scholarly journals Structural insights into acyl-ACP selective recognition by the Aeromonas hydrophila AHL synthase AhyI

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Lei Jin ◽  
Jingjiao Bao ◽  
Yu Chen ◽  
Wenge Yang ◽  
Wenyi Du
Keyword(s):  
Aeromonas Hydrophila ◽  
Colorimetric Assay ◽  
Acyl Chain ◽  
Homoserine Lactone ◽  
Selective Recognition ◽  
Structural Basis ◽  
Acyl Donor ◽  
E Coli ◽  
Long Chain

Abstract 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.

scholarly journals Structural Insight Into Aeromonas Hydrophila AHL Synthase AhyI Driving Acyl-ACP Selective Recognition

2021 ◽  
Author(s):  
Lei Jin ◽  
Yu chen ◽  
Wenge Yang
Keyword(s):  
Aeromonas Hydrophila ◽  
Acyl Chain ◽  
Virulence Gene ◽  
Kinetic Studies ◽  
Homoserine Lactone ◽  
Selective Recognition ◽  
Acyl Donor ◽  
Virulence Gene Expression ◽  
Long Chain

Abstract Background: The gram-negative bacterium Aeromonas hydrophila as the major causative agent of the fish disease motile aeromonad septicemia, uses N-acyl-homoserine lactone quorum sensing signals to coordinate biofilm formation, motility and virulence gene expression in pathogens. Thus, AHL signaling pathway is considered as a therapeutic target against pathogenic A. hydrophila infection. AHL autoinducers biosynthesis in A. hydrophila are specifically catalyzed by an ACP-dependent AHL synthase AhyI using SAM and acyl-ACP as the precursors. Our previously reported AhyI protein heterologously expressed in E. coli strain showed the production characteristics of medium-long chain AHLs, although AhyI was only considered as a short-chain C4/C6-HSL synthase during the past two decades.Results: In this study, we carried out the in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs. These resulting data all indicate that C4/C6-ACP are the native acyl substrates for AhyI against acyl-ACPs with longer linear chains as the non-native acyl donor. In an effort to further understand AhyI acyl-donor substrates preferences, 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) as part of the acyl-chain binding pocket that confer AhyI to selectively recognize native C4/C6-ACP substrates. The predictions were further supported by computational Ala mutation assay.Conclusions: Our current studies redefined AhyI protein that is a multiple short- to long-chain AHL molecules synthase with longer acyl-ACPs (C8~C14) as the non-native substrates, and we also theorized that with knowledge of the key residues in AHL signal synthase AhyI to drive acyl-ACP selective recognition.

scholarly journals Structural basis for transcription inhibition by E. coli SspA

2020 ◽  
Vol 48 (17) ◽  
pp. 9931-9942 ◽  
Author(s):  
Fulin Wang ◽  
Jing Shi ◽  
Dingwei He ◽  
Bei Tong ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Acid Tolerance ◽  
In Vitro Transcription ◽  
Nucleotide Metabolism ◽  
Structural Basis ◽  
E Coli ◽  
Promoter Escape ◽  
Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid

2018 ◽  
Vol 475 (8) ◽  
pp. 1473-1489 ◽  
Author(s):  
Yang Xu ◽  
Roman Holic ◽  
Darren Li ◽  
Xue Pan ◽  
Elzbieta Mietkiewska ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linolenic Acid ◽  
Seed Oil ◽  
Diacylglycerol Acyltransferase ◽  
Acyl Donor ◽  
Flax Seed ◽  
Chain Acyl ◽  
Long Chain

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis) or linoleic acid (18:2Δ9cis,12cis). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

scholarly journals Reversible Acyl-Homoserine Lactone Binding to Purified Vibrio fischeri LuxR Protein

2004 ◽  
Vol 186 (3) ◽  
pp. 631-637 ◽  
Author(s):  
M. L. Urbanowski ◽  
C. P. Lostroh ◽  
E. P. Greenberg
Keyword(s):  
Quorum Sensing ◽  
Promoter Region ◽  
Vibrio Fischeri ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
E Coli ◽  
High Affinity ◽  
Founding Member ◽  
Luxr Homolog

