Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

ABSTRACT Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophicEnterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 106 CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains ofEnterobacteriaceae stored at 5°C under an N2atmosphere when mean cell densities increased to 106 CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenousEnterobacteriaceae reached 106 CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.

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Production of acylated homoserine lactones byAeromonasandPseudomonasstrains isolated from municipal activated sludge

Canadian Journal of Microbiology ◽

10.1139/w05-077 ◽

2005 ◽

Vol 51 (11) ◽

pp. 924-933 ◽

Cited By ~ 30

Author(s):

Fernando Morgan-Sagastume ◽

Nico Boon ◽

Sofie Dobbelaere ◽

Tom Defoirdt ◽

Willy Verstraete

Keyword(s):

Activated Sludge ◽

Homoserine Lactone ◽

Homoserine Lactones ◽

Pseudomonas Spp ◽

Signal Production ◽

Reporter Strains ◽

Overlay Technique ◽

Different Strains ◽

Acylated Homoserine Lactones ◽

Layer Chromatography

Up to now, the production and role of N-acyl homoserine lactones (AHLs) in activated sludge have been poorly understood. In this study, cross-feeding assays with the reporter strains Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 were used to investigate AHL signal production by municipal activated sludge samples. AHL signal production was consistently detected from municipal activated sludge when different samples were incubated on nutrient media. From one municipal activated sludge sample, 10 strains producing AHL-like auto inducers were isolated by an overlay technique. 16S rDNA-based phylogenetic analysis showed that eight of the isolates belonged to Aeromonas spp. and two to Pseudomonas spp. Box-PCR indicated that six of these Aeromonas isolates were different strains and the two Pseudomonas strains were identical. The production of AHL or AHL-like compounds by these strains was confirmed by thin layer chromatography and biosensor overlays. The six different Aeromonas strains were found to produce the same set of AHLs, including N-hexanoyl-L-homoserine lactone. These results may indicate the possible presence of AHLs in municipal activated sludge. The potential roles of AHL in this eco system are briefly discussed.Key words: municipal activated sludge, acylated homoserine lactones, Aeromonas spp., bioaggregates, Pseudomonas spp., AHL biosensors.

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A Marine Mesorhizobium sp. Produces Structurally Novel Long-Chain N-Acyl-l-Homoserine Lactones

Applied and Environmental Microbiology ◽

10.1128/aem.02344-06 ◽

2007 ◽

Vol 73 (11) ◽

pp. 3587-3594 ◽

Cited By ~ 29

Author(s):

Anja Krick ◽

Stefan Kehraus ◽

Leo Eberl ◽

Kathrin Riedel ◽

Heidrun Anke ◽

...

Keyword(s):

Quorum Sensing ◽

Solid Phase ◽

Homoserine Lactone ◽

Signal Molecules ◽

Cytotoxic Activities ◽

Rrna Gene ◽

Liquid Chromatography Mass Spectrometry ◽

Homoserine Lactones ◽

Chain Lengths ◽

Acylated Homoserine Lactones

ABSTRACT Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C10 to C16. Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C12-homoserine lactone (5-cis-3-oxo-C12-HSL) (compound 1) and 5-cis-C12-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.

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Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3174-3179.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3174-3179 ◽

Cited By ~ 119

Author(s):

S. A. Borchardt ◽

E. J. Allain ◽

J. J. Michels ◽

G. W. Stearns ◽

R. F. Kelly ◽

...

Keyword(s):

Cell Signaling ◽

Marine Alga ◽

Defense Mechanism ◽

Microbial Control ◽

Homoserine Lactone ◽

Signaling Molecules ◽

Chromobacterium Violaceum ◽

Homoserine Lactones ◽

Acylated Homoserine Lactone ◽

Acylated Homoserine Lactones

ABSTRACT Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817239116 ◽

2018 ◽

Vol 116 (1) ◽

pp. 245-254 ◽

Cited By ~ 16

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.

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Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl)homoserine Lactone

Applied and Environmental Microbiology ◽

10.1128/aem.71.4.2113-2120.2005 ◽

2005 ◽

Vol 71 (4) ◽

pp. 2113-2120 ◽

Cited By ~ 32

Author(s):

L. R. Flodgaard ◽

P. Dalgaard ◽

J. B. Andersen ◽

K. F. Nielsen ◽

M. Givskov ◽

...

