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PubMed2006

Environmental biology of the marine Roseobacter lineage.

The Roseobacter lineage is a phylogenetically coherent, physiologically heterogeneous group of alpha-Proteobacteria comprising up to 25% of marine microbial communities, especially in coastal and polar oceans, and it is the only lineage in which cultivated bacteria are closely related to environmental clones. Currently 41 subclusters are described, covering all major marine ecological niches (seawater, algal blooms, microbial mats, sediments, sea ice, marine invertebrates). Members of the Roseobacter lineage play an important role for the global carbon and sulfur cycle and the climate, since they have the trait of aerobic anoxygenic photosynthesis, oxidize the greenhouse gas carbon monoxide, and produce the climate-relevant gas dimethylsulfide through the degradation of algal osmolytes. Production of bioactive metabolites and quorum-sensing-regulated control of gene expression mediate their success in complex communities. Studies of representative isolates in culture, whole-genome sequencing, e.g., of Silicibacter pomeroyi, and the analysis of marine metagenome libraries have started to reveal the environmental biology of this important marine group.

Anti-Bacterial Agents↗

PubMed2006

Effect of phosphorus limitation on microbial floc structure and gene expression in activated sludge.

The effect of limiting phosphorus (P) in activated sludge was investigated in laboratory-scale sequencing batch reactors (SBRs). Correlative microscopy revealed that P-limitation (COD:N:P = 100:5:0.05) leads to morphological changes in floc structure and the composition of extracellular polymeric substances (EPS). This was found to be accompanied by expression of quorum-sensing in an acyl homoserine lactone bioassay. Differential gene expression in relation to P-limitation was examined in a global profile using the Affymetrix Escherichia coli antisense genomic microarray. Three separate experiments were conducted where the impact of P-limitation was examined under batch conditions and in SBRs at stable operating conditions and within 3-7 days following a down-shift in P. Significant changes in open reading frames (ORF) and intergenic regions based on the E. coli microarray were observed. Several genes associated with cell structure, including slt, wbbH, fimH, amB, rfaJ and slp were found to be expressed. Quorum regulated genes were also found to be expressed including psiF which is known to be induced by P-starvation (92% confidence level; 1.45 log ratio).

Escherichia coli↗

PubMed2026

Acetylcholine signaling regulates osmotic stress adaptation in the phytopathogen Dickeya solani.

Plants impose strong selective pressures that shape both the composition and functional potential of plant microbiomes. The adaptation of plant-associated bacteria to their hosts relies on an extensive repertoire of signal transduction systems that sense plant-derived molecules and dynamically adjust bacterial physiology and metabolism within the holobiont. These signals include key plant signaling compounds that regulate processes essential for plant-microbe interactions. Among them, acetylcholine is emerging as an important signaling molecule in both plants and bacteria. Here, we demonstrate that acetylcholine regulates the expression of the osmotic stress response betIBA gene cluster in the important phytopathogen Dickeya solani, where it plays an important role in osmoprotection. We show that the TetR-family transcriptional regulator associated with this pathway, BetIDs, recognizes acetylcholine as well as choline and trimethylamine. These three ligands differentially induce betIBA transcription in a manner that correlates with their binding affinities. Ligand binding does not affect BetIDs binding to the bet promoter or its oligomeric state. Instead, it induces pronounced changes in the secondary structure of BetIDs, with the magnitude of these conformational changes being ligand-dependent. We further show that quorum sensing modulates osmotic stress tolerance in D. solani by regulating the expression of the Bet pathway. The Bet system is required for the full virulence of D. solani, particularly in chemically complex plant tissues. Phylogenetic analyses reveal that the BetIBA system is widely distributed among plant-associated Pseudomonadota, collectively supporting its importance for bacterial survival and adaptation in plant-related environments.

Osmotic Pressure↗

PubMed2006

Convergence of hormones and autoinducers at the host/pathogen interface.

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed "quorum sensing" (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host's immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others' soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.

4-Butyrolactone↗

PubMed2026

Metagenome-resolved evidence that soluble factors in granular activated carbon-amended reactor effluent reprogram propionate metabolism and methanogenic pathways.

