Abstract:Many bacteria use
membrane-diffusible small molecule quorum signals
to coordinate gene transcription in response to changes in cell density,
known as quorum sensing (QS). Among these, acyl-homoserine lactones
(AHL) are widely distributed in Proteobacteria and
are involved in controlling the expression of virulence genes and
biofilm formation in pathogens, such as Pseudomonas aeruginosa. AHL molecules are specifically biosynthesized by the cognate LuxI
type AHL synthases using S-adenosylmethionine (SAM)
and eit… Show more
“…Residue variations at these positions provided a relatively reasonable explanation for how AhyI can utilize non-native acyl-ACP substrates (C8~C14) with a limited acyl-chain pocket volume. A similar tunnel prediction had been recently verified by the observation of the increase in C 4 -HSL production and decrease in C 12 -HSL after a corresponding residue T105Y mutation in MplI [36]. Dong et al [19,36] showed that the acyl-substrate tolerance of some CoA-based LuxI synthases is likely to depend on the volume of binding pocket, because residues important for acyl group binding were occuping the position that branched or linear alkyl-group of acyl-CoA substrates would be in.…”
Section: Discussionsupporting
confidence: 80%
“…Residue variations at these positions provided a relatively reasonable explanation for how AhyI can utilize non-native acyl-ACP substrates (C8 ~ C14) with a limited acyl-chain pocket volume. A similar tunnel prediction had been recently verified by the observation of the increase in C 4 -HSL production and decrease in C 12 -HSL after a corresponding residue T105Y mutation in MplI [ 36 ]. Fig.…”
Section: Discussionsupporting
confidence: 76%
“…4 B), shows that AhyI lacks the canonical “indole platform”, resulting in a binding pocket that is more suitable for an acyl-ACP substrate, rather than an acyl-CoA one. Two Trp residues in BjaI (W142, W143) and RpaI (W146, W147) establish the “indole platform” which provides the basis for acyl-CoA preferences [ 19 , 36 ]. Hydrogen-bond formation between the side chain of Q124 in RpaI and the hydroxyl group of p -coumarate is critical for p C-CoA binding, reflecting the difference in aryl- and alkyl-CoA substrate preference between RpaI and BjaI [ 36 ].…”
Section: Discussionmentioning
confidence: 99%
“…Two Trp residues in BjaI (W142, W143) and RpaI (W146, W147) establish the “indole platform” which provides the basis for acyl-CoA preferences [ 19 , 36 ]. Hydrogen-bond formation between the side chain of Q124 in RpaI and the hydroxyl group of p -coumarate is critical for p C-CoA binding, reflecting the difference in aryl- and alkyl-CoA substrate preference between RpaI and BjaI [ 36 ]. The C3 carbonyl of 3-oxo-acyl ACP participates in a critical hydrogen bond with the hydroxyl of a hydrophilic residue in EsaI (T140) [ 21 ] and LasI (T142) [ 24 ].…”
Section: Discussionmentioning
confidence: 99%
“…Dong et al [ 19 , 36 ] showed that the acyl-substrate tolerance of some CoA-based LuxI synthases is likely to depend on the volume of binding pocket, because residues important for acyl group binding were occuping the position that branched or linear alkyl-group of acyl-CoA substrates would be in. To further test the relationship between pocket size and acyl-ACP substrate tolerance, we carried out computational alanine mutation using AutoDock to compare the autodock-score values.…”
Background
Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase.
Results
In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay.
Conclusions
In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.
