Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters

Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested.     

Identification of lactobacilli residing in chicken ceca with antagonism against Campylobacter

Bacteriocins produced by Lactobacillus salivarius have been recently recognized as a natural means to control Campylobacter and Salmonella in live poultry. This finding is of relevance since Campylobacter jejuni and Campylobacter coli are the predominant species isolated from poultry that are associated with human campylobacteriosis. In the present work, lactic acid bacteria (LAB) isolated from the cecum of twenty Tunisian chickens were identified and those isolates with antagonism against Campylobacter were further characterized. Following their preliminary confirmation as LAB, 150 strains were identified by combining morphological criteria, biochemical tests, and molecular methods, the latter inluding intergenic 16S- 23S PCR, specific lactobacilli PCR, and a biphasic approach. Most of the LAB isolated belonged to the genus Lactobacillus, among them Lb. sakei (33.3%), Lb. salivarius (19.4%), Lb. reuteri (8.6%), and Lb. curvatus (8.6%). The other LAB strains included those of the genus Weissella (16.7%), Enterococcus faecalis (5.3%), Leuconostoc mesenteroides (2.7%), Lactococcus graviae (2.7%), and Streptococcus sp. (2.7%). The Lactobacilli strains were tested for their antagonism against C. jejuni and C. coli. The activity of three of them, Lb. salivarius SMXD51, Lb. salivarius MMS122, and Lb. salivarius MMS151, against the aforementioned target strains could be ascribed to the production of bacteriocins.     

甲烷古菌群感效应信号分子的检测

竹节状甲烷鬃菌(Methanosaeta harundinacea)6Ac是本实验室分离自厌氧颗粒污泥中的甲烷古菌新种。该菌具有短杆(3μm-5μm)和长链状(>200μm)两种细胞形态,且与细胞密度相关,暗示该菌可能存在群感效应调控的细胞形态变化。【目的】验证该菌存在群感效应信号分子并与细胞形态变化相关。【方法】用高丝氨酸内酯指示菌Agrobacterium tumefaciens NTL4检测菌株6Ac的培养液,并用购买的高丝氨酸内酯标准品加入短杆菌株6Ac检测形态变化。【结果】菌株6Ac的培养液中含有高丝氨酸内酯类物质。实验证明化学合成的高丝氨酸内酯N-(β-酮基)辛酰高丝氨酸内酯能够促进竹节状甲烷鬃菌的长链细胞形成。而且在马氏甲烷八叠球菌(Methanosarcina mazei)、热自养甲烷杆菌(Methanothermobacter thermautotrophicus)和甲酸甲烷杆菌(Methanobacterium formicicum)的培养液中也检测到了高丝氨酸内酯。【结论】多种甲烷古菌可以产生高丝氨酸内酯类物质,并可能以此类物质作为群感效应的信号分子。     

Comprehensive computational analysis of Hmd enzymes and paralogs in methanogenic Archaea

Background  

Methanogenesis is the sole means of energy production in methanogenic Archaea. H₂-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) catalyzes a step in the hydrogenotrophic methanogenesis pathway in class I methanogens. At least one hmd paralog has been identified in nine of the eleven complete genome sequences of class I hydrogenotrophic methanogens. The products of these paralog genes have thus far eluded any detailed functional characterization.     

A novel pathway of peptide biosynthesis found in methanogenic Archaea

The peptide subunits of the pseudomurein, the cell-wall peptidoglycan of some methanogens, are usually composed of glutamic acid, alanine and lysine. In order to get a more detailed picture of the biosynthetic pathway of the peptide subunit, we performed in vitro assays. Starting from glutamic acid a pentapeptide was obtained in seven steps:

Identification of microRNAs from different tissues of chicken embryo and adult chicken

We report for the first time the identification of 25 microRNAs from tissues originating from chicken embryo and adult chicken. Most of the cloned microRNAs are expressed in both adult chickens and chicken embryos. Fourteen were identified without any prior prediction. One microRNA, miR-757, is thought to be chicken-specific. Three of the microRNAs appear to be extremely tissue specific.     

