Quorum Sensing

LuxR solo types. Here we describe steps to distinguish and meth-

ods to identify AHL-binding LuxR solos,plant-associatedbacterial

(PAB) LuxR solos, and non-AHL-binding LuxR solos by in silico

analyses.

1. Go to Clustal W [31–33] and align the protein sequence of the
   LuxR solo of interest against the TraR amino acid sequence.


2. Inspect the alignment and evaluate the presence or absence of
   invariant amino acids at the designated positions of the LuxR
   solo protein sequence. No change indicates that the LuxR solo
   likely binds AHLs.


3. Further inspect the alignment at positions W57 and Y61 (with
   respect to TraR). LuxR solos of PAB are known to carry sub-
   stitutions at these positions (seeNote 8).


4. Next, note the conservation of amino acids at Y61 and D70
   (with respect to TraR). These are the only two amino acids at
   the AHL-binding domain that remain invariant in the non-
   AHL-binding LuxR solos.


5. Having determined tentatively the LuxR solo type, analyze the
   genomic locus adjacent to the gene encoding the LuxR solo.
   LuxR solos and non-AHL LuxR proteins are known to regulate
   adjacent target genes, which encode for proteins that synthe-
   size secondary metabolites or enzymes. Identify any genes/
   operons using FGENESB and analyze promoter regions
   using prokaryotic promoter search software such as BPROM
   [30]. Retrieve the promoter region of the adjacent locus and
   ascertain if it has the 20-bp palindromicluxbox using tools
   available at MEME [34]. Examine the annotated genes to
   determine if they encode protein(s) responsible for the synthe-
   sis of any secondary metabolite. The promoter analysis infor-
   mation and the identity of the adjacent gene may be used for
   wet-lab validation of in silico predictions.

3.2 Studies on the
Ligand of the LuxR
Solo/Orphan

QS-LuxRs are highly insoluble when overexpressed; however, in

the presence of their cognate AHL molecule, which they usually

bind with very high affinity, they often become soluble (seeNote 9).

Cognate AHL is required for the proper folding of the nascent

protein, for formation of homomultimers, and for protection from

proteases [23, 35–38]. AHL-free QS-LuxRs instead misfold, and

are highly unstable and prone to proteolysis. Similarly, it has been

observed, for the subfamily of LuxR solos that bind a plant-derived

molecule, that plant extracts can solubilize the protein [27, 39], the

reason being that instead of binding AHLs they bind a plant low-

molecular-weight compound. This clear biochemical feature can

therefore be exploited to indirectly demonstrate that a LuxR solo

binds AHLs or a plant-derived molecule. This methodology con-

sists of a simple and fast protocol for the expression and purification

Solo/Orphan QS Receptors 151