity binding sites will be regulated first during the transition from LCD to HCD. For example, VIBHAR_05222
is repressed 2-fold by LuxR at LCD and 7-fold at HCD. Thus, because LuxR has a high binding affinity for the binding site at P05222
, it is repressed even by the low concentrations of LuxR present at LCD. In contrast, luxC
is one of the final genes to be activated in response to quorum sensing (data not shown), which fits with our observation that LuxR has a weak affinity for PluxC
site 2. Thus, DNA binding affinity, coupled to other features, results in a finely choreographed pattern of gene expression. We propose that LuxR, although structurally similar to TetR, has evolved unique characteristics that enable it to differentially control the genes in the quorum-sensing regulon in response to quorum-sensing cues.