Presence of tRNA-dependent pathways correlates with high cysteine content in methanogenic Archaea

Archeal proteomes can be clustered into two groups based on their cysteine content. One group of proteomes displays a low cysteine content ( approximately 0.7% of the entire proteome), whereas the second group contains twice as many cysteines as the first ( approximately 1.3%). All cysteine-rich organisms belong to the methanogenic Archaea, which generates special cysteine clusters associated with primitive metabolic reactions. Our findings suggest that cysteine plays an important role in early forms of life.     

Morphological and physiological characteristics of Gemmiger formicilis isolated from chicken ceca.

Morphological and physiological studies were made on chicken cecal isolates of the strictly anaerobic bacterial species Gemmiger formicilis. Structural features (phase-contrast and electron microscopy) of these microorganisms indicate they (i) are highly pleomorphic, (ii) possess a trilaminar cell wall like gram-negative bacteria, (iii) exhibit an unusual growth process characterized by polar swelling (resembling budding bacteria), and (iv) grow into elongated cells when exposed to a subinhibitory concentration of penicillin. The morphological data presented suggest that this species has a rod-shaped structure. These bacteria ferment a variety of sugars to produce formic, butyric, and lactic acids. There appear to be two groups of Gemmiger, one producing primarily lactate and the other producing formate as major fermentation metabolites. Growth of six strains in a basal medium, consisting of Trypticase, minerals, carbohydrate, Na2CO3 buffer, and cysteine as reducing agent, was stimulated by rumen fluid and yeast extract. Volatile fatty acids partially replaced the requirement for rumen fluid with some strains. Single deletions of vitamins (from a defined vitamin mixture) indicated that pantothenate, riboflavin, and thiamine were highly stimulatory to growth of the organism in a medium containing rumen fluid and Trypticase as source of vitamins. Other vitamin requirements were not studied.     

Localization and quantification of extracellular polymers in methanogenic granular sludge

Summary Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed. Offsprint requests to: A. J. B. Zehnder     

Identification of an androgen receptor in the adult chicken oviduct

The chicken oviduct androgen receptor was characterized by sucrose density gradient centrifugation, Scatchard analysis, competition studies, and affinity labeled with dihydrotestosterone 17 beta-bromoacetate. A specific 8.5 S peak was seen on 0.01 M KCl sucrose density gradients when the receptor was labeled with [3H]5 alpha-dihydrotestosterone. Specific 4.6 S peaks were seen when receptor labeled with [3H]5 alpha-dihydrotestosterone or [3H]dihydrotestosterone 17 beta-bromoacetate was analyzed on 0.3 M KCl sucrose density gradients. Scatchard analysis of [3H]5 alpha-dihydrotestosterone binding by oviduct cytosol was consistent with two binding sites. A Kd of 0.13 nM was found for the high affinity androgen receptor. Competition studies showed the following order of ligand affinity: 5 alpha-dihydrotestosterone greater than dihydrotestosterone 17 beta-bromoacetate greater than progesterone greater than estradiol. A 61.2 kDa protein was specifically covalently labeled with [3H]dihydrotestosterone 17 beta-bromoacetate. The chicken oviduct androgen receptor possesses characteristics similar to other androgen receptors, and provides a good source of androgen receptor for physicochemical studies of the native receptor protein.     

Identification of Protein-Tyrosine Phosphatases in Archaea

Protein-tyrosine dephosphorylation is a major mechanism in cellular regulation. A large number of protein-tyrosine phosphatases is known from Eukarya, and more recently bacterial homologues have also been identified. By employing conserved sequence patterns from both eukaryotic and bacterial protein-tyrosine phosphatases, we have identified three homologous sequences in two of the four complete archaeal genomes. Two hypothetical open reading frames in the genome of Methanococcus jannaschii (MJ0215 and MJECL20) and one in the genome of Pyrococcus horikoshii (PH1732) clearly bear all the conserved residues of this family. No homologues were found in the genomes of Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. This is the first report of protein-tyrosine phosphatase sequences in Archaea. Received: 29 April 1998 / Accepted: 27 November 1998     

Identification of acetate-utilizing Bacteria and Archaea in methanogenic profundal sediments of Lake Kinneret (Israel) by stable isotope probing of rRNA

