scholarly journals Chitinolytic Activity in Chromobacterium violaceum: Substrate Analysis and Regulation by Quorum Sensing

1998 ◽  
Vol 180 (17) ◽  
pp. 4435-4441 ◽  
Author(s):  
Leonid S. Chernin ◽  
Michael K. Winson ◽  
Jacquelyn M. Thompson ◽  
Shoshan Haran ◽  
Barrie W. Bycroft ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Specificity ◽  
Homoserine Lactone ◽  
Chromobacterium Violaceum ◽  
Chitinolytic Activity ◽  
Gram Negative ◽  
Molecular Masses ◽  
Chitinase Production

ABSTRACT Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHLN-hexanoyl-l-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers ofN-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant ofC. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 μM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

scholarly journals Effect of New Analogs of Hexyloxy Phenyl Imidazoline on Quorum Sensing in Chromobacterium violaceum and In Silico Analysis of Ligand-Receptor Interactions

2020 ◽  
Vol 2020 ◽  
pp. 1-18 ◽  
Author(s):  
José Luis Herrera-Arizmendi ◽  
Everardo Curiel-Quesada ◽  
José Correa-Basurto ◽  
Martiniano Bello ◽  
Alicia Reyes-Arellano
Keyword(s):  
Quorum Sensing ◽  
In Silico Analysis ◽  
Binding Mode ◽  
Homoserine Lactone ◽  
Chromobacterium Violaceum ◽  
Public Health Issue ◽  
Resistant Bacteria ◽  
Attractive Alternative ◽  
Allosteric Site ◽  
Gram Negative

The increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. There are currently some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9) demonstrated 71% inhibitory activity of QS at 100 μM (IC50 = 90.9 μM) in Chromobacterium violaceum, a Gram-negative bacterium. The aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13–15) and three 2-oxazolines (16–18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. The binding mode of the test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-pentyloxyphenyl-2-imidazoline (13) reduced the production of violacein (IC50 = 56.38 μM) without affecting bacterial growth, suggesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C6-AHL) on the CviR protein. Ten amino acid residues in the active binding site of C6-AHL in the Cvi receptor interacted with 13, and 7 of these are the same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and 9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C6-AHL for the orthosteric site.

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.

scholarly journals Analysis of LuxR Regulon Gene Expression during Quorum Sensing in Vibrio fischeri

2007 ◽  
Vol 189 (11) ◽  
pp. 4127-4134 ◽  
Author(s):  
Nan Qin ◽  
Sean M. Callahan ◽  
Paul V. Dunlap ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Vibrio Fischeri ◽  
Stationary Phases ◽  
Regulatory Element ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Homoserine Lactone ◽  
Mobility Shift

ABSTRACT The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-l-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position −40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth.

scholarly journals β-Cyclodextrin Interaction with N-Hexanoyl Homoserine Lactone as Quorum Sensing Signal Produced in Gram-Negative Bacteria

2012 ◽  
Vol 37 (2) ◽  
pp. 315-318 ◽  
Author(s):  
Chigusa Okano ◽  
Marina Arai ◽  
Eri Nasuno ◽  
Ken-ichi Iimura ◽  
Tomohiro Morohoshi ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Gram Negative Bacteria ◽  
Gram Negative

Variations on a Theme: Diverse N-Acyl Homoserine Lactone-Mediated Quorum Sensing Mechanisms in Gram-Negative Bacteria

2006 ◽  
Vol 89 (3-4) ◽  
pp. 167-211 ◽  
Author(s):  
Debra Smith ◽  
Jin-Hong Wang ◽  
Jane E. Swatton ◽  
Peter Davenport ◽  
Bianca Price ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Variations On A Theme

scholarly journals Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

2007 ◽  
Vol 73 (20) ◽  
pp. 6339-6344 ◽  
Author(s):  
Tomohiro Morohoshi ◽  
Toshitaka Shiono ◽  
Kiyomi Takidouchi ◽  
Masashi Kato ◽  
Norihiro Kato ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Serratia Marcescens ◽  
Biofilm Formation ◽  
Acyl Chain ◽  
Regulatory System ◽  
Homoserine Lactone ◽  
Signal Molecule ◽  
Swarming Motility ◽  
Gram Negative ◽  
Acylhomoserine Lactone