Granular activated carbon (GAC) enhances anaerobic digestion performance, yet the mechanisms underlying reactor-scale improvements remain incompletely understood, particularly how GAC affects biomass not attached to its surface. Here, sludge from a non-GAC up-flow anaerobic sludge blanket reactor was incubated with 0.45-μm-filtered effluents from non-GAC and GAC-amended reactors under repeated propionate loading, followed by genome-resolved metagenomics. GAC-reactor effluent increased methane yield from 64 ± 3% to 76 ± 3% (p < 0.01) in the absence of GAC particles. A non-redundant catalog of 170 quality-filtered metagenome-assembled genomes (MAGs) was recovered, enabling pathway- and gene-set quantification. Genomic potential for both major propionate-oxidation routes increased in the GAC-effluent group relative to the non-GAC group, with a larger increase for the methylmalonyl-CoA (MMC) route than for the dismutation route (1.289- versus 1.221-fold). Accordingly, the MMC-to-dismutation preference ratio was 5.60% higher in the GAC-effluent group, alongside a broader carrier base. Cobamide potential shifted toward remodeling and cobamide-dependent use rather than increased de novo corrin-ring synthesis. Candidate electron-transfer architectures were also rebalanced: PilA-associated carriers became less prominent, whereas maturation-supported multiheme cytochrome carriers increased from 22.96% to 34.90% of community abundance, although H2/formate-module carriers remained prevalent. Quorum-sensing systems underwent pathway- and carrier-specific redistribution, while all eight curated extracellular-polysaccharide modules showed higher mean gene abundance in the GAC-effluent composite. These findings show that a filter-passing effluent fraction can extend GAC-associated effects beyond direct particle contact and link enhanced methanogenesis to a broader, redistributed network of metabolic, redox, and coordination capacities. This expands the mechanistic framework of conductive-material-assisted anaerobic digestion and provides a basis for harnessing GAC-derived functions throughout the reactor.

Extracellular polymeric substances (EPS)↗

PubMed2006

N-acyl homoserine lactones are degraded via an amidolytic activity in Comamonas sp. strain D1.

Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.

4-Butyrolactone↗

PubMed2006

Biofilms, a new approach to the microbiology of dental plaque.

Dental plaque has the properties of a biofilm, similar to other biofilms found in the body and the environment. Modern molecular biological techniques have identified about 1000 different bacterial species in the dental biofilm, twice as many as can be cultured. Oral biofilms are very heterogeneous in structure. Dense mushroom-like structures originate from the enamel surface, interspersed with bacteria-free channels used as diffusion pathways. The channels are probably filled with an extracellular polysaccharide (EPS) matrix produced by the bacteria. Bacteria in biofilms communicate through signaling molecules, and use this "quorum-sensing" system to optimize their virulence factors and survival. Bacteria in a biofilm have a physiology different from that of planktonic cells. They generally live under nutrient limitation and often in a dormant state. Such "sleepy" bacteria respond differently to antibiotics and antimicrobials, because these agents were generally selected in experiments with metabolically active bacteria. This is one of the explanations as to why antibiotics and antimicrobials are not as successful in the clinic as could be expected from laboratory studies. In addition, it has been found that many therapeutic agents bind to the biofilm EPS matrix before they even reach the bacteria, and are thereby inactivated. Taken together, these fundings highlight why the study of bacteria in the oral cavity is now taken on by studying the biofilms rather than individual species.

Bacteriological Techniques↗

PubMed2006

[Construction of Streptococcus mutans luxS gene allelic exchange plasmid].

OBJECTIVE: It is reported that Streptococcus mutans luxS gene may have an important role in the interspecies quorum sensing system. To construct the S. mutans luxS gene knockout mutant, this research aim to construct the luxS gene allelic exchange plasmid. METHODS: The upstream and downstream flank DNA fragments of S. mutans luxS gene (Xup, Xdn)and the E. coli kanamycin resistance gene (Kana) were enriched by pfu DNA polymerase with "nest PCR" methods. These fragments were ligated into pBluescript SK (+) Phagemids vector with double endonuclease reaction sequentially. RESULTS: With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Xukd-pbsk, was constructed correctively and the kanamycin resistance gene could be expressed in vitro. CONCLUSION: The S. mutans luxS gene allelic exchange plasmid is constructed correctively in this research and can be used in the future research of S. mutans luxS gene knockout mutant.

