scholarly journals Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

2020 ◽  
Vol 295 (37) ◽  
pp. 12993-13007 ◽  
Author(s):  
Raphaël Billot ◽  
Laure Plener ◽  
Pauline Jacquet ◽  
Mikael Elias ◽  
Eric Chabrière ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Communication Process ◽  
Dependent Manner ◽  
Biotechnological Potential ◽  
Bacterial Communication ◽  
Homoserine Lactones ◽  
A Cell ◽  
Eukaryotic Organisms ◽  
Bacterial Control

Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density–dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

scholarly journals Exploring the transcriptome of luxI- and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida

2018 ◽  
Author(s):  
Miriam Khider ◽  
Hilde Hansen ◽  
Jostein A. Johansen ◽  
Erik Hjerde ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Transcriptome Profiling ◽  
Homoserine Lactone ◽  
Colony Morphology ◽  
Phenotypic Traits ◽  
Dependent Manner ◽  
Rna Seq ◽  
Homoserine Lactones ◽  
A Cell ◽  
The Impact

Background. The marine bacterium A. salmonicida uses the quorum sensing (QS) systems, AinS/R and LuxI/R to produce eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. Biofilm formation is one of the QS regulated phenotypes, which requires the expression of exopolysaccharides (EPS).We previously demonstrated that inactivation of LitR, the master regulator of QS in A. salmonicida resulted in biofilm formation, which was, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI-.In this work, we have identified genes regulated by AinS and LuxI using RNA sequensing (RNA-Seq), and studied their role in biofilm formation, colony morphology and motility. We have also studied the effect of two AHLs on the biofilm formation. Results.The transcriptome profiling of ΔainS and luxI- mutants allowed us to identify essential genes regulated by QS in A. salmonicida. Relative to the wild-type, the ΔainS and luxI- mutants revealed 40 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI-) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI- mutant was supplemented with N-3-oxo-hexanoyl-L- homoserine lactone (3OC6-HSL) and N-3-hydroxy-decanoyl-L-homoserine lactone(3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and repression for EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor. Conclusion.These findings imply that LuxI and AinS synthases have a critical contribution to the QS-dependent regulation on gene expression and the phenotypic traits related to it.

scholarly journals Exploring the transcriptome of luxI- and ΔainS mutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation in Aliivibrio salmonicida

2018 ◽  
Author(s):  
Miriam Khider ◽  
Hilde Hansen ◽  
Jostein A. Johansen ◽  
Erik Hjerde ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Transcriptome Profiling ◽  
Homoserine Lactone ◽  
Colony Morphology ◽  
Phenotypic Traits ◽  
Dependent Manner ◽  
Rna Seq ◽  
Homoserine Lactones ◽  
A Cell ◽  
The Impact

Background. The marine bacterium A. salmonicida uses the quorum sensing (QS) systems, AinS/R and LuxI/R to produce eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. Biofilm formation is one of the QS regulated phenotypes, which requires the expression of exopolysaccharides (EPS).We previously demonstrated that inactivation of LitR, the master regulator of QS in A. salmonicida resulted in biofilm formation, which was, similar to the biofilm formed by the AHL deficient mutant ΔainSluxI-.In this work, we have identified genes regulated by AinS and LuxI using RNA sequensing (RNA-Seq), and studied their role in biofilm formation, colony morphology and motility. We have also studied the effect of two AHLs on the biofilm formation. Results.The transcriptome profiling of ΔainS and luxI- mutants allowed us to identify essential genes regulated by QS in A. salmonicida. Relative to the wild-type, the ΔainS and luxI- mutants revealed 40 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation of luxI but not ainS genes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI-) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when the ΔainSluxI- mutant was supplemented with N-3-oxo-hexanoyl-L- homoserine lactone (3OC6-HSL) and N-3-hydroxy-decanoyl-L-homoserine lactone(3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and repression for EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor. Conclusion.These findings imply that LuxI and AinS synthases have a critical contribution to the QS-dependent regulation on gene expression and the phenotypic traits related to it.

scholarly journals Analysis of Quorum-Sensing-Dependent Control of Rhizosphere-Expressed (rhi) Genes in Rhizobium leguminosarum bv. viciae

