A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.

The hydroxylamine oxidoreductase/hydrazine dehydrogenase (HAO/HDH) protein family constitutes an important group of octaheme cytochromes c (OCCs). The majority of these proteins form homotrimers, with their subunits being covalently attached to each other via a rare cross-link between the catalytic heme moiety and a conserved tyrosine residue in an adjacent subunit. This covalent cross-link has been proposed to modulate the active-site heme towards oxidative catalysis by distorting the heme plane. In this study, the crystal structure of a stable complex of an HAO homologue (KsHAOr) with its diheme cytochrome c redox partner (KsDH) from the anammox bacterium Kuenenia stuttgartiensis was determined. KsHAOr lacks the tyrosine cross-link and is therefore tuned to reductive catalysis. The molecular model of the KsHAOr-KsDH complex at 2.6 Šresolution shows a heterododecameric (α6ß6) assembly, which was also shown to be the oligomeric state in solution by analytical ultracentrifugation and multi-angle static light scattering. The 60-heme-containing protein complex reveals a unique extended electron transfer pathway and provides deeper insights into catalysis and electron transfer in reductive OCCs.

Ultrafast fabrication of thermally stable protein-coated silver iodide nanoparticles for solid-state superionic conductors.

Solid-state ionic conductor is an essential and critical part of electrochemical devices such as batteries and sensors. Nano-sized silver iodide (AgI) is the most promising ionic conductor due to its superionic conductivity at room temperature. In recent years, proteins have been used as organic templates to obtain high-performance solid-state ionic conductors as well as to extend their applications in a biosensor. Here, we report the unprecedented ultrafast synthesis of thermally stable protein-coated AgI nanoparticles (NPs) through the photo-irradiation method for solid-state electrolyte. The synthesis was performed using a hyperthermostable bacterial ß-glucosidase. The protein-coated AgI NPs with an approximate diameter of 13 nm showed that the controllable transition from the α- to ß-/γ-phase was drastically suppressed down to 41 °C in the cooling process. After drying, the product represents a thermally stable organic-inorganic hybrid system with superionic conductivity. It is noteworthy that the superionic conductivity (σ ˜ 0.14 S/cm at 170 °C) of thermally stable protein-coated AgI NPs is maintained during several thermal cycles (25-170 °C). To our knowledge, this is the first report showing the diffusion of mobile Ag+ ions on the surface of the AgI NPs through a protein matrix. The facile synthesis method and high performance of the protein-coated AgI NPs may provide a latent application in the mass production of nanobatteries and other technological applications.

Characterization of a novel arabinose-tolerant α-L-arabinofuranosidase with high ginsenoside Rc to ginsenoside Rd bioconversion productivity.

AIMS: (i) To investigate the enzymatic characterization of α-L-arabinofuranosidase from Thermotoga thermarum DSM5069. (ii) To evaluate the performance of its excellent properties on converting ginsenoside Rc to ginsenoside Rd. METHODS AND RESULTS: The thermostable α-L-arabinofuranosidase (Tt-Afs) gene from T. thermarum DSM5069 was cloned and overexpressed. Recombinant Tt-Afs was purified, and its molecular weight was approx. 55 kDa. Its optimal activity was at pH 5·0 and 95°C. It has high selectivity for cleaving the outer arabinofuranosyl moieties at the C-20 carbon of ginsenoside Rc and its sugar-tolerance makes Tt-Afs a promising candidate for the production of ginsenoside Rd. In a reaction at 85°C and pH 5·0, 25 g l(-1) of ginsenoside Rc was transformed into 21·8 g l(-1) of Rd within 60 min, with a corresponding molar conversion of 99·4% and a high ginsenoside Rd productivity of 21800 mg l(-1) h(-1). CONCLUSIONS: We have successfully cloned and overexpressed the novel α-l-arabinofuranosidase from T. thermarum DSM5069. The high ginsenoside Rd productivity and detailed characterization of recombinant Tt-Afs was provided. SIGNIFICANCE AND IMPACT OF THE STUDY: The result shows a high productivity on the bioconversion from high concentration ginsenoside Rc to ginsenoside Rd, which also give rise to a potential commercial enzyme application.

Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.

A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and ß-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.

Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease.

A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8-10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.

Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4.

Family A DNA polymerase (K4PolI) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4PolI showed thermostable DNA-dependent DNA polymerase activity with 3'-5' exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated by in silico modeling to understand the binding situation between K4PolI and template DNA. Nine amino acids in the 3'-5' exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2' hydroxy group of ribose. To allow K4PolI to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2' hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3'-5' exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3'-5' exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-ß-1,3-glucanase from Thermotoga petrophila.

1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal ß-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

Role of Tannerella forsythia NanH sialidase in epithelial cell attachment.

