Prebiotics and Probiotics: Healthy Biotools for Molecular Integrative and Modulation Approaches 2.0.

The current version (2 [...].

Characterization of an extremophile bacterial acid phosphatase derived from metagenomics analysis.

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100-fold more efficient than with p-nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family.

A perspective current and past modes of inhalation therapy.

Inhalation is the preferred route of delivery for anti-asthma and chronic obstructive pulmonary disease (COPD) drugs. The use of this route has demonstrated efficacy in these and other conditions, it offers rapid onset of action, and is associated with minimal systemic exposure, thereby reducing the risk of adverse effects. Therefore, the current brief covers an interesting collection of inhaler action modes, shedding light on their molecular mechanisms and clinical applications for anti-asthma, COPD and antibacterial inhalation therapy. Hence, not only enriches our understanding of inhalation therapy molecular intricacies but also provides a comprehensive overview of the evolving landscape in clinical and antibacterial inhalation therapy. In doing so, it underscores the pivotal role of microbiology and biotechnology in advancing therapeutic approaches that harness the power of inhalation.

Prebiotics and Probiotics: Healthy Biotools for Molecular Integrative and Modulation Approaches.

The scope of this Special Issue is to highlight and expand our knowledge on the molecular mechanisms of prebiotics and probiotics, as well as to offer a broad overview of current advancements and future directions in this research field [...].

Corrigendum: A standardized extract of Lentinula edodes cultured mycelium inhibits Pseudomonas aeruginosa infectivity mechanisms.

[This corrects the article DOI: 10.3389/fmicb.2022.814448.].

Rio Tinto as a niche for acidophilus enzymes of industrial relevance.

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rio Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5-5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

A Standardized Extract of Lentinula edodes Cultured Mycelium Inhibits Pseudomonas aeruginosa Infectivity Mechanisms.

The priority pathogen list of the World Health Organization classified Pseudomonas aeruginosa as the second top critical pathogen. Hence, the development of novel antibacterial strategies to tackle this bacterium is highly necessary. Herein we explore the potential antibacterial effect of a standardized extract of cultured mycelium of Lentinula edodes (AHCC®) on P. aeruginosa. AHCC® was found to inhibit the growth rate and biofilm formation of strain PAO1. No change in swarming was observed, but AHCC® hampered swimming and twitching motility. In accordance, a decreased expression of metabolism, growth, and biofilm formation genes was shown. AHCC® also diminished the levels of exotoxin A and bacteria inside IEC18 cells and the secretion of IL-6, IL-10 and TNF by infected macrophages. This effect was related to a reduced phosphorylation of MAPKs and to bacteria internalization. Taken together, our data suggest that AHCC® has a potential role to prevent P. aeruginosa infections and may lead to the development of new therapies.

Influence of the FCGR2A rs1801274 and FCGR3A rs396991 Polymorphisms on Response to Abatacept in Patients with Rheumatoid Arthritis.

Abatacept (ABA) is an immunosuppressant indicated for treatment of rheumatoid arthritis (RA). Effectiveness might be influenced by clinical RA variants and single-nucleotide polymorphisms (SNPs) in genes encoding protein FCGR2A (His131Arg) and FCGR3A (Phe158Val) involved in pharmacokinetics of ABA. An observational cohort study was conducted in 120 RA Caucasian patients treated with ABA for 6 and 12 months. Patients with the FCGR2A rs1801274-AA genotype (FCGR2A-p.131His) showed a better EULAR response (OR = 2.43; 95% CI = 1.01-5.92) at 12 months and low disease activity (LDA) at 6 months (OR = 3.16; 95% CI = 1.19-8.66) and 12 months (OR = 6.62; 95% CI = 1.25-46.89) of treatment with ABA. A tendency was observed towards an association between the FCGR3A rs396991-A allele (FCGR3A-p.158Phe) and better therapeutic response to ABA after 12 months of treatment (p = 0.078). Moreover, we found a significant association between the low-affinity FCGR2A/FCGR3A haplotypes variable and LDA after 12 months of ABA treatment (OR = 1.59; 95% CI = 1.01-2.58). The clinical variables associated with better response to ABA were lower age at starting ABA (OR = 1.06; 95% CI = 1.02-1.11) and greater duration of ABA treatment (OR = 1.02; 95% CI = 1.01-1.04), lower duration of previous biological therapies (OR = 0.99; 95% CI = 0.98-0.99), non-administration of concomitant disease-modifying antirheumatic drugs (DMARDs) (OR = 24.53; 95% CI = 3.46-523.80), non-use of concomitant glucocorticoids (OR = 0.12; 95% CI = 0.02-0.47), monotherapy (OR = 19.22; 95% CI = 2.05-343.00), lower initial patient's visual analogue scale (PVAS) value (OR = 0.95; 95% CI = 0.92-0.97), and lower baseline ESR (OR = 0.92; 95% CI = 0.87-0.97). This study showed that high-affinity FCGR2A-p.131His variant, low-affinity FCGR3A-p.158Phe variant, and combined use of FCGR2A/FCGR3A genetic variations could affect ABA effectiveness. Further studies will be required to confirm these results.