1999 ◽  
Vol 181 (12) ◽  
pp. 3816-3823 ◽  
Author(s):  
Belen Rodelas ◽  
James K. Lithgow ◽  
Florence Wisniewski-Dye ◽  
Andrea Hardman ◽  
Adam Wilkinson ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Culture Supernatant ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Chemical Analyses ◽  
Wild Type ◽  
Homoserine Lactones ◽  
Acyl Homoserine Lactones ◽  
A Cell ◽  
Culture Supernatants

ABSTRACT The rhi genes of Rhizobium leguminosarumbiovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372–376, 1996) therhiABC operon had been shown to be regulated by RhiR and to be induced by addedN-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3OH,C14:1-HSL). Mutagenesis of a cosmid carrying the rhiABC and rhiR gene region identified a gene (rhiI) that affects the level of rhiAexpression. Mutation of rhiI slightly increased the number of nodules formed on the pea. The rhiI gene is (likerhiA) regulated by rhiR in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of N-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces N-(hexanoyl)-l-homoserine lactone (C6-HSL) andN-(octanoyl)-l-homoserine lactone (C8-HSL). Both of these AHLs induced rhiA-lacZand rhiI-lacZ expression on plasmids introduced into anAgrobacterium strain that produces no AHLs, showing thatrhiI is positively regulated by autoinduction. However, in this system no induction of rhiA or rhiI with 3OH,C14:1-HSL was observed. Analysis of the spent culture supernatant of the wild-type R. leguminosarum bv. viciae revealed that at least seven different AHLs are made. Mutation ofrhiI decreased the amounts of C6-HSL and C8-HSL but did not block their formation, and in this background the rhiI mutation did not significantly affect the expression levels of the rhiI gene orrhiABC genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in R. leguminosarum bv. viciae and that they affect rhiI and rhiABC expression. We postulate that the previously observed induction of rhiA by 3OH,C14:1-HSL may be due to an indirect effect caused by induction of other AHL production loci.

scholarly journals Exploring the transcriptome ofluxI−andΔainSmutants and the impact of N-3-oxo-hexanoyl-L- and N-3-hydroxy-decanoyl-L-homoserine lactones on biofilm formation inAliivibrio salmonicida

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6845
Author(s):  
Miriam Khider ◽  
Hilde Hansen ◽  
Erik Hjerde ◽  
Jostein A. Johansen ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Expression Patterns ◽  
Critical Role ◽  
Transcriptome Profiling ◽  
Global Gene Expression ◽  
Gene Clusters ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Homoserine Lactones ◽  
The Impact

BackgroundBacterial communication through quorum sensing (QS) systems has been reported to be important in coordinating several traits such as biofilm formation. InAliivibrio salmonicidatwo QS systems the LuxI/R and AinS/R, have been shown to be responsible for the production of eight acyl-homoserine lactones (AHLs) in a cell density dependent manner. We have previously demonstrated that inactivation of LitR, the master regulator of the QS system resulted in biofilm formation, similar to the biofilm formed by the AHL deficient mutantΔainSluxI−. In this study, we aimed to investigate the global gene expression patterns ofluxIandainSautoinducer synthases mutants using transcriptomic profiling. In addition, we examined the influence of the different AHLs on biofilm formation.ResultsThe transcriptome profiling ofΔainSandluxI−mutants allowed us to identify genes and gene clusters regulated by QS inA. salmonicida. Relative to the wild type, theΔainSandluxI−mutants revealed 29 and 500 differentially expressed genes (DEGs), respectively. The functional analysis demonstrated that the most pronounced DEGs were involved in bacterial motility and chemotaxis, exopolysaccharide production, and surface structures related to adhesion. Inactivation ofluxI, but notainSgenes resulted in wrinkled colony morphology. While inactivation of both genes (ΔainSluxI−) resulted in strains able to form wrinkled colonies and mushroom structured biofilm. Moreover, when theΔainSluxI−mutant was supplemented with N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), the biofilm did not develop. We also show that LuxI is needed for motility and for repression of EPS production, where repression of EPS is likely operated through the RpoQ-sigma factor.ConclusionThese findings imply that the LuxI and AinS autoinducer synthases play a critical role in the regulation of biofilm formation, EPS production, and motility.

scholarly journals The Complex Quorum Sensing Circuitry of Burkholderia thailandensis Is Both Hierarchically and Homeostatically Organized

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Servane Le Guillouzer ◽  
Marie-Christine Groleau ◽  
Eric Déziel
Keyword(s):  
Quorum Sensing ◽  
Regulatory Network ◽  
Target Genes ◽  
Wide Spectrum ◽  
Homoserine Lactone ◽  
Growth Stages ◽  
Dependent Manner ◽  
Burkholderia Thailandensis ◽  
Bacterial Communication ◽  
A Cell