“…Residue variations at these positions provided a relatively reasonable explanation for how AhyI can utilize non-native acyl-ACP substrates (C8~C14) with a limited acyl-chain pocket volume. A similar tunnel prediction had been recently verified by the observation of the increase in C 4 -HSL production and decrease in C 12 -HSL after a corresponding residue T105Y mutation in MplI [36]. Dong et al [19,36] showed that the acyl-substrate tolerance of some CoA-based LuxI synthases is likely to depend on the volume of binding pocket, because residues important for acyl group binding were occuping the position that branched or linear alkyl-group of acyl-CoA substrates would be in.…”
Section: Discussionsupporting
confidence: 80%
“…Residue variations at these positions provided a relatively reasonable explanation for how AhyI can utilize non-native acyl-ACP substrates (C8 ~ C14) with a limited acyl-chain pocket volume. A similar tunnel prediction had been recently verified by the observation of the increase in C 4 -HSL production and decrease in C 12 -HSL after a corresponding residue T105Y mutation in MplI [ 36 ]. Fig.…”
Section: Discussionsupporting
confidence: 76%
“…4 B), shows that AhyI lacks the canonical “indole platform”, resulting in a binding pocket that is more suitable for an acyl-ACP substrate, rather than an acyl-CoA one. Two Trp residues in BjaI (W142, W143) and RpaI (W146, W147) establish the “indole platform” which provides the basis for acyl-CoA preferences [ 19 , 36 ]. Hydrogen-bond formation between the side chain of Q124 in RpaI and the hydroxyl group of p -coumarate is critical for p C-CoA binding, reflecting the difference in aryl- and alkyl-CoA substrate preference between RpaI and BjaI [ 36 ].…”
Section: Discussionmentioning
confidence: 99%
“…Two Trp residues in BjaI (W142, W143) and RpaI (W146, W147) establish the “indole platform” which provides the basis for acyl-CoA preferences [ 19 , 36 ]. Hydrogen-bond formation between the side chain of Q124 in RpaI and the hydroxyl group of p -coumarate is critical for p C-CoA binding, reflecting the difference in aryl- and alkyl-CoA substrate preference between RpaI and BjaI [ 36 ]. The C3 carbonyl of 3-oxo-acyl ACP participates in a critical hydrogen bond with the hydroxyl of a hydrophilic residue in EsaI (T140) [ 21 ] and LasI (T142) [ 24 ].…”
Section: Discussionmentioning
confidence: 99%
“…Dong et al [ 19 , 36 ] showed that the acyl-substrate tolerance of some CoA-based LuxI synthases is likely to depend on the volume of binding pocket, because residues important for acyl group binding were occuping the position that branched or linear alkyl-group of acyl-CoA substrates would be in. To further test the relationship between pocket size and acyl-ACP substrate tolerance, we carried out computational alanine mutation using AutoDock to compare the autodock-score values.…”
Background
Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase.
Results
In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay.
Conclusions
In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.
Programmable intercellular signaling using components of naturally occurring quorum sensing can allow for coordinated functions to be engineered in microbial consortia. LuxR-type transcriptional regulators are widely used for this purpose and are activated by homoserine lactone (HSL) signals. However, they often suffer from imperfect molecular discrimination of structurally similar HSLs, causing misregulation within engineered consortia containing multiple HSL signals. Here, we studied one such example, the regulator LasR from Pseudomonas aeruginosa. We elucidated its sequence−function relationship for ligand specificity using targeted protein engineering and multiplexed high-throughput biosensor screening. A pooled combinatorial saturation mutagenesis library (9,486 LasR DNA sequences) was created by mutating six residues in LasR's β5 sheet with single, double, or triple amino acid substitutions. Sort-seq assays were performed in parallel using cognate and noncognate HSLs to quantify each corresponding sensor's response to each HSL signal, which identified hundreds of highly specific variants. Sensor variants identified were individually assayed and exhibited up to 60.6-fold (p = 0.0013) improved relative activation by the cognate signal compared to the wildtype. Interestingly, we uncovered prevalent mutational epistasis and previously unidentified residues contributing to signal specificity. The resulting sensors with negligible signal crosstalk could be broadly applied to engineer bacteria consortia.
Programmable intercellular signaling using components of naturally-occurring quorum sensing can allow for coordinated functions to be engineered in microbial consortia. LuxR-type transcriptional regulators are widely used for this purpose and are activated by homoserine lactone (HSL) signals. However, they often suffer from imperfect molecular discrimination of structurally similar HSLs, causing misregulation within engineered consortia containing multiple HSL signals. Here, we studied one such example, the regulator LasR fromPseudomonas aeruginosa. We elucidated its sequence-function relationship for ligand specificity using targeted protein engineering and multiplexed high-throughput biosensor screening. A pooled combinatorial saturation mutagenesis library (9,486 LasR DNA sequences) was created by mutating six residues in LasR’s β5 sheet with single, double, or triple amino acid substitutions. Sort-seq assays were performed in parallel using cognate and non-cognate HSLs to quantify each corresponding sensor’s response to each HSL signal, which identified hundreds of highly specific variants. Sensor variants identified were individually assayed and exhibited up to 60.6-fold (p= 0.0013) improved relative activation by the cognate signal compared to the wildtype. Interestingly, we uncovered prevalent mutational epistasis and previously unidentified residues contributing to signal specificity. The resulting sensors with negligible signal crosstalk could be broadly applied to engineer bacteria consortia.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
