Acyl-homoserine Lactones Receptors Research Articles

Cross-species activation of hydrogen cyanide production by a promiscuous quorum-sensing receptor promotes Chromobacterium subtsugae competition in a dual-species model.

Many saprophytic bacteria have LuxR-I-type acyl-homoserine lactone (AHL) quorum-sensing systems that may be important for competing with other bacteria in complex soil communities. LuxR AHL receptors specifically interact with cognate AHLs to cause changes in expression of target genes. Some LuxR-type AHL receptors have relaxed specificity and are responsive to non-cognate AHLs. These promiscuous receptors might be used to sense and respond to AHLs produced by other bacteria by eavesdropping. We are interested in understanding the role of eavesdropping during interspecies competition. The soil saprophyte Chromobacterium subtsugae has a single AHL circuit, CviR-I, which produces and responds to N-hexanoyl-HSL (C6-HSL). The AHL receptor CviR can respond to a variety of AHLs in addition to C6-HSL. In prior studies we have utilized a coculture model with C. subtsugae and another soil saprophyte, Burkholderia thailandensis. Using this model, we previously showed that promiscuous activation of CviR by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Here, we show that B. thailandensis AHLs activate transcription of dozens of genes in C. subtsugae, including the hcnABC genes coding for production of hydrogen cyanide. We show that hydrogen cyanide production is population density-dependent and demonstrate that the cross-induction of hydrogen cyanide by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Our results provide new information on C. subtsugae quorum sensing and are the basis for future studies aimed at understanding the role of eavesdropping in interspecies competition.

DOP50 Impact of Inflammatory Bowel Disease associated dysbiosis on bacterial quorum sensing mediated by Acyl-homoserine Lactone in human gut microbiota

Abstract Background Intestinal dysbiosis is a key feature in the pathogenesis of inflammatory bowel diseases (IBD). Bacterial quorum sensing mediated by acyl-homoserine lactones (AHL) might play a role in the dialogue between the gut microbiota and the host. The main objective of our study was to investigate the presence and expression of AHL synthase and receptor genes in the human gut ecosystem during IBD. Methods To confirm the presence of AHL in the gut, mass spectrometric detection was performed on stool samples from IBD patients and non-IBD subjects. Then, by an in silico approach, we exploited the open access database: Inflammatory Bowel Disease Multi’omics Database, an American cohort with bacterial metagenomes and metatranscriptomes data of stool samples from non-IBD and IBD subjects. To characterise gut dysbiosis, the most discriminating bacterial species between non-IBD and IBD patients were identified by multivariate analysis and allowed us to define two groups (dysbiosis/non-dysbiosis). The search for AHL synthase (luxI) and receptor (luxR) known homolog genes, was performed using Basic Local Alignment Search Tool (BLAST) from previously assembled gene files (presence/absence) as well as raw data sequencing files (relative abundance and expression). Results Mass spectrometry confirmed a higher concentration of AHL molecules in healthy subjects than in relapsing IBD. Regarding in silico analysis, 103 subjects were selected including 50 with Crohn’s disease (CD), 27 with ulcerative colitis (UC), and 26 non-IBD subjects. No luxI-like synthase genes were retrieved by BLAST searches. However, several homologs of receptor genes were identified: sdiA gene from Escherichia coli (7/103 patients) and luxR-like homologs from Bacteroides fragilis and Bacteroides dorei present in all patients. According to disease, only one luxR-like gene from Bacteroides dorei was under-expressed in IBD patients (p = 0.02) compared to non-IBD, especially in CD (p = 0.02) (Figure 1). In dysbiosis situation, one luxR receptor gene from Bacteroides fragilis appeared to be over-expressed (p = 0.04) compared to non-dysbiotic patients (Figure 2). Conclusion Through this computational approach, AHL-synthesising bacteria have not been found. However, the expression of quorum sensing receptor genes appears to be modulated by IBD-associated gut dysbiosis. The role of LuxR receptors, especially in Bacteroides species, should be investigated to understand its impact on gut microbiota (Figure 3). Targeting LuxR receptors of bacterial quorum sensing might represent a new approach to modulate the gut microbiota in IBD.

