A Mutation in the luxS Gene Influences Listeria monocytogenes Biofilm Formation

Using a Vibrio harveyi reporter strain, we demonstrated that Listeria monocytogenes secretes a functional autoinducer 2 (AI-2)-like signal. A luxS-deficient mutant produced a denser biofilm and attached to a glass surface 19-fold better than the parent strain. Exogenous AI-2 failed to restore the wild-type phenotype to the mutant. It seems that an intact luxS gene is associated with repression of components required for attachment and biofilm formation.     

Changes in physico-chemical characteristics and viable bacterial communities during fermentation of alfalfa silages inoculated with Lactobacillus plantarum

Sulfate-reducing bacteria (SRB) are culprits for microbiologically influenced corrosion, and biofilms are believed to play essential roles in the corrosion induced by SRB. However, little is known about the regulation of SRB biofilms. Quorum sensing signal molecules acyl-homoserine lactones (AHLs) and autoinducer-2 (AI-2) regulate biofilm formation of many bacteria. In this study, the production of AHLs and AI-2 by one SRB strain, Desulfovibrio sp. Huiquan2017, was detected, and the effect of exogenous AI-2 on bacterial biofilm formation was discussed. It was found that the cell-free supernatants of Desulfovibrio sp. Huiquan2017 induced luminescence in a ?luxS mutant strain Vibrio harveyi BB170, indicating the production of functional AI-2 by the bacterium. In the presence of exogenous AI-2, the growth of Desulfovibrio sp. Huiquan2017 and early biofilm formation were not affected, but the later stage of biofilm development was inhibited significantly. The biofilms became looser, smaller, and thinner, and contained less bacteria and extracellular polymeric substances (EPS). The inhibition effect of AI-2 on the biofilm development of Desulfovibrio sp. Huiquan2017 was mainly achieved through reducing the amount of EPS in biofilms. These findings shed light on the biofilm regulation of SRB.

     

Diversity and functional analysis of luxS genes in Vibrios from marine sponges Mycale laxissima and Ircinia strobilina

Sponges harbor highly diverse and dense microbial communities, providing an environment in which bacterial signaling may be important. Quorum sensing (QS) is a cell density-dependent signaling process that bacteria employ to coordinate and regulate their gene expression. Previous studies have found that bacteria isolated from sponges are able to produce acyl-homoserine lactones (AHLs), an important class of QS molecules found in proteobacteria. Autoinducer-2 (AI-2) is a second class of QS molecule, and is considered to be an interspecies signal. However, AI-2 signaling has not been reported in sponge bacterial symbionts. In this study, degenerate primers were designed based on known Vibrio luxS sequences to amplify the luxS genes encoding AI-2 synthases of several Vibrio isolates from marine sponges Mycale laxissima and Ircinia strobilina. All the vibrios isolated from these two sponges had luxS genes and were able to produce signals with AI-2 activity as detected using a biological reporter. A novel group of luxS sequences was found, thus extending the known diversity of luxS genes. One isolate was chosen for further analysis of its luxS gene by expression of the gene in Escherichia coli DH5α and by characterization of the profile of AI-2 activity. This work provides the first information about luxS genes and AI-2 activity in sponge-associated bacterial communities.     

A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi

Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-l-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~ 38 kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~ 28 kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two flurophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN⁻ (defective in AI-1), luxS⁻ (defective in BAI-2), and luxN⁻/luxS⁻ (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS⁻ mutant (MM30) and luxN⁻/luxS⁻ double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating extracellular protease production in V. harveyi.     

Quorum Sensing and Production of Autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica Serovar Typhimurium in Foods

Autoinducer molecules are utilized by gram-negative and gram-positive bacteria to regulate density-dependent gene expression by a mechanism known as quorum sensing. PCR and DNA sequencing results showed that Campylobacter jejuni and Campylobacter coli possessed luxS, which is responsible for autoinducer-2 (AI-2) production. Using a Vibrio harveyi luminescence assay, the production of AI-2 was observed in milk, chicken broth, and brucella broth by C. coli, C. jejuni, Salmonella enterica serovar Typhimurium, and Escherichia coli O157:H7 under different conditions.     

Assessment of the Roles of LuxS, S-Ribosyl Homocysteine, and Autoinducer 2 in Cell Attachment during Biofilm Formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5α, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10°C, 15°C, 25°C, and 42°C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.     

Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lactone signaling molecules, while dioxazaborocanes structurally resemble previously reported oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all compounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC₅₀ values in the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein. The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.     

Type 2 Quorum Sensing Monitoring,Inhibition and Biofilm Formation in Marine Microrganisms

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 μM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 μM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.     