Impact of Single-Nucleotide Polymorphisms of CTLA-4, CD80 and CD86 on the Effectiveness of Abatacept in Patients with Rheumatoid Arthritis.

Abatacept (ABA) is used as a first-line treatment in patients diagnosed with moderate and severe rheumatoid arthritis (RA). The interindividual response to ABA therapy is very variable in these patients. The objective of our study was therefore to investigate the role of polymorphisms of the CTLA-4, CD80 and CD86 genes, as well as that of clinical factors of the disease, in the response to ABA in patients with RA. A retrospective cohort study was carried out in 109 patients receiving treatment with ABA and diagnosed with RA. The genetic variables were analyzed using real-time PCR with TaqMan® probes. The patients were classified according to the European League Against Rheumatism (EULAR) criteria at 6 and 12 months from start of treatment. The independent variables associated with higher EULAR response were lower duration of previous biologic disease-modifying anti-rheumatic drugs and lower baseline values of the disease activity score 28 after 6 months of ABA treatment; and lower baseline patient's visual analogue scale (PVAS) after 12 months. In addition, a significant association was found between duration of ABA treatment, non-administration of concomitant glucocorticoids and lower baseline values of the number of inflamed joints and erythrocyte sedimentation rate clinical variables, with remission of the disease after 6 months' treatment with ABA. Finally, remission of the disease after 12 months' treatment with ABA was associated with earlier age at start of ABA therapy and lower number of previous biologic therapies (BTs). The CTLA-4rs5742909-T allele and the CTLA-4rs231775-G allele were found to be associated with satisfactory EULAR response and low disease activity (LDA) after 12 months' treatment with ABA (CTLA-4rs5742909 T vs. CC; OR = 5.88; CI95% = 1.48-23.29 and OR = 4.75; CI95% = 1.35-17.94, respectively, and CTLA-4rs231775 G vs. AA, OR = 3.48; CI95% = 1.20-10.09 and OR = 4.68; CI95% = 1.49-17.94, respectively). In conclusion, patients with RA treated with ABA showed better EULAR response and LDA rate when they had the CTLA-4 rs5742909-T or CTLA-4 rs231775-G polymorphisms; furthermore, this remission rate increased in patients that began ABA treatment earlier, those with a lower number of previous BTs and those with a lower PVAS value.

Developing robust protein analysis profiles to identify bacterial acid phosphatases in genomes and metagenomic libraries.

Phylogenetic analysis of more than 4000 annotated bacterial acid phosphatases was carried out. Our analysis enabled us to sort these enzymes into the following three types: (1) class B acid phosphatases, which were distantly related to the other types, (2) class C acid phosphatases and (3) generic acid phosphatases (GAP). Although class B phosphatases are found in a limited number of bacterial families, which include known pathogens, class C acid phosphatases and GAP proteins are found in a variety of microbes that inhabit soil, fresh water and marine environments. As part of our analysis, we developed three profiles, named Pfr-B-Phos, Pfr-C-Phos and Pfr-GAP, to describe the three groups of acid phosphatases. These sequence-based profiles were then used to scan genomes and metagenomes to identify a large number of formerly unknown acid phosphatases. A number of proteins in databases annotated as hypothetical proteins were also identified by these profiles as putative acid phosphatases. To validate these in silico results, we cloned genes encoding candidate acid phosphatases from genomic DNA or recovered from metagenomic libraries or genes synthesized in vitro based on protein sequences recovered from metagenomic data. Expression of a number of these genes, followed by enzymatic analysis of the proteins, further confirmed that sequence similarity searches using our profiles could successfully identify previously unknown acid phosphatases.

Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide.