ABSTRACT The Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorum-sensing transcription factors. Previous genetic evidence indicates that in the presence of its quorum-sensing signal, N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), LuxR binds to lux box DNA within the promoter region of the luxI gene and activates transcription of the luxICDABEG luminescence operon. We have purified LuxR from recombinant Escherichia coli. Purified LuxR binds specifically and with high affinity to DNA containing a lux box. This binding requires addition of 3OC6-HSL to the assay reactions, presumably forming a LuxR-3OC6-HSL complex. When bound to the lux box at the luxI promoter in vitro, LuxR-3OC6-HSL enables E. coli RNA polymerase to initiate transcription from the luxI promoter. Unlike the well-characterized LuxR homolog TraR in complex with its signal (3-oxo-octanoyl-HSL), the LuxR-30C6-HSL complex can be reversibly inactivated by dilution, suggesting that 3OC6-HSL in the complex is not tightly bound and is in equilibrium with the bulk solvent. Thus, although LuxR and TraR both bind 3-oxoacyl-HSLs, the binding is qualitatively different. The differences have implications for the ways in which these proteins respond to decreases in signal concentrations or rapid drops in population density.

scholarly journals Short-chain cyanoacrylates and long-chain cyanoacrylates (Dermabond) have different antimicrobial effects

2021 ◽  
Vol 6 (1) ◽  
pp. e000591
Author(s):  
Richard Yudi Hida ◽  
Cely Barreto Silva ◽  
Ivana Lopes Romero-Kusabara ◽  
Lycia Mara Jenne Mimica
Keyword(s):  
Short Chain ◽  
Klebsiella Pneumonia ◽  
Bacteriostatic Activity ◽  
Bacterial Strains ◽  
Total Size ◽  
Filter Paper Disc ◽  
E Coli ◽  
Ethyl Cyanoacrylate ◽  
Long Chain

ObjectiveTo compare the antimicrobial effect in vitro of a short-chain cyanoacrylate with a long-chain cyanoacrylate (Dermabond, Ethicon, Johnson and Johnson, USA) against bacterial strains.Methods and analysisThe following bacterial strains were analysed: Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. For each microorganism, standardised sterile discs (6 mm) containing 10 µL of ethyl-cyanoacrylate and 2-octyl cyanoacrylate were applied to the plate. All plates received a blank filter-paper disc with no adhesive (control). All plates were incubated for 24 hours, after which the bacterial inhibitory halos, if present, were measured in millimetres in its greater length.ResultsInhibitory halos were observed for both adhesives for S. aureus. Inhibition halos were observed only for ethyl-cyanoacrylate for K. pneumoniae and E. coli. No inhibition halo was observed for P. aeruginosa in any sample. The relationship between the total size of the inhibition halos and the diameter of the paper filter for S. aureus was statistically significant compared with 2-octyl cyanoacrylate.ConclusionData shown conclude that ethyl-cyanoacrylate showed in vitro bacteriostatic activity for S. aureus, E. coli and K. pneumoniae. 2-Octyl cyanoacrylate showed in vitro lower bacteriostatic activity only against S. aureus when compared with ethyl-cyanoacrylate. No in vitro bactericidal activity of ethyl-cyanoacrylate or 2-octyl cyanoacrylate was observed.

scholarly journals Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria

2018 ◽  
Vol 14 ◽  
pp. 1309-1316 ◽  
Author(s):  
Lisa Ziesche ◽  
Jan Rinkel ◽  
Jeroen S Dickschat ◽  
Stefan Schulz
Keyword(s):  
Quorum Sensing ◽  
Chain Length ◽  
Acyl Chain ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Incubation Experiments ◽  
Dinoroseobacter Shibae ◽  
A Cell ◽  
Acyl Coenzyme A

N-Acylhomoserine lactones (AHLs) are important bacterial messengers, mediating different bacterial traits by quorum sensing in a cell-density dependent manner. AHLs are also produced by many bacteria of the marine Roseobacter group, which constitutes a large group within the marine microbiome. Often, specific mixtures of AHLs differing in chain length and oxidation status are produced by bacteria, but how the biosynthetic enzymes, LuxI homologs, are selecting the correct acyl precursors is largely unknown. We have analyzed the AHL production in Dinoroseobacter shibae and three Phaeobacter inhibens strains, revealing strain-specific mixtures. Although large differences were present between the species, the fatty acid profiles, the pool for the acyl precursors for AHL biosynthesis, were very similar. To test the acyl-chain selectivity, the three enzymes LuxI1 and LuxI2 from D. shibae DFL-12 as well as PgaI2 from P. inhibens DSM 17395 were heterologously expressed in E. coli and the enzymes isolated for in vitro incubation experiments. The enzymes readily accepted shortened acyl coenzyme A analogs, N-pantothenoylcysteamine thioesters of fatty acids (PCEs). Fifteen PCEs were synthesized, varying in chain length from C4 to C20, the degree of unsaturation and also including unusual acid esters, e.g., 2E,11Z-C18:2-PCE. The latter served as a precursor of the major AHL of D. shibae DFL-12 LuxI1, 2E,11Z-C18:2-homoserine lactone (HSL). Incubation experiments revealed that PgaI2 accepts all substrates except C4 and C20-PCE. Competition experiments demonstrated a preference of this enzyme for C10 and C12 PCEs. In contrast, the LuxI enzymes of D. shibae are more selective. While 2E,11Z-C18:2-PCE is preferentially accepted by LuxI1, all other PCEs were not, except for the shorter, saturated C10–C14-PCEs. The AHL synthase LuxI2 accepted only C14 PCE and 3-hydroxydecanoyl-PCE. In summary, chain-length selectivity in AHLs can vary between different AHL enzymes. Both, a broad substrate acceptance and tuned specificity occur in the investigated enzymes.