Impact of IBD-Associated Dysbiosis on Bacterial Quorum Sensing Mediated by Acyl-Homoserine Lactone in Human Gut Microbiota

Intestinal dysbiosis is a key feature in the pathogenesis of inflammatory bowel disease (IBD). Acyl-homoserine lactones (AHL) are bacterial quorum-sensing metabolites that may play a role in the changes in host cells-gut microbiota interaction observed during IBD. The objective of our study was to investigate the presence and expression of AHL synthases and receptor genes in the human gut ecosystem during IBD. We used an in silico approach, applied to the Inflammatory Bowel Disease Multi'omics Database comprising bacterial metagenomic and metatranscriptomic data from stools of patients with Crohn's disease (CD) (n = 50), ulcerative colitis (UC) (n = 27) and non-IBD controls (n = 26). No known putative AHL synthase gene was identified; however, several putative luxR receptors were observed. Regarding the expression of these receptor genes, the luxR gene from Bacteroides dorei was under-expressed in IBD patients (p = 0.02) compared to non-IBD patients, especially in CD patients (p = 0.02). In the dysbiosis situation, one luxR receptor gene from Bacteroides fragilis appeared to be over-expressed (p = 0.04) compared to that of non-dysbiotic patients. Targeting LuxR receptors of bacterial quorum sensing might represent a new approach to modulate the gut microbiota in IBD.

Molecular Mechanisms and Applications of N-Acyl Homoserine Lactone-Mediated Quorum Sensing in Bacteria.

Microbial biodiversity includes biotic and abiotic components that support all life forms by adapting to environmental conditions. Climate change, pollution, human activity, and natural calamities affect microbial biodiversity. Microbes have diverse growth conditions, physiology, and metabolism. Bacteria use signaling systems such as quorum sensing (QS) to regulate cellular interactions via small chemical signaling molecules which also help with adaptation under undesirable survival conditions. Proteobacteria use acyl-homoserine lactone (AHL) molecules as autoinducers to sense population density and modulate gene expression. The LuxI-type enzymes synthesize AHL molecules, while the LuxR-type proteins (AHL transcriptional regulators) bind to AHLs to regulate QS-dependent gene expression. Diverse AHLs have been identified, and the diversity extends to AHL synthases and AHL receptors. This review comprehensively explains the molecular diversity of AHL signaling components of Pseudomonas aeruginosa, Chromobacterium violaceum, Agrobacterium tumefaciens, and Escherichia coli. The regulatory mechanism of AHL signaling is also highlighted in this review, which adds to the current understanding of AHL signaling in Gram-negative bacteria. We summarize molecular diversity among well-studied QS systems and recent advances in the role of QS proteins in bacterial cellular signaling pathways. This review describes AHL-dependent QS details in bacteria that can be employed to understand their features, improve environmental adaptation, and develop broad biomolecule-based biotechnological applications.

Inhibition of biofilm formation and quorum sensing by soy isoflavones in Pseudomonas aeruginosa

The potential of soybean isoflavones (SI) as a quorum sensing (QS) inhibitor against Pseudomonas aeruginosa was investigated in the present study. The inhibitory effects of SI on violacein production, biofilm formation, motility, and virulence factor production were evaluated. The acyl-homoserine lactones (AHLs) were quantified by liquid chromatography-mass spectrometry (LC-MS). The QS-related genes were analyzed by qRT-PCR. The molecular docking analysis was employed to investigate the anti-QS mechanism of the SI. Results demonstrated that the SI exhibited significant inhibition ( p < 0.05) in violacein production, biofilm formation, motility, virulence factors, AHLs production, and expression levels of QS-related genes in P. aeruginosa . Additionally, the result of molecular docking analysis revealed that SI had a promising competitive interaction with AHLs. Hence, the study provided a deep insight into SI as a QS inhibitor against P. aeruginosa . • SI significantly ( p < 0.05) inhibited the violacein production in C. violaceum 026. • SI significantly ( p < 0.05) inhibited the biofilm and virulence factor production. • SI significantly ( p < 0.05) inhibited the expression levels of QS-related genes. • The competitive binding of SI with AHLs receptors inhibited the initiation of QS.