Bacterial Proteins↗

PubMed2026

Pseudomonas aeruginosa adaptation and persistence in the aspergilloma microbiome revealed by integrated multi-omics.

Chronic pulmonary aspergillosis involves the formation of a fungal ball (aspergilloma) in lung cavities. Pseudomonas aeruginosa commonly co-colonizes these lesions; however, the in vivo mechanisms underlying its persistence are unknown. Using a multi-omics approach on resected aspergillomas, we defined the genomic, transcriptional, and metabolic adaptations of P. aeruginosa within this polymicrobial niche. We reconstructed high-quality P. aeruginosa genomes and identified a conserved core genome, along with accessory genes for secondary metabolism, virulence, and antimicrobial resistance. Phylogenomics revealed heterogeneous evolutionary paths among co-colonizing strains. Metatranscriptomics showed stark physiological heterogeneity, from metabolically aggressive to stress-adapted states. High expression of phenazine, quorum-sensing (PQS), siderophore, and secretion-system operons was corroborated by metabolomic detection of phenazine-1-carboxylic acid and 2-heptylquinolin-4(1H)-one, confirming active bacterial antagonism in vivo. Concurrent Aspergillus fumigatus transcriptomics revealed the activation of oxidative stress responses, secondary metabolism (eg fumagillin), and iron scavenging, demonstrating reciprocal competition. Host transcriptomics revealed patient-specific immune signatures that correlated with the metabolic activity of the co-colonizers. This work provides an integrated systems-level analysis of the tri-kingdom aspergilloma ecosystem. P. aeruginosa persistence is driven by genomic plasticity and context-dependent expression of competitive pathways, shaped within a chronic inflammatory environment. These findings redefine aspergillomas as active polymicrobial consortia, establishing a framework for targeting resilient microbial communities in chronic lung disease.

Multiomics↗

PubMed2007

Genetic variation in comC, the gene encoding competence-stimulating peptide (CSP) in Streptococcus mutans.

The genetic variability in comC, the gene encoding the quorum-sensing molecule, competence-stimulating peptide (CSP) in Streptococcus mutans is reported. Seven comC alleles encoding three distinct mature CSPs were identified among 36 geographically diverse strains, although, compared with Streptococcus pneumoniae, the amount of predicted amino acid sequence variation is low. In agreement with other studies, significant variation was found in the natural competence for DNA uptake in these strains. However, there was no correlation between the CSP genotype and the ability to transform these strains. Representative strains encoding each of the CSP variants became competent in response to synthetic CSPs of each type. Therefore, in contrast to S. pneumoniae, comC alleles in S. mutans are functionally equivalent and there is no evidence of pherotype specificity.

Alleles↗

PubMed2006

Induction of systemic resistance in tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria.

N-acyl-L-homoserine lactone (AHL) signal molecules are utilized by Gram-negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density-dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL-negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL-producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL-negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA- and ethylene-dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.

4-Butyrolactone↗

PubMed2006

Occurrence of N-acyl-L-homoserine lactones in extracts of some Gram-negative bacteria evaluated by gas chromatography-mass spectrometry.

Acylated homoserine lactones (AHLs) are self-generated signal molecules that mediate population density-dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria. These signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. In selected foods AHLs contribute to product spoilage. As different bacterial species produce AHL analogs that differ in length of the N-acyl chain, ranging from 4 to 14 carbons and in the substitution at the C-3 position of the side chain (i.e., oxo or hydroxyl group), the suitability and applicability of a gas chromatography-mass spectrometry direct method for characterizing trace amounts of AHLs was evaluated using N-heptanoyl-homoserine lactone as internal standard. Crude cell-free supernatants of bacterial cultures of Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia enterocolitica, and Serratia liquefaciens were screened for AHL production in selected ion monitoring mode, using the prominent fragment at m/z 143. The observed profiles of distinguishable N-acyl-homoserine lactones occurring in bacterial extracts were compared and discussed. The presence of a labile 3-oxo-hexanoylhomoserine lactone was evidenced but serious difficulties arose in estimating its concentration as thermal degradation occurs during the gas chromatographic separation. Its electron impact mass spectra was, however, given and interpreted.