scholarly journals Conversion of the Vibrio fischeriTranscriptional Activator, LuxR, to a Repressor

2000 ◽  
Vol 182 (3) ◽  
pp. 805-811 ◽  
Author(s):  
Kristi A. Egland ◽  
E. P. Greenberg
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Vibrio Fischeri ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Mutant Proteins ◽  
E Coli ◽  
Polymerase Binding ◽  
Regulatory Dna

ABSTRACT The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with ς70 RNA polymerase it can activate transcription of the lux operon. Both the truncated LuxR and RNA polymerase are required for binding tolux regulatory DNA in vitro. We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus −35 and −10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiplelux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind tolux boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a nativelux operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.

scholarly journals A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Miglena Manandhar ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Chemical Synthesis ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Bacillus Sphaericus ◽  
Content Type ◽  
E Coli

ABSTRACTBioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed,in vitrotheEscherichia coliandBacillus sphaericusenzymes have been shown to condense pimeloyl-CoA withl-alanine in a pyridoxal 5′-phosphate-dependent reaction with concomitant CoA release and decarboxylation ofl-alanine. However, recentin vivostudies ofE. coliandBacillus subtilissuggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in thatE. coliBioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast,B. subtilisBioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic andin vitrodata demonstrating thatB. subtilisBioF specifically utilizes pimeloyl-CoA.IMPORTANCEBiotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of theBacillus subtilispathway and shows that it differs from that ofEscherichia coliin the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis.

scholarly journals UJI IN VITRO BAKTERI ANTI QUORUM SENSING PENDEGRADASI ACYL HOMOSERINE LACTONE Aeromonas hydrophila

2017 ◽  
Vol 11 (3) ◽  
pp. 291
Author(s):  
Hessy Novita ◽  
Iman Rusmana ◽  
Munti Yuhana ◽  
Fachriyan Hasmi Pasaribu ◽  
Angela Mariana Lusiastuti
Keyword(s):  
Quorum Sensing ◽  
Fresh Water ◽  
Virulence Factors ◽  
Aeromonas Hydrophila ◽  
Homoserine Lactone ◽  
In Vitro Assay ◽  
Acyl Homoserine Lactone ◽  
Bacillus Sp ◽  
Vitro Assay

Anti quorum sensing (AQS) adalah proses inaktivasi atau degradasi molekul sinyal quorum sensing (QS) yaitu acyl homoserine lactone (AHL) tanpa memengaruhi pertumbuhan bakteri. Tujuan dari penelitian ini adalah untuk melakukan uji kultur bersama dan uji penghambatan faktor virulensi secara in vitro antara bakteri AQS dengan Aeromonas hydrophila sebagai patogen yang menyebabkan Motile Aeromonad Septicaemia (MAS) pada ikan air tawar. Hasil penelitian menunjukkan bahwa uji in vitro dengan kultur bersama antara bakteri AQS Bacillus sp. dan A. hydrophila tidak ada penghambatan pertumbuhan pada kedua bakteri, tetapi bakteri AQS dapat menghambat produksi faktor virulensi dari A. hydrophila yaitu protease dan hemolisin. AQS merupakan salah satu strategi yang potensial untuk diaplikasikan dalam pengendalian penyakit infeksius atau bakteri patogen resisten antibiotik pada budidaya ikan air tawar.Anti quorum sensing (AQS) was process of inactivation or degradation of Quorum sensing signal molecules of acyl homoserine lactone (AHL) without affecting growth of the bacteria. The aim of the reseach was to study in vitro assay of co-culture and inhibition of virulence factors between AQS bacteria which Aeromonas hydrophila as pathogen caused motile aeromonad septicaemia (MAS) in fresh water fish. The result showed that in vitro assay of co culture between AQS bacteria Bacillus sp. and A. hydrophila without inhibited of growth in both bacteria but bacteria AQS could suppressed production A. hydrophila virulence factors, protease, and hemolysin. The AQS is one of potential strategies to inhibit QS for application to control of infectious diseases or antibiotic resistant bacterial pathogens in fresh water aquaculture.

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