Characterization of differentiated autoregulation of LuxI/LuxR-type quorum sensing system in Pseudoalteromonas

Gram-negative bacteria usually use acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR-type quorum sensing (QS) systems for cell-cell cooperation and/or bacteria-environment communication. LuxI and LuxR are AHLs synthase and receptor, respectively. These two parts could form a positive regulatory feedback loop, controlling various types of group behaviors. However, the autoregulation mechanisms between them are fragmented and could be highly differentiated in different bacteria. Here, we clarified the autoregulation mechanism between LuxI and LuxR in Pseudoalteromonas sp. R3. YasI (LuxI in strain R3) synthesizes two types of AHLs, C8-HSL and 3-OH-C8-HSL. It is worth noting that YasR (LuxR in strain R3) only responds to C8-HSL rather than 3-OH-C8-HSL. YasR-C8HSL can activate the yasI transcription by recognizing "lux box" at yasI upstream. Interestingly, YasR can directly promote the yasR expression with AHL-independent manner, but AHL absence caused by the yasI-deficiency led to the significant decrease in the yasR expression. Further study demonstrated that the yasI-deficiency can result in the decrease in the yasR mRNA stability. Notably, both yasI-deficiency and yasR-deficiency led to the significant decrease in the expression of hfq encoding RNA chaperone. Therefore, it was speculated that not only YasR itself can directly regulate the yasR transcription, but YasR-C8HSL complex indirectly affects the yasR mRNA stability by regulating Hfq.

Quorum sensing-1 signaling of N-hexanoyl-L-homoserine lactone contributes to virulence in avian pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) caused avian colibacillosis is mostly common in poultry industry worldwide. APEC virulence factors lead to pathogenesis and the quorum sensing (QS) system is actively involved in the regulation of these virulence factors. Signaling molecules in QS are known as autoinducers (AIs). In QS-1, E. coli encodes a single LuxR homolog, i.e., SdiA, but does not express the LuxI homolog, an acyl-homoserine lactone (AHL) synthase of producing AI-1. Avian pathogenic E. coli (APEC) regulates its virulence genes expression in response to exogenous AHLs, but regulatory mechanisms of AHL and QS-1 are still unknown. This study targeted the APEC CE129 isolate as the reference strain, and the Yersinia enterocolitica yenI gene was expressed into APEC CE129. CE129/pyenI was conferred the ability to produce AHL signal. The CE129 SdiA mutant strain with an in-frame sdiA (AHL receptor) gene deletion was constructed by a λRed recombination system, which lost the ability to sense AHL. The goal of this study was to explore the function of QS-1 upon virulence and elucidate the regulatory effect of QS-1/AHL signals in the APEC strain. Adherence and invasion assays revealed that QS-1 affected APEC adherence and survival ability. APEC biofilm formation was also suppressed under C6HSL. Interestingly, APEC exhibited different phenotypes of acid tolerance and flagella expression when compared to enterotoxigenic E. coli or enterohemorrhagic E. coli (ETEC and EHEC, respectively). These findings enhance our understanding of the QS mechanism.

A widespread response of Gram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces γ-butyrolactone signaling molecules.

Cell-cell communication is critical for bacterial survival in natural habitats, in which miscellaneous regulatory networks are encompassed. However, elucidating the interaction networks of a microbial community has been hindered by the population complexity. This study reveals that γ-butyrolactone (GBL) molecules from Streptomyces species, the major antibiotic producers, can directly bind to the acyl-homoserine lactone (AHL) receptor of Chromobacterium violaceum and influence violacein production controlled by the quorum sensing (QS) system. Subsequently, the widespread responses of more Gram-negative bacterial AHL receptors to Gram-positive Streptomyces signaling molecules are unveiled. Based on the cross-talk between GBL and AHL signaling systems, combinatorial regulatory circuits (CRC) are designed and proved to be workable in Escherichia coli (E. coli). It is significant that the QS systems of Gram-positive and Gram-negative bacteria can be bridged via native Streptomyces signaling molecules. These findings pave a new path for unlocking the comprehensive cell-cell communications in microbial communities and facilitate the exploitation of innovative regulatory elements for synthetic biology.