Pseudomonas aeruginosa is an ubiquitous gram-negative opportunistic human pathogen which is not considered part of the human commensal gut microbiota. However, depletion of the intestinal microbiota (Dysbiosis) following antibiotic treatment facilitates the colonization of the intestinal tract by Multidrug-Resistant P. aeruginosa. One possible strategy is based on the use of functional foods with prebiotic activity. The bifidogenic effect of the prebiotic inulin and its hydrolyzed form (fructooligosaccharide: FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and to a greater extent FOS reduce growth and biofilm formation, which was found to be due to a decrease in motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNA-sequence analysis. Changes in the transcript level induced by inulin and FOS were similar, but a set of transcript levels were increased in response to inulin and reduced in the presence of FOS. In the presence of inulin or FOS, 260 and 217 transcript levels, respectively, were altered compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analyses of differentially expressed genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, ß-lactamase resistance, and in the modulation of type III and VI secretion systems; results that have been partially verified by real time quantitative PCR measurements. Moreover, we have identified a genomic island formed by a cluster of 15 genes, encoding uncharacterized proteins, which were repressed in the presence of FOS. The analysis of isogenic mutants has shown that genes of this genomic island encode proteins involved in growth, biofilm formation and motility. These results indicate that FOS selectively modulates bacterial pathogenicity by interfering with different signaling pathways.

Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins.

Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for γ-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.

Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato.

Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity l-Asp, l-Glu, and d-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of d-Asp also reduced bacterial virulence.IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of l- and d-Asp and l-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of d-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.

FCGR2A/FCGR3A Gene Polymorphisms and Clinical Variables as Predictors of Response to Tocilizumab and Rituximab in Patients With Rheumatoid Arthritis.

We evaluated the influence of clinical, biochemical, and genetic factors on response in 142 patients diagnosed with rheumatoid arthritis, of whom 87 patients were treated with tocilizumab (61.26%) and 55 patients were treated with rituximab (38.7%;) according to the variables European League Against Rheumatism (EULAR) response, remission, low disease activity, and improvement in Disease Activity Score, 28 joints (DAS28) at 6, 12, and 18 months. A retrospective prospective cohort study was conducted. Patients carrying the FCGR3A rs396991-TT genotype treated with tocilizumab showed higher EULAR response (OR, 5.075; 95%CI, 1.20-21.33; P = .027) at 12 months, those who were naive for biological disease-modifying antirheumatic drugs (bDMARDs) at the beginning of treatment showed satisfactory EULAR response, higher remission, and greater improvement in DAS28 at 6 months. Younger age at start of tocilizumab treatment was associated with satisfactory EULAR response at 18 months and greater remission at 6 and 18 months. Subcutaneous tocilizumab administration was associated with higher remission at 6 months and improved low disease activity rate at 12 months. In patients treated with rituximab, carriers of the FCGR2A rs1801274-TT genotype had higher EULAR response at 6 months (OR, 4.861; 95%CI, 1.11-21.12; P = .035), 12 months (OR, 4.667; p = 0.066, 95%CI, 0.90-24.12; P = .066), and 18 months (OR, 2.487; 95%CI, 0.35-17.31; P = .357), higher remission (OR: 10.625; p = 0.044, CI95% : 1.07, 105.47) at 6 months, and greater improvement in DAS28 at 12 months (B = 0.782; 95%CI, -0.15 to 1.71; P = .098) and 18 months (B = 1.414; 95%CI, 0.19-2.63; P = .025). The FCGR3A rs396991-G allele was associated with improved low disease activity rate (OR, 4.904; 95%CI, 0.84-28.48; P = .077) and greater improvement in DAS28 (B = -1.083; 95%CI, -1.98 to -0.18; P = .021) at 18 months. Patients with a lower number of previous biological therapies had higher remission at 12 months. We suggest that the FCGR3A rs396991-TT genotype, higher baseline value of DAS28, subcutaneous tocilizumab administration, younger age at the beginning of treatment, and being bDMARD naive are associated with better response to tocilizumab. In patients treated with rituximab, we found better response in those patients with the FCGR2A rs1801274-TT genotype, the FCGR3A rs396991-G allele, and lower number of previous biological therapies.

An auxin controls bacterial antibiotics production.