Keyword(s):

High Performance ◽

High Resolution Mass Spectrometry ◽

Cell Communication ◽

Homoserine Lactone ◽

Photobacterium Phosphoreum ◽

Relative Mobility ◽

Homoserine Lactones ◽

Communication Signal ◽

Culture Supernatants ◽

Acylated Homoserine Lactones

ABSTRACT Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.

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Long- and Short-Chain Plant-Produced Bacterial N-Acyl-Homoserine Lactones Become Components of Phyllosphere, Rhizosphere, and Soil

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0227 ◽

2006 ◽

Vol 19 (3) ◽

pp. 227-239 ◽

Cited By ~ 43

Author(s):

Russell A. Scott ◽

Jason Weil ◽

Phuong T. Le ◽

Paul Williams ◽

Rupert G. Fray ◽

...

Keyword(s):

Leaf Tissue ◽

Homoserine Lactone ◽

Wild Type ◽

Fresh Weight ◽

Liquid Chromatograph ◽

Homoserine Lactones ◽

Transgenic Lines ◽

Axenic Conditions ◽

Root Tissues ◽

Layer Chromatography

Two N-acyl-homoserine lactone (acyl-HSL) synthase genes, lasI from Pseudomonas aeruginosa and yenI from Yersinia enterocolitica, were introduced into tobacco, individually and in combination. Liquid chromatograph-tandem mass spectrometry and thin-layer chromatography confirmed products of lasI and yenI activity in single and cotransformants. Cotransformants expressing plastid-localized LasI and YenI synthases produced the major acyl-HSLs for each synthase in all tissues tested. Total acyl-HSL signals accumulated in leaf tissue up to 3 pmol/mg of fresh weight, half as much in stem tissue, and approximately 10-fold less in root tissues. Acyl-HSLs were present in aqueous leaf washes from greenhouse-grown transgenic plants. Transgenic lines grown for 14 days under axenic conditions produced detectable levels of acyl-HSLs in root exudates. Ethyl acetate extractions of rhizosphere and nonrhizosphere soil from transgenically grown plants contained active acyl-HSLs, whereas plant-free soil or rhizosphere and nonrhizosphere soil from wild-type plants lacked detectable amounts of acyl-HSLs. This work shows that bioactive acyl-HSLs are exuded from leaves and roots and accumulate in the phytosphere of plants engineered to produce acyl-HSLs. These data further suggest that plants that are bioengineered to synthesize acyl-HSLs can foster beneficial plant-bacteria communications or deter deleterious interactions. Therefore, it is feasible to use bioengineered plants to supplement soils with specific acyl-HSLs to modulate bacterial phenotypes and plant-associated bacterial community structures.

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Biofilms on Indwelling Urethral Catheters Produce Quorum-Sensing Signal Molecules In Situ and In Vitro

Applied and Environmental Microbiology ◽

10.1128/aem.64.9.3486-3490.1998 ◽

1998 ◽

Vol 64 (9) ◽

pp. 3486-3490 ◽

Cited By ~ 146

Author(s):

David J. Stickler ◽

Nicola S. Morris ◽

Robert J. C. McLean ◽

Clay Fuqua

Keyword(s):

Quorum Sensing ◽

Signal Molecules ◽

Gram Negative Bacteria ◽

Feeding Assay ◽

Gram Negative ◽

Homoserine Lactones ◽

Acylated Homoserine Lactones

ABSTRACT Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for isolates of five other species. P. aeruginosa biofilms were then produced on catheters in a physical model of the bladder. Sections of colonized all-silicone catheters gave positive reactions for the quorum-sensing signal molecules as did sections that had been cleaned of biofilm and autoclaved. Control sections of unused catheters were negative in the tests. Sections from four of nine catheters that had been freshly removed from patients gave positive reactions for AHLs. Cleaned autoclaved sections of three of these catheters also gave strongly positive reactions for AHLs. These results demonstrate that AHLs are produced by biofilms as they develop on the catheters both in vitro in the model and in vivo in the patient’s bladder. They represent the first demonstration of AHL production by biofilms in a clinical setting.

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