Hfq and sRNA00002 positively regulate the LuxI/LuxR-type quorum sensing system in Pseudoalteromonas

Pseudoalteromonas spp. are Gram-negative bacteria which are ubiquitous in marine environments. Our previous work found that there is a classic LuxI/LuxR-type quorum sensing (QS) system which was named YasI/YasR in Pseudoalteromonas sp. R3, but the factors that control QS in strain R3 are unclear yet. Here, we found that the deficiency of hfq encoding RNA chaperon Hfq down-regulated the transcription levels of yasI encoding acyl-homoserine lactones (AHLs) synthase and yasR encoding AHLs receptor in strain R3. The assay based on fusion reporter of yasI-lacZ showed that Hfq regulates the expression of yasR at both transcriptional and translational levels. In addition, Hfq affects the expression of yasI via yasR. Further analysis indicated that the 5′UTR region of yasR is necessary for Hfq to control QS. In addition, the deletion of hfq increases the unstability of the target yasR mRNA. Based on transcriptome sequencing and bioinformatic analysis together with molecular experiments, Hfq-dependent sRNA00002 was identified to be involved in positively regulating QS in Pseudoalternas sp. R3. It was found that sRNA00002 deficiency causes the decrease in expression of yasI and yasR, and thus abolishes the production of AHLs in strain R3. It was concluded that Hfq-dependent sRNA00002 regulates yasR expression by base-pairing with target yasR mRNA at 5′UTR region and altering the stability of yasR mRNA. Our work paves the way for understanding the regulation mechanism of Hfq-dependent sRNAs on QS in Pseudoalteromonas.

A covariation analysis reveals elements of selectivity in quorum sensing systems.

Many bacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). In these systems, a signal synthase produces an AHL to which its paired receptor selectively responds. Selectivity is fundamental to cell signaling. Despite its importance, it has been challenging to determine how this selectivity is achieved and how AHL QS systems evolve and diversify. We hypothesized that we could use covariation within the protein sequences of AHL synthases and receptors to identify selectivity residues. We began by identifying about 6000 unique synthase-receptor pairs. We then used the protein sequences of these pairs to identify covariation patterns and mapped the patterns onto the LasI/R system from Pseudomonas aeruginosa PAO1. The covarying residues in both proteins cluster around the ligand-binding sites. We demonstrate that these residues are involved in system selectivity toward the cognate signal and go on to engineer the Las system to both produce and respond to an alternate AHL signal. We have thus demonstrated that covariation methods provide a powerful approach for investigating selectivity in protein-small molecule interactions and have deepened our understanding of how communication systems evolve and diversify.

Identification of a solo acylhomoserine lactone synthase from the myxobacterium Archangium gephyra

Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Identification and characterization of a LuxI/R-type quorum sensing system in Pseudoalteromonas

Bacteria usually produce, release and detect quorum sensing (QS)-based signal molecules, and successively orchestrate gene expression in respond to environmental changes. Pseudoalteromonas are typical marine bacteria, but knowledge on their QS systems is extremely fragmentary. In this study, genome sequencing of Pseudoalteromonas sp. R3 was performed. Accordingly, a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3. Among them, the typical LuxI/R-type QS system using acyl-homoserine lactones (AHLs) as signal molecules was characterized. Sequence similarity analysis indicated luxI encoding AHLs synthase is novel. The luxR encoding AHLs receptor is directly adjacent to luxI downstream. Notably, mutagenesis demonstrated LuxI and LuxR affect each other at transcriptional level, and both control the AHLs formation. Interestingly, it was found that LuxI/R-type QS system positively involves resistance to streptomycin. Thin-layer chromatography analysis showed strain R3 can produce 3-OH-C6-HSL and C8-HSL, which was supported by heterologous expression of LuxI in Escherichia coli. Sequence alignment analysis indicated that the N-terminal region of LuxI is more conservative than the C-terminal region, revealing the importance of N-terminal region in AHLs synthesis. The obtained findings enrich our knowledge on LuxI/R-type QS system in Pseudoalteromonas and its regulation on adaptation to marine environments.