The majority of clinically used antibiotics originate from bacteria. As the need for new antibiotics grows, large-scale genome sequencing and mining approaches are being used to identify novel antibiotics. However, this task is hampered by the fact that many antibiotic biosynthetic clusters are not expressed under laboratory conditions. One strategy to overcome this limitation is the identification of signals that activate the expression of silent biosynthetic pathways. Here, we report the use of high-throughput screening to identify signals that control the biosynthesis of the acetyl-CoA carboxylase inhibitor antibiotic andrimid in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. We reveal that the pathway-specific transcriptional activator AdmX recognizes the auxin indole-3-acetic acid (IAA). IAA binding causes conformational changes in AdmX that result in the inhibition of the expression of the andrimid cluster and the suppression of antibiotic production. We also show that IAA synthesis by pathogenic and beneficial plant-associated bacteria inhibits andrimid production in A153. Because IAA is a signalling molecule that is present across all domains of life, this study highlights the importance of intra- and inter-kingdom signalling in the regulation of antibiotic synthesis. Our discovery unravels, for the first time, an IAA-dependent molecular mechanism for the regulation of antibiotic synthesis.

Regulation of carbohydrate degradation pathways in Pseudomonas involves a versatile set of transcriptional regulators.

Bacteria of the genus Pseudomonas are widespread in nature. In the last decades, members of this genus, especially Pseudomonas aeruginosa and Pseudomonas putida, have acquired great interest because of their interactions with higher organisms. Pseudomonas aeruginosa is an opportunistic pathogen that colonizes the lung of cystic fibrosis patients, while P. putida is a soil bacterium able to establish a positive interaction with the plant rhizosphere. Members of Pseudomonas genus have a robust metabolism for amino acids and organic acids as well as aromatic compounds; however, these microbes metabolize a very limited number of sugars. Interestingly, they have three-pronged metabolic system to generate 6-phosphogluconate from glucose suggesting an adaptation to efficiently consume this sugar. This review focuses on the description of the regulatory network of glucose utilization in Pseudomonas, highlighting the differences between P. putida and P. aeruginosa. Most interestingly, It is highlighted a functional link between glucose assimilation and exotoxin A production in P. aeruginosa. The physiological relevance of this connection remains unclear, and it needs to be established whether a similar relationship is also found in other bacteria.

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with ß-xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and ß-xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active ß-xylosidase was at least 10-fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue.

High-Throughput Screening to Identify Chemoreceptor Ligands.

The majority of bacterial chemoreceptors remain functionally un-annotated. The knowledge of chemoreceptor function, however, is indispensable to understanding the evolution of the chemotaxis system in bacteria with different lifestyles. Significant progress in the annotation of chemoreceptor function has been made using experimental strategies that are based on the individual, genetically engineered ligand binding domain (LBD) of chemoreceptors. There is now evidence that all major classes of LBDs can be produced as individual domains that retain their ligand binding activity. Here, we provide a protocol for the combined use of high-throughput ligand screening using Differential Scanning Fluorimetry followed by Isothermal Titration Calorimetry to identify and characterize ligands that bind to recombinant chemoreceptor LBDs. This approach has been shown to be very efficient for determining the function of novel chemoreceptors.

Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440.

The genome of Pseudomonas putida KT2440 contains two open reading frames (ORFs), PP_3722 and PP_5269, that encode proteins with a Pyridoxal phosphate binding motif and a high similarity to alanine racemases. Alanine racemases play a key role in the biosynthesis of D-alanine, a crucial amino acid in the peptidoglycan layer. For these ORFs, we generated single and double mutants and found that inactivation of PP_5269 resulted in D-alanine auxotrophy, while inactivation of PP_3722 did not. Furthermore, as expected, the PP_3722/PP_5269 double mutant was a strict auxotroph for D-alanine. These results indicate that PP_5269 is an alr allele and that it is the essential alanine racemase in P. putida. We observed that the PP_5269 mutant grew very slowly, while the double PP_5269/PP_3722 mutant did not grow at all. This suggests that PP_3722 may replace PP_5269 in vivo. In fact, when the ORF encoding PP_3772 was cloned into a wide host range expression vector, ORF PP_3722 successfully complemented P. putida PP_5269 mutants. We purified both proteins to homogeneity and while they exhibit similar KM values, the Vmax of PP_5269 is fourfold higher than that of PP_3722. Here, we propose that PP_5269 and PP_3722 encode functional alanine racemases and that these genes be named alr-1 and alr-2 respectively.

Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures.

Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 µM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.