4-Butyrolactone↗

PubMed2006

Direct quantification of N-(3-oxo-hexanoyl)-L-homoserine lactone in culture supernatant using a whole-cell bioreporter.

The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) plays a significant role in the quorum-sensing system of the marine bacterium Vibrio fischeri. Upon forming a transcriptional activation complex with LuxR, 3-oxo-C6-HSL induces transcription of the luxICDABEG operon, leading to the increased production of both the 3-oxo-C6-HSL synthase (LuxI) and the bioluminescent proteins. In order to quantitatively analyze this regulatory mechanism, a novel approach was developed to measure 3-oxo-C6-HSL concentrations in V. fischeri cell culture supernatant. A bioluminescent strain of Escherichia coli that responds to 3-oxo-C6-HSL was used as a bioreporter. Although a linear response of the bioreporter to exogenously added synthetic 3-oxo-C6-HSL was found over several orders of magnitude, we show that bioreporter performance was dramatically impacted by variations in the supernatants using samples from a V. fischeri LuxI- strain. However, when maintained in the same supernatant background, the normalized peak bioluminescence maintained a linear response to 3-oxo-C6-HSL concentrations. Therefore, a standard additions technique was developed in which a known concentration of 3-oxo-C6-HSL was added to supernatant samples from wild-type V. fischeri cultures, and the incremental increase of the normalized peak bioluminescence relative to the untreated sample was determined. The concentration of 3-oxo-C6-HSL in the supernatant of the unknown sample was then quantified from the slope of the response between the normalized bioluminescent peaks with and without the addition of 3-oxo-C6-HSL. Advantages of this method are that it is rapid, does not require concentration or extraction, uses a small sample volume (ca. 2 ml), and accounts for effects caused by the composition of the supernatant. Furthermore, the findings can be broadly applicable to other bioreporter systems involving variable background conditions.

4-Butyrolactone↗

PubMed2006

Functional analysis of luxS in the probiotic strain Lactobacillus rhamnosus GG reveals a central metabolic role important for growth and biofilm formation.

Quorum sensing is involved in the regulation of multicellular behavior through communication via small molecules. Given the high number and diversity of the gastrointestinal microbiota, it is postulated that members of this community communicate to coordinate a variety of adaptive processes. AI-2 is suggested to be a universal bacterial signaling molecule synthesized by the LuxS enzyme, which forms an integral part of the activated methyl cycle. We have previously reported that the well-documented probiotic strain Lactobacillus rhamnosus GG, a human isolate, produces AI-2-like molecules. In this study, we identified the luxS homologue of L. rhamnosus GG. luxS seems to be located in an operon with a yxjH gene encoding a putative cobalamin-independent methionine synthase. In silico analysis revealed a methionine-specific T box in the leader sequence of the putative yxjH-luxS operon. However, transcriptional analysis showed that luxS is expressed mainly as a monocistronic transcript. Construction of a luxS knockout mutant confirmed that the luxS gene is responsible for AI-2 production in L. rhamnosus GG. However, this mutation also resulted in pleiotropic effects on the growth of this fastidious strain. Cysteine, pantothenate, folic acid, and biotin could partially complement growth, suggesting a central metabolic role for luxS in L. rhamnosus GG. Interestingly, the luxS mutant also showed a defect in monospecies biofilm formation. Experiments with chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione, coculture with the wild type, and nutritional complementation suggested that the main cause of this defect has a metabolic nature. Moreover, our data indicate that suppressor mutations are likely to occur in luxS mutants of L. rhamnosus GG. Therefore, results of luxS-related studies should be carefully interpreted.

Bacterial Proteins↗

PubMed2002

Toward an understanding of microbial communities through analysis of communication networks.