AI-2 does not function as a quorum sensing molecule inCampylobacter jejuniduring exponential growthin vitro

Background  

Campylobacter jejunicontains a homologue of theluxSgene shown to be responsible for the production of the signalling molecule autoinducer-2 (AI-2) inVibrio harveyiandVibrio cholerae. The aim of this study was to determine whether AI-2 acted as a diffusible quorum sensing signal controllingC. jejunigene expression when it is produced at high levels during mid exponential growth phase.     

Ecological Behavior of Lactobacillus reuteri 100-23 Is Affected by Mutation of the luxS Gene

The luxS gene of Lactobacillus reuteri 100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for the luxS gene in L. reuteri 100-23C, a luxS mutant was constructed by insertional mutagenesis. The luxS mutant did not produce autoinducers AI-2 or AI-3. Complementation of the luxS mutation by a plasmid construct containing luxS restored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in the luxS mutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of the luxS mutant was, however, 65% of that of wild-type cells. Biofilms formed by the luxS mutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex-Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of the luxS mutant, when in competition with L. reuteri strain 100-93 in the mouse cecum, was reduced compared to that of a xylA mutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes of L. reuteri 100-23C, the consequences of which are niche specific.     

Inhibition of Pseudomonas aeruginosa quorum sensing by AI-2 analogs

Autoinducer-2 (AI-2) has been suggested to serve as a universal interspecies quorum sensing signaling molecule. We have synthesized a set of AI-2 analogs with small incremental changes in alkyl substitution on C-2 and evaluated them for their agonistic and antagonistic potential as quorum sensing (QS) attenuators in two different bacterial species: Pseudomonas aeruginosa and Vibrio harveyi. Unexpectedly, several of the analogs were found to function as synergistic QS agonists in V. harveyi, while two of these analogs inhibit QS in P. aeruginosa.     

Autoinducer 2 Affects Biofilm Formation by Bacillus cereus

Cell-free supernatants from growing Bacillus cereus strain ATCC 10987 induced luminescence in a Photorhabdus luminescens ΔluxS mutant, indicating the production of functional autoinducer 2 (AI-2). The exogenous addition of in vitro synthesized AI-2 had an inhibitory effect on biofilm formation by B. cereus and promoted release of the cells from a preformed biofilm.     

Isolate-Specific Effects of Patulin, Penicillic Acid and EDTA on Biofilm Formation and Growth of Dental Unit Water Line Biofilm Isolates

Dental unit water line (DUWL) contamination by opportunistic pathogens has its significance in nosocomial infection of patients, health care workers, and life-threatening infections to immunocompromized persons. Recently, the quorum sensing (QS) system of DUWL isolates has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. In this study, the effect of two quorum-sensing inhibitory compounds (patulin; PAT, penicillic acid; PA) and EDTA on planktonic growth, AI-2 signalling and in vitro biofilm formation of Pseudomonas aeruginosa, Achromobacter xylosoxidans and Achromobacter sp. was monitored. Vibrio harveyi BB170 bioassay and crystal violet staining methods were used to detect the AI-2 monitoring and biofilm formation in DUWL isolates, respectively. The V. harveyi BB170 bioassay failed to induce bioluminescence in A. xylosoxidans and Achromobacter sp., while P. aeruginosa showed AI-2 like activity suggesting the need of some pretreatments prior to bioassay. All strains were found to form biofilms within 72 h of incubation. The QSIs/EDTA combination have isolate-specific effects on biofilm formation and in some cases it stimulated biofilm formation as often as it was inhibited. However, detailed information about the anti-biofilm effect of these compounds is still lacking.     

长双歧杆菌NCC2705群体感应系统信号分子AI-2的检测

目的:用生物学方法检测长双歧杆菌NCC2705是否产生群体感应系统信号分子AI-2。方法:将长双歧杆菌NCC2705不同时间点的培养上清分别加至AI-2特异报告系统哈氏弧菌BB170中,以空白培养基上清为对照,用荧光光度计对哈氏弧菌发光强度进行计量,推测出长双歧杆菌NCC2705上清中是否含有分泌的AI-2,并由此推断AI-2的活性。结果:通过微孔板检测系统对加入长双歧杆菌NCC2705培养上清的哈氏弧菌BB170进行检测,发现双歧杆菌上清的加入增强了哈氏弧菌BB170发出的荧光强度。结论:长双歧杆菌NCC2705中存在依赖于luxS/AI-2的群体感应系统,并能够分泌有活性的AI-2,为进一步研究长双歧杆菌NCC2705AI-2及luxS基因的功能打下基础。     