Tannerella forsythia is a Gram-negative oral anaerobe which contributes to the development of periodontitis, an inflammatory disease of the tooth-supporting tissues leading to tooth loss. The mechanisms by which this bacterium colonizes the oral cavity are poorly understood. The bacterium has been shown to express two distinct sialidases, namely, SiaHI and NanH, with the latter being the major sialidase. Bacterial sialidases can play roles in pathogenesis by cleaving sialic acids on host glycoproteins, destroying their integrity, and/or unmasking hidden epitopes on host surfaces for colonization. In the present study, we investigated the roles of the SiaHI and NanH sialidases by generating and characterizing specific deletion mutants. Our results showed that the NanH deficiency resulted in a total loss of sialidase activity associated with the outer-membrane and secreted fractions. On the other hand, the SiaHI deficiency resulted in only a slight reduction in the total sialidase activity, with no significant differences in the levels of sialidase activity in the outer membrane or secreted fractions compared to that in the wild-type strain. The results demonstrated that NanH is both surface localized and secreted. The NanH-deficient mutant but not the SiaHI-deficient mutant was significantly attenuated in epithelial cell binding and invasion abilities compared to the wild-type strain. Moreover, the NanH-deficient mutant alone was impaired in cleaving surface sialic acids on epithelial cells. Thus, our study suggests that NanH sialidase might play roles in bacterial colonization by exposing sialic acid-hidden epitopes on epithelial cells.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of Pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99 percent 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97 percent, 98 percent and 99 percent 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

Avaliou-se a biodiversidade e a importância industrial de bactérias indígenas da mina de sal Khewra, Paquistão. Efetuou-se a amplificação do 16S rDNA dos isolados por PCR empregando-se os iniciadores universais FD1 e rP1, e os produtos foram seqüenciados comercialmente. Essas seqüências de genes foram comparadas com outras seqüências disponíveis no GenBank a fim de encontrar seqüências relacionadas, construindo-se uma árvore filogenética para essas bactérias. Os genes foram depositados no GenBank obtendo-se os números de acesso. A maioria dos isolados pertenceu a diferentes espécies do gênero Bacillus, apresentando 92-99 por cento de identidade de 16S rDNA com a respectiva cepa de referencia. Outros isolados apresentaram alta similaridade com Escherichia coli, Staphylococcus arlettae e Staphylococcus gallinarum, com 97 por cento, 98 por cento e 99 por cento de similaridade de16S rDNA, respectivamente. A capacidade dos isolados produzirem enzimas industriais (amilase, carboximetilcelulase, xilanase, celulase e protease) foi verificada. Todos os isolados foram testados em placas quanto a degradação de amido, carboximetilcelulose, xilana, celulose e caseína. Os isolados BPT-5, 11, 18, 19 e 25 produziram grandes quantidades de enzimas degradadoras de carboidratos e proteínas. Conclui-se que a mina de Sal Khewra apresenta diferentes grupos de bactérias, que são fontes potenciais de enzimas industriais de aplicação comercial.

Detection of extra-cellular enzymes of anaerobic gram-negative bacteria from clinically diseased and healthy sites.

Anaerobic gram-negative bacteria (AGNB) produce enzymes that play a significant role in the development of disease. We tested 50 AGNB isolates, 25 each from clinically diseased and healthy human sites for in vitro production of caseinase, collagenase, etc. Majority of the isolates were Bacteroides fragilis and Porphyromonas gingivalis, which more commonly produced collagenase and haemolysin. Comparatively larger number of clinical AGNB produced collagenase (P = 0.004). No such difference was observed with other enzymes. Hence, collagenase is probably one of the key virulence markers of pathogenic AGNB, and the inhibitors targeting collagenases might help in the therapy of anaerobic infections.

Activity, stability and structural studies of lactate dehydrogenases adapted to extreme thermal environments.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.

Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea.

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 degrees C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO(2), H(2), L-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 +/- 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 +/- 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe(2+). The enzyme was thermostable with a half-life of about 10 h at 70 degrees C, and its catalytic activity increased along with the rise of temperature up to 95 degrees C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K (m) values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.

Growth and pectate-lyase activity of the ruminal bacterium Lachnospira multiparus in the presence of short-chain organic acids.

AIMS: Acetic, propionic, butyric and lactic acids are end products of feed fermentation by rumen microbes. The effects of these short chain acids on growth and pectate-lyase (PL) activity of Lachnospira multiparus were studied. METHODS AND RESULTS: The bacterial strain used was L. multiparus D32. Acids were tested between 50 and 300 mmol l(-1). Growth and PL activity were measured by the increase in total protein content and by the increase in absorbance at 235 nm in the reaction medium respectively. With the exception of lactic acid, all acids decreased bacterial growth rates; generally, these effects were more pronounced at higher concentrations and with acids of longer chains. PL activity was inhibited by all the acids except by butyric acid at 50 and 100 mmol l(-1). Enzyme inhibition increased with the concentrations of the acids and lactic acid was the most inhibitory. CONCLUSIONS: High concentrations of short chain acids can differentially inhibit the growth rate and the PL activity of L. multiparus. SIGNIFICANCE AND IMPACT OF THE STUDY: Products of fermentation generated by the ruminal microbiota could modify the degradation of pectic substances by this bacterium.