Purification and enzymatic assay of BmaI1, a quorum sensing signal synthase

Quorum sensing is a bacterial cell‐cell communication system that functions through the synthesis, secretion, and detection of signaling molecules called autoinducers. Quorum sensing enables bacteria to assess their cell density and coordinate group behaviors that are advantageous at high cell density. Many bacteria that cause acute and chronic infections in humans use quorum sensing to control expression of virulence factors and formation of biofilms, so inhibitors of quorum sensing hold significant promise as novel antibacterial agents. By targeting bacterial virulence as opposed to growth and survival, quorum sensing inhibitors avoid the selective pressure for drug resistance that is inherent to traditional antibiotics.The main quorum sensing system in Gram‐negative bacteria is based on the synthesis and detection of acyl homoserine lactone (AHL) autoinducers. Efforts to inhibit AHL‐mediated quorum sensing have focused on developing synthetic AHL analogs that act as antagonists of AHL receptors. These compounds have been shown to block virulence controlled by quorum sensing, validating quorum sensing as an antibacterial drug target.The overall goal of the project is to develop inhibitors of AHL synthase enzymes, an alternative quorum sensing drug target that remains under‐explored. Towards this goal, we cloned and purified BmaI1, an AHL synthase from Burkholderia mallei. AHL synthases use S‐adenosyl methionine and acylated ACP protein as substrates. Generating Acyl‐ACP requires purification of ACP, and several sequential enzymatic modifications to make it suitable as an AHL synthase substrate. We have developed a small, synthetic substitute for acyl ACP that is utilized by BmaI1, avoiding the difficult and time‐consuming production of acyl‐ACP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cloning, purification, and enzymatic activity of the quorum sensing signal synthase RhlI

Quorum sensing is a bacterial cell‐cell communication system that functions through the synthesis, secretion, and detection of signaling molecules called autoinducers. Quorum sensing enables bacteria to assess their cell density and coordinate group behaviors that are advantageous at high cell density. Many bacteria that cause acute and chronic infections in humans use quorum sensing to control expression of virulence factors and formation of biofilms, so inhibitors of quorum sensing hold significant promise as novel antibacterial agents. By targeting bacterial virulence as opposed to growth and survival, quorum sensing inhibitors avoid the selective pressure for drug resistance that is inherent to traditional antibiotics.The main quorum sensing system in Gram‐negative bacteria is based on the synthesis and detection of acyl homoserine lactone (AHL) autoinducers. Efforts to inhibit AHL‐mediated quorum sensing have focused on developing synthetic AHL analogs that act as antagonists of AHL receptors. These compounds have been shown to block virulence controlled by quorum sensing, validating quorum sensing as an antibacterial drug target.The overall goal of the project is to develop inhibitors of AHL synthase enzymes, an alternative quorum sensing drug target that remains under‐explored. Towards this goal, we have cloned and purified RhlI from Pseudomonas aeruginosa and established its enzymatic activity as an assay platform for candidate synthetic inhibitors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Specific quorum sensing molecules of ammonia oxidizers and their role during ammonium metabolism in Zhalong wetland, China

The primary challenge of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) surviving in wetlands are the rapid and unpredictable environmental changes. To adapt to a fluctuant environment, ammonia oxidizers have to communicate with each other via acyl-homoserine lactones (AHLs). In this study, AOA and AOB in the soil samples taken from Zhalong wetland were incubated. Dynamics of AHLs during the incubation of ammonia oxidizers were measured. Then, the specific AHLs of AOA and AOB were identified, respectively. The results showed that AOA secreted N-butyryl-dl-homoserine lactone (C4-HSL) and N-octanoyl-l-homoserine lactone (C8-HSL) to cope with nitrite accumulation, while they secreted N-(3-oxododecanoyl)-dl-homoserine lactone (OXOC12-HSL) to regulate their ammonium metabolism activity. AOB secreted N-hexanoyl-dl-homoserine lactone (C6-HSL), N-dodecanoyl-l-homoserine lactone (C12-HSL), N-tetradecanoyl-dl-homoserine lactone (C14-HSL) and N-(3-oxododecanoyl)-tetradecanoyl-dl-homoserine lactone (OXOC14-HSL) only to enhance the metabolism activity. The dominant AOA belonged to the Nitrososphaera lineage, while the dominant AOB grouped into the Nitrosomonas lineage. The AHLs receptor homologs were identified in both AOA and AOB, which confirmed that AOA and AOB had the QS system. The present work was the first study that elucidated the QS system of AOA and AOB in multidimensional, and confirmed the role of QS system in ammonia oxidizers' metabolism.