禽致病性大肠杆菌安徽分离株luxS和pfs基因的克隆、表达与细胞外合成AI-2 活性检测

【目的】LuxS/AI-2型密度感应系统存在于革兰氏阴性和阳性菌中,可产生用于细菌种间交流的通用自诱导信号分子AI-2(Autoinducer-2,AI-2),细菌许多生理功能都受此系统的调节。本研究开展对禽致病性大肠杆菌(Avian Pathogenic Escherichia coli,APEC)自诱导信号分子AI-2的检测和建立体外合成、定量的方法,为进一步研究APEC的AI-2调控作用奠定基础。【方法】利用哈维弧菌BB170(Vibrio harveyi BB170)开展对APEC AI-2的检测;利用表达、纯化的LuxS和Pfs在体外催化S-腺苷同型半胱氨酸(Sadenosylhomocysteine,SAH),进行AI-2的体外合成。【结果】APEC能产生自诱导信号分子AI-2;成功表达可用于AI-2合成的可溶性重组蛋白LuxS和Pfs;纯化的重组蛋白LuxS和Pfs与SAH同时作用后,合成了浓度为300μmol/L的AI-2;运用哈维弧菌BB170对合成的AI-2活性检测表明,其活性是阴性对照的700倍。【结论】APEC存在LuxS/AI-2型密度感应系统,APEC的LuxS和Pfs可以在体外催化SAH生成有活性的AI-2分子。本研究为进一步研究APEC的AI-2的调控作用奠定基础。     

In vitro biosynthesis of autoinducer 2 of Steptococcus suis Serotype 2 using recombinant LuxS and Pfs

Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules, called autoinducers. LuxS and Pfs catalyze synthesis of the quorum-sensing signaling molecule autoinducer 2 (AI-2), which has been shown to control a variety of cellular processes. We studied the cloning, expression, and purification of LuxS and Pfs from Streptococcus suis Serotype 2 strain HA9801 (SS2); the two enzymes gave an apparent single protein band, and revealed a molecular mass of 21.74 and 28.44 kDa on an SDS-PAGE, respectively. Expressed and purified LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH). The reaction products were able to induce luminescence of Vibrio harveyi BB170, clearly demonstrating that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. Optimum pH and temperature for biosynthesis AI-2 in vitro were 8.0 and 37 °C, respectively. Biosynthesis AI-2 in vitro was stimulated by Cr³⁺, Al³⁺, and Ba²⁺and was inhibited by Fe²⁺ and Ni²⁺, respectively. It was strongly inhibited by Hg²⁺, Cu²⁺, and Mn²⁺, while enzyme activity was not affected by Li⁺, Mg²⁺, and Zn²⁺. In this study, we cloned, expressed, and purified LuxS and Pfs, identified the pathway of AI-2 synthesis in SS2, and analyzed the impact factor of AI-2 synthesis in vitro, which provided a solid basis for future research concerning the role of AI-2 in SS2.     

Phenotypes of Campylobacter jejuni luxS Mutants Are Depending on Strain Background,Kind of Mutation and Experimental Conditions

Since the discovery that Campylobacter (C.) jejuni produces Autoinducer 2 (AI-2), various studies have been conducted to explore the function and role of AI-2 in C. jejuni. However, the interpretation of these analyses has been complicated by differences in strain backgrounds, kind of mutation and culture conditions used. Furthermore, all research on AI-2 dependent phenotypes has been conducted with AI-2 synthase (luxS) mutants. This mutation also leads to a disruption of the activated-methyl-cycle. Most studies lack sufficient complementation resulting in not knowing whether phenotypes of luxS mutants depend on disrupted metabolism or lack of AI-2. Additionally, no AI-2 receptor has been found yet. All this contributes to an intensive discussion about the exact role of AI-2 in C. jejuni. Therefore, we examined the impact of different experiment settings on three different C. jejuni luxS mutants on growth and motility (37°C and 42°C). Our study showed that differing phenotypes of C. jejuni luxS mutants depend on strain background, mutation strategy and culture conditions. Furthermore, we complemented experiments with synthetic AI-2 or homocysteine as well as the combination of both. Complementation with AI-2 and AI-2+homocysteine significantly increased the cell number of C. jejuni NCTC 11168ΔluxS in stationary phase compared to the non-complemented C. jejuni NCTC 11168ΔluxS mutant. Genetic complementation of both C. jejuni 81-176 luxS mutants resulted in wild type comparable growth curves. Also swarming ability could be partially complemented. While genetic complementation restored swarming abilities of C. jejuni 81-176ΔluxS, it did not fully restore the phenotype of C. jejuni 81-176::luxS, which indicates that compensatory mutations in other parts of the chromosome and/or potential polar effects may appear in this mutant strain. Also with neither synthetic complementation, the phenotype of the wild type-strains was achieved, suggesting yet another reason for differing phenotypes other than communication and methionine metabolism for C. jejuni luxS mutants.