ABSTRACT The genome of the bacterium Burkholderia thailandensis encodes three complete LuxI/LuxR-type quorum sensing (QS) systems: BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). The LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3 modulate the expression of target genes in association with various N-acyl-l-homoserine lactones (AHLs) as signaling molecules produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3. We have systematically dissected the complex QS circuitry of B. thailandensis strain E264. Direct quantification of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), the primary AHLs produced by this bacterium, was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in QS deletion mutants. This was compared to the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, the levels of expression of btaR1, btaR2, and btaR3 were monitored by quantitative reverse transcription-PCR (qRT-PCR). We observed that C8-HSL, 3OHC10-HSL, and 3OHC8-HSL are differentially produced over time during bacterial growth and correlate with the btaI1, btaI2, and btaI3 gene expression profiles, revealing a successive activation of the corresponding QS systems. Moreover, the transcription of the btaR1, btaR2, and btaR3 genes is modulated by cognate and noncognate AHLs, showing that their regulation depends on themselves and on other QS systems. We conclude that the three QS systems in B. thailandensis are interdependent, suggesting that they cooperate dynamically and function in a concerted manner in modulating the expression of QS target genes through a successive regulatory network. IMPORTANCE Quorum sensing (QS) is a widespread bacterial communication system coordinating the expression of specific genes in a cell density-dependent manner and allowing bacteria to synchronize their activities and to function as multicellular communities. QS plays a crucial role in bacterial pathogenicity by regulating the expression of a wide spectrum of virulence/survival factors and is essential to environmental adaptation. The results presented here demonstrate that the multiple QS systems coexisting in the bacterium Burkholderia thailandensis, which is considered the avirulent version of the human pathogen Burkholderia pseudomallei and thus commonly used as an alternative study model, are hierarchically and homeostatically organized. We found these QS systems to be finely integrated into a complex regulatory network, including transcriptional and posttranscriptional interactions, and further incorporating growth stages and temporal expression. These results provide a unique, comprehensive illustration of a sophisticated QS network and will contribute to a better comprehension of the regulatory mechanisms that can be involved in the expression of QS-controlled genes, in particular those associated with the establishment of host-pathogen interactions and acclimatization to the environment. IMPORTANCE Quorum sensing (QS) is a widespread bacterial communication system coordinating the expression of specific genes in a cell density-dependent manner and allowing bacteria to synchronize their activities and to function as multicellular communities. QS plays a crucial role in bacterial pathogenicity by regulating the expression of a wide spectrum of virulence/survival factors and is essential to environmental adaptation. The results presented here demonstrate that the multiple QS systems coexisting in the bacterium Burkholderia thailandensis, which is considered the avirulent version of the human pathogen Burkholderia pseudomallei and thus commonly used as an alternative study model, are hierarchically and homeostatically organized. We found these QS systems to be finely integrated into a complex regulatory network, including transcriptional and posttranscriptional interactions, and further incorporating growth stages and temporal expression. These results provide a unique, comprehensive illustration of a sophisticated QS network and will contribute to a better comprehension of the regulatory mechanisms that can be involved in the expression of QS-controlled genes, in particular those associated with the establishment of host-pathogen interactions and acclimatization to the environment.

scholarly journals Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi ProducesN-Acyl-Homoserine Lactone Autoinducers

2015 ◽  
Vol 81 (17) ◽  
pp. 5917-5926 ◽  
Author(s):  
Brett L. Mellbye ◽  
Peter J. Bottomley ◽  
Luis A. Sayavedra-Soto
Keyword(s):  
Mass Spectrometry ◽  
Chemostat Culture ◽  
Rhodopseudomonas Palustris ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Acyl Homoserine Lactone ◽  
Dependent Manner ◽  
Content Type ◽  
Genes Encoding ◽  
A Cell

ABSTRACTNitrobacter winogradskyiis a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functionalN-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. TheN. winogradskyigenome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626,nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627,nwiR) with amino acid sequences 38 to 78% identical to those inRhodopseudomonas palustrisand otherRhizobiales. Expression ofnwiIandnwiRcorrelated with acyl-HSL production during culture.N. winogradskyiproduces two distinct acyl-HSLs,N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

scholarly journals Structural determinants driving homoserine lactone ligand selection in thePseudomonas aeruginosaLasR quorum-sensing receptor