Acyl-homoserine-lactones receptor LuxR of Shewanella baltica involved in the development of microbiota and spoilage of refrigerated shrimp.

Numerous bacterial species utilize quorum sensing molecules acyl-homoserine-lactones (AHLs) to communicate, however, crosstalk often complicates the dynamics and behaviors of mixed populations. In this study, we developed a luxR mutant of wild type Shewanella baltica SA03 (WT SA03), and aimed to investigate the role of S. baltica LuxR (AHLs receptor) involved in the spoilage of refrigerated shrimp (Litopenaeus vannamei) by inoculating WT SA03 and luxR mutant of S. baltica SA03 (ΔluxR SA03), respectively. The results indicated the maximum growth rate of total viable bacteria in shrimp inoculated with ΔluxR SA03 was 73.34% lower than that of WT SA03. The lag time of total bacteria in shrimp treated with ΔluxR SA03 were 87.6h, significantly longer than that of WT SA03. Meanwhile, the total volatile basic nitrogen concentrations of shrimp treated with WT SA03 were significantly higher than that of ΔluxR SA03 after 2days of storage, which were in agreement with the decrease of the content of AHLs of the shrimp. The results indicated S. baltica might utilize AHLs produced by other bacteria and accelerate the shrimp spoilage process through LuxR receptor system.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, Nitrobacter, and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-l-homoserine lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-l-homoserine lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria.IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification.

Crystal structure of the N-terminal domain of VqsR from Pseudomonas aeruginosa at 2.1 Å resolution.

VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las, rhl and pqs) by directly downregulating the expression of qscR in Pseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix-turn-helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1-193) was determined at a resolution of 2.1 Å. The structure is folded into a regular α-β-α sandwich topology, which is similar to the ligand-binding domain (LBD) of the LuxR-type QS receptors. Although their sequence similarity is very low, structural comparison reveals that VqsR-N has a conserved enclosed cavity which could recognize acyl-homoserine lactones (AHLs) as in other LuxR-type AHL receptors. The structure suggests that VqsR could be a potential AHL receptor.

Novel insights from molecular docking of SdiA from Salmonella Enteritidis and Escherichia coli with quorum sensing and quorum quenching molecules

Quorum sensing is a cell-to-cell communication mechanism leading to differential gene expression in response to high population density. The autoinducer-1 (AI-1) type quorum sensing system is incomplete in Escherichia coli and Salmonella due to the lack of the AI-1 synthase (LuxI homolog) responsible for acyl homoserine lactone (AHL) synthesis. However, these bacteria encode the AHL receptor SdiA (a LuxR homolog) leading to gene regulation in response to AI-1 produced by other bacteria. This study aimed to model the SdiA protein of Salmonella enterica serovar Enteritidis PT4 578 based on three crystallized SdiA structures from Enterohemorrhagic E. coli (EHEC) with different ligands. Molecular docking of these predicted structures with AHLs, furanones and 1-octanoyl-rac-glycerol were also performed. The available EHEC SdiA structures provided good prototypes for modeling SdiA from Salmonella. The molecular docking of these proteins showed that residues Y63, W67, Y71, D80 and S134 are common binding sites for different quorum modulating signals, besides being conserved among other LuxR type proteins. We also show that AHLs with twelve carbons presented better binding affinity to SdiA than AHLs with smaller side chains in our docking analysis, regardless of the protein structures used. Interestingly, the conformational changes provided by AHL binding resulted in structural models with increased affinities to brominated furanones. These results suggest that the use of brominated furanones to inhibit phenotypes controlled by quorum sensing in Salmonella and EHEC may present a good strategy since these inhibitors seem to specifically compete with AHLs for binding to SdiA in both pathogens.