Impact of antimicrobial exposure and beta-lactamase-producing bacteria on salivary beta-lactamase activity in infancy.

Beta-lactamase production by oral bacteria is common in infancy and is associated with use of antimicrobial agents in infants. The present longitudinal study aimed to examine the frequency of salivary beta-lactamase activity (SbetaA), to compare SbetaA with the presence of beta-lactamase-producing (beta+) aerobic and anaerobic species in saliva, and to estimate the impact of antimicrobial exposure on the emergence of SbetaA in healthy infants during their first year of life. At 6 months, SbetaA was detected in 46% infants; 89% SbetaA-positive infants and 55% SbetaA-negative infants harboured beta+ species at this time (OR 7.08; CI 1.31-38.34). At 12 months, SbetaA was detected in 54% infants. Exposure to antimicrobials during the first year of life increased the risk (OR 2.60; CI 0.72-9.36) of having SbetaA.

The role of the flavodiiron proteins in microbial nitric oxide detoxification.

The flavodiiron proteins (first named as A-type flavoproteins) constitute a large superfamily of enzymes, widespread among anaerobic and facultative anaerobic prokaryotes, from both the Archaea and Bacteria domains. Noticeably, genes encoding for homologous enzymes are also present in the genomes of some pathogenic and anaerobic amitochondriate protozoa. The fingerprint of this enzyme family is the conservation of a two-domain structural core, built by a metallo-beta-lactamase-like domain, at the N-terminal region, harbouring a non-heme diiron site, and a flavodoxin-like domain, containing one FMN moiety. These enzymes have a significant nitric oxide reductase activity, and there is increasing evidence that they are involved in microbial resistance to nitric oxide. In this review, we will discuss available data for this novel family of enzymes, including their physicochemical properties, structural and phylogenetic analyses, enzymatic properties and the molecular genetic approaches so far used to tackle their function.

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur.

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.

Crystal structure of the carboxyltransferase subunit of the bacterial sodium ion pump glutaconyl-coenzyme A decarboxylase.

Glutaconyl-CoA decarboxylase is a biotin-dependent ion pump whereby the free energy of the glutaconyl-CoA decarboxylation to crotonyl-CoA drives the electrogenic transport of sodium ions from the cytoplasm into the periplasm. Here we present the crystal structure of the decarboxylase subunit (Gcdalpha) from Acidaminococcus fermentans and its complex with glutaconyl-CoA. The active sites of the dimeric Gcdalpha lie at the two interfaces between the mono mers, whereas the N-terminal domain provides the glutaconyl-CoA-binding site and the C-terminal domain binds the biotinyllysine moiety. The Gcdalpha catalyses the transfer of carbon dioxide from glutaconyl-CoA to a biotin carrier (Gcdgamma) that subsequently is decarboxylated by the carboxybiotin decarboxylation site within the actual Na(+) pump (Gcdbeta). The analysis of the active site lead to a novel mechanism for the biotin-dependent carboxy transfer whereby biotin acts as general acid. Furthermore, we propose a holoenzyme assembly in which the water-filled central channel of the Gcdalpha dimer lies co-axial with the ion channel (Gcdbeta). The central channel is blocked by arginines against passage of sodium ions which might enter the central channel through two side channels.

Complete DNA sequence of the atp operon of the sodium-dependent F1Fo ATP synthase from Ilyobacter tartaricus and identification of the encoded subunits.

The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F(1)F(o) ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6,992 base pairs with a GC content of 38.1%. The F(1)F(o) ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4,810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na(+)-translocating F(1)F(o) ATP synthases, especially in the F(o) subunits a and c which are directly involved in ion translocation.

Evidence of quorum sensing in the rumen ecosystem: detection of N-acyl homoserine lactone autoinducers in ruminal contents.

Acyl-homoserine lactone (AHL) based quorum-sensing systems are widespread among gram-negative bacteria, particularly in association with plants and animals. As yet, there have been no reports of AHL signaling in the anaerobic rumen environment, an ecosystem of great complexity in which cell-cell signaling is likely to occur. We detected multiple AHL autoinducers in the rumen contents of 6 out of 8 cattle fed a representative selection of diets. The signals were not associated with feed. Surprisingly, no pure cultures produced AHLs in vitro when grown under the laboratory conditions we tested. Our observations suggest that either (a) a factor specific to the rumen ecosystem is required for the rumen isolates we tested to produce AHLs or (b) a strain (or strains) that we were not able to culture but which grows to a high cell density in the rumen produces the AHLs we detected.