2018 ◽  
Vol 116 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Amelia R. McCready ◽  
Jon E. Paczkowski ◽  
Brad R. Henke ◽  
Bonnie L. Bassler
Keyword(s):  
Quorum Sensing ◽  
Ligand Binding ◽  
Homoserine Lactone ◽  
Communication Process ◽  
Signal Molecules ◽  
Structural Determinants ◽  
Homoserine Lactones ◽  
Ligand Selectivity ◽  
Ligand Promiscuity

Quorum sensing is a cell–cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use thePseudomonas aeruginosaLasR quorum-sensing receptor to explore signal discrimination. The cognate autoinducer, 3OC12homoserine lactone (3OC12HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC12HSL, while enhancing discrimination against noncognate autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC12HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate autoinducers. Comparison with existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits.

scholarly journals Two Dissimilar N-Acyl-Homoserine Lactone Acylases of Pseudomonas syringae Influence Colony and Biofilm Morphology

2008 ◽  
Vol 75 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Ryan W. Shepherd ◽  
Steven E. Lindow
Keyword(s):  
Pseudomonas Syringae ◽  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Associated Bacteria ◽  
Homoserine Lactones ◽  
Amide Bonds ◽  
Acyl Homoserine Lactones ◽  
Complex Habitat ◽  
Long Chain

ABSTRACT Plant aerial surfaces comprise a complex habitat for microorganisms, and many plant-associated bacteria, such as the pathogen Pseudomonas syringae, exhibit density-dependent survival on leaves by utilizing quorum sensing (QS). QS is often mediated by diffusible signals called N-acyl-homoserine lactones (AHLs), and P. syringae utilizes N-3-oxo-hexanoyl-dl-homoserine lactone (3OC6HSL) to control traits influencing epiphytic fitness and virulence. The P. syringae pathovar syringae B728a genome sequence revealed two putative AHL acylases, termed HacA (Psyr_1971) and HacB (Psyr_4858), which are N-terminal nucleophile hydrolases that inactivate AHLs by cleaving their amide bonds. HacA is a secreted AHL acylase that degrades only long-chain (C ≥ 8) AHLs, while HacB is not secreted and degrades all tested AHLs. Targeted disruptions of hacA, hacB, and hacA and hacB together do not alter endogenous 3OC6HSL levels under the tested conditions. Surprisingly, targeted disruptions of hacA alone and hacA and hacB together confer complementable phenotypes that are very similar to autoaggregative phenotypes seen in other species. While AHL acylases might enable P. syringae B728a to degrade signals of competing species and block expression of their QS-dependent traits, these enzymes also play fundamental roles in biofilm formation.

scholarly journals Disrupting quorum sensing alters social interactions in Chromobacterium violaceum

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Sonia Mion ◽  
Nathan Carriot ◽  
Julien Lopez ◽  
Laure Plener ◽  
Annick Ortalo-Magné ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Social Interactions ◽  
Antibiotic Production ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Chromobacterium Violaceum ◽  
Dependent Manner ◽  
Gram Negative ◽  
A Cell ◽  
Microbial Population Dynamics

AbstractQuorum sensing (QS) is a communication system used by bacteria to coordinate a wide panel of biological functions in a cell density-dependent manner. The Gram-negative Chromobacterium violaceum has previously been shown to use an acyl-homoserine lactone (AHL)-based QS to regulate various behaviors, including the production of proteases, hydrogen cyanide, or antimicrobial compounds such as violacein. By using combined metabolomic and proteomic approaches, we demonstrated that QS modulates the production of antimicrobial and toxic compounds in C. violaceum ATCC 12472. We provided the first evidence of anisomycin antibiotic production by this strain as well as evidence of its regulation by QS and identified new AHLs produced by C. violaceum ATCC 12472. Furthermore, we demonstrated that targeting AHLs with lactonase leads to major QS disruption yielding significant molecular and phenotypic changes. These modifications resulted in drastic changes in social interactions between C. violaceum and a Gram-positive bacterium (Bacillus cereus), a yeast (Saccharomyces cerevisiae), immune cells (murine macrophages), and an animal model (planarian Schmidtea mediterranea). These results underscored that AHL-based QS plays a key role in the capacity of C. violaceum to interact with micro- and macroorganisms and that quorum quenching can affect microbial population dynamics beyond AHL-producing bacteria and Gram-negative bacteria.

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