ABSTRACT Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C6-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C9-CPA), had a strong inhibitory effect on prodigiosin production. C9-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C9-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C6-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C9-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.

scholarly journals Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium byLeuconostoc mesenteroides NRRL B-1299

1998 ◽  
Vol 64 (4) ◽  
pp. 1298-1302 ◽  
Author(s):  
Marguerite Dols ◽  
M. Remaud-Simeon ◽  
R. M. Willemot ◽  
M. Vignon ◽  
P. Monsan
Keyword(s):  
Gel Electrophoresis ◽  
Leuconostoc Mesenteroides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Traditional Culture ◽  
Culture Conditions ◽  
Acceptor Reaction ◽  
Sucrose Medium ◽  
Molecular Masses

ABSTRACT When grown in glucose or fructose medium in the absence of sucrose,Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.

scholarly journals Eight Gram-negative bacteria are 10 000 times more sensitive to cationic detergents than to anionic detergents

2003 ◽  
Vol 49 (12) ◽  
pp. 775-779 ◽  
Author(s):  
Soumitra Rajagopal ◽  
Nicole Eis ◽  
Kenneth W Nickerson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Cetyltrimethylammonium Bromide ◽  
Liquid Culture ◽  
Sodium Dodecyl ◽  
Homoserine Lactone ◽  
Luria Bertani ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cationic Detergents ◽  
Dodecyl Sulfate

In liquid culture, eight typical Gram-negative bacteria were ca. 10 000-fold more sensitive to cationic detergents than to the anionic detergent sodium dodecyl sulfate. Cetyltrimethylammonium bromide (CTAB) was inhibitory at concentrations ranging from 0.0006% to 0.01%. Four pseudomonads able to form biofilms were ca. 1000-fold more resistant to CTAB on Luria–Bertani agar plates than they were in liquid culture. A lasI mutant of Pseudomonas aerugi nosa was only able to tolerate 0.1% CTAB on Luria–Bertani agar plates but could tolerate 5% CTAB when supplemented with homoserine lactone containing culture supernatants.Key words: sodium dodecyl sulfate, cetyltrimethylammonium bromide, bacterial detergent resistance, homoserine lactones, Pseudomonas biofilms.

Protein Expression in Vibrio parahaemolyticus 690 Subjected to Sublethal Heat and Ethanol Shock Treatments

2008 ◽  
Vol 71 (11) ◽  
pp. 2289-2294 ◽  
Author(s):  
MING-LUN CHIANG ◽  
WEI-LI HO ◽  
ROCH-CHUI YU ◽  
CHENG-CHUN CHOU
Keyword(s):  
Heat Shock ◽  
Vibrio Parahaemolyticus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Test Organism ◽  
Protein Patterns ◽  
One Dimensional ◽  
Sds Page ◽  
Molecular Masses ◽  
Stressed Cells

Cells of Vibrio parahaemolyticus 690 were subjected either to heat shock at 42°C for 45 min or to ethanol shock in the presence of 5% ethanol for 60 min. The protein profiles of the unstressed and stressed V. parahaemolyticus cells were compared. Additionally, the induction of DnaK- and GroEL-like proteins in the unstressed and stressed cells of V. parahaemolyticus was also examined. Analysis with one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) indicated that three proteins with molecular masses of 93, 77, and 58 kDa were induced by both heat shock and ethanol shock. The protein patterns revealed by two-dimensional electrophoresis were more detailed than those revealed by one-dimensional SDS-PAGE. It was found that heat shock and ethanol shock affected the expression of a total of 28 proteins. Among them, four proteins with molecular masses of 94, 32.1, 26.7, and 25.7 kDa were enhanced by both heat shock and ethanol shock. Furthermore, immunoblot analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the test organism, with the heat-shocked and ethanol-shocked cells producing a GroEL-like protein in a larger quantity than the unstressed cells. However, DnaK-like protein was not detectable in either the unstressed or the stressed cells.

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