Bacteria receive signals from diverse members of their biotic environment. They sense their own species through the process of quorum sensing, which detects the density of bacterial cells and regulates functions such as bioluminescence, virulence, and competence. Bacteria also respond to the presence of other microorganisms and eukaryotic hosts. Most studies of microbial communication focus on signaling between the microbe and one other organism for empirical simplicity and because few experimental systems offer the opportunity to study communication among various types of organisms. But in the real biological world, microorganisms must carry on multiple molecular conversations simultaneously between diverse organisms, thereby constructing communication networks. We propose that biocontrol of plant disease, the process of suppressing disease through application of a microorganism, offers a model for the study of communication among multiple organisms. Successful biocontrol requires the sending and receiving of signals between the biocontrol agent and the pathogen, plant host, and microbial community surrounding the host. We are using Bacillus cereus, a biocontrol agent, and the organisms it must interact with, to dissect a communication network. This system offers an excellent starting point for study because its members are defined and well studied. An understanding of signaling in the B. cereus biocontrol system may provide a model for network communication among organisms that share a habitat and provide a new angle of analysis for understanding the interconnections that define communities.

Bacillus cereus↗

PubMed2005

Effect of sdiA on biosensors of N-acylhomoserine lactones.

Many gram-negative bacteria synthesize N-acylhomoserine lactones (AHLs) and then use transcription factors of the LuxR family to sense and respond to AHL accumulation in the environment; this phenomenon is termed quorum sensing. Bacteria produce a variety of AHLs, and numerous bacterial reporter strains, or biosensors, that can detect subsets of these molecules have been constructed. Many of these are based on Escherichia coli because this species does not produce AHLs. However, both Escherichia and Salmonella spp. contain a LuxR homolog named SdiA that can detect exogenous AHL synthesized by other microbial species. In this study we have determined that sdiA of E. coli and Salmonella spp. can activate an RhlR-based biosensor plasmid in response to AHLs other than what the biosensor was designed to detect. SdiA does not activate LuxR-, LasR-, or AhyR-based biosensor plasmids, although the presence of sdiA in E. coli does interfere with the function of the AhyR-based biosensor. Because sdiA interferes with the function of two of the four reporters, we have constructed a set of E. coli biosensor strains that lack sdiA. The set includes control reporters that allow the luxR dependence of responses to be determined.

4-Butyrolactone↗

PubMed2002

Controlling infection by tuning in and turning down the volume of bacterial small-talk.

As the prevalence of bacterial resistance to multiple antibiotics increases it is becoming progressively more difficult to treat infections and, in many cases, the available therapeutic options are severely limited. Hence, there is a growing urgency to the search for novel targets and the development of new antimicrobials. To infect a host and cause disease bacteria produce an array of virulence determinants that contribute to pathogenesis. It is now known that many different Gram-positive and Gram-negative pathogens communicate via the production and sensing of small, diffusible signal molecules, to coordinate virulence determinant production. As a consequence, this event, now termed quorum sensing, represents a novel therapeutic target offering the opportunity to attenuate virulence, and thus control infection, by blocking cell-to-cell communication.

4-Butyrolactone↗

PubMed2004

Biofilm formation and cell-to-cell signalling in Gram-negative bacteria isolated from a food processing environment.

AIMS: To investigate the biofilm-forming capacity and the production of quorum signals in Gram-negative bacteria isolated from a food production environment, and the possible correlation between both phenotypes. METHODS AND RESULTS: Sixty-eight Gram-negative bacteria were isolated from equipment and working surfaces in a raw vegetable processing line, and tested for biofilm-forming capacity using an in vitro microplate assay. All isolates showed significantly higher biofilm-forming capacity than Escherichia coli laboratory strain DH5alpha, which was included as a negative control, and differed up to 56-fold in relative biofilm-forming capacity. Various assays based on reporter bacteria were used to detect quorum signals produced by the isolates. Twenty-six isolates produced autoinducer-2, five isolates produced N-acyl-homoserine lactones (AHLs), and none produced the Pseudomonas quinolone signal. CONCLUSIONS: No correlation was found between in vitro biofilm-forming capacity and production of quorum signalling molecules among the 68 strains isolated from the raw vegetable processing line. SIGNIFICANCE AND IMPACT OF THE STUDY: Several recent studies have shown a role of AHL-based quorum sensing in biofilm formation of specific Gram-negative bacterial strains. The current work shows that production of AHL and other quorum signals is not widespread in Gram-negative isolates from a raw vegetable processing line, and is not a general requirement for biofilm formation, at least in vitro.

4-Butyrolactone↗