Acetate is an important intermediate in the decomposition of organic matter in anoxic freshwater sediments. Here, we identified distinct microorganisms active in its oxidation and transformation to methane in the anoxic methanogenic layers of Lake Kinneret (Israel) profundal sediment by rRNA-based stable isotope probing (RNA-SIP). After 18 days of incubation with amended [U-(13)C]acetate we found that archaeal 16S rRNA was (13)C-labelled to a far greater extent than bacterial rRNA. We identified acetoclastic methanogens related to Methanosaeta concilii as being most active in the degradation and assimilation of acetate. Oxidation of the acetate-methyl group played only a minor role, but nevertheless 'heavy'(13)C-labelled bacterial rRNA templates were identified. 'Heavy' bacteria were mainly affiliated with the Betaproteobacteria (mostly Rhodocyclales and Nitrosomonadales), the Nitrospira phylum (related to 'Magnetobacterium bavaricum' and Thermodesulfovibrio yellowstonii), and also with the candidate phylum 'Endomicrobia'. However, the mode of energy gain that allowed for the assimilation of (13)C-acetate by these bacterial groups remains unknown. It may have involved syntrophic oxidation of acetate, reduction of chlorinated compounds, reduction of humic substances, fermentation of organic compounds, or even predation of (13)C-labelled Methanosaeta spp. In summary, this SIP experiment shows that acetate carbon was predominantly consumed by acetoclastic methanogens in profundal Lake Kinneret sediment, while it was also utilized by a small and heterogeneous community of bacteria.     

Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log(10) 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log(10) 6.9 CFU/g).     

Molecular detection and direct enumeration of methanogenic Archaea and methanotrophic Bacteria in domestic solid waste landfill soils

Methane oxidizing and producing activities of cover soil (10, 30 cm depth) and burial waste (1, 3 m depth) were evaluated: top cover soil (10 cm) had the highest methane oxidizing activity, while 1 m depth buried waste showed the highest methane producing potential. All the sequences of the 1 m sample were found to be closely related to 16S rDNAs of mainly hydrogenotrophic methanogens known, such as genera Methanosarcina, Methanoculleus, and Methanobacterium. We developed a modified fluorescence in situ hybridization (FISH) direct counting method for landfill samples, resulting in the detection of approx. 1% of total cells as archaeal cells (presumably methanogens). However, probe-positive cells could not be found with probes for methanotrophs by the methods.     

Effect of ammonia on the methanogenic activity of methylaminotrophic methane producing Archaea enriched biofilm

Ammonia is a metabolic product in the decomposition of protein wastes, and has a recognized inhibitory effect on methanogenesis; this effect has been slightly quantified on methanogenic biofilms and particularly those populated by methanogenic Archaea which produce ammonia as a catabolic product from methylated amines. This paper presents studies on the effect of ammonia on maximum methanogenic activity of anaerobic biofilms enriched by methylaminotrophic methane producing Archaea (mMPA). The effect of unionized free ammonia on the specific maximum methanogenic activity of a mMPA enriched biofilm was studied, using 250 mL flasks containing ceramic rings colonized by 30 day-old experimental biofilm and adding 48.8 (control system), 73.8, 98.8, 148.8, 248.8, 448.8 and 848.8 mg NH(3)-N/L. The systems were maintained for ten days at a pH of 7.5 and temperature of 37 degrees C. The results showed that at 848.8 mg NH(3)-N/L, biofilm methane production required 36 h adaptation period, prior to entering into maximum production phase. The highest maximum methanogenic activity reached a value of 2.337+/-0.213 g COD methane/g VSS *day when 48.8 mg NH(3)-N/L was added, and inhibition was clearly observed in those systems above 148.8 mg NH(3)-N/L, producing under 1.658+/-0.185 g COD methane/g VSS *day. The lowest methanogenic activity reached was 0.639+/-0.162 g COD methane/g VSS *day at the system added with 848.8 mg NH(3)-N/L. When applying the Luong and non-competitive inhibition models, the best fit was obtained with the non-competitive model, which predicted 50% inhibition of methanogenic activity at 365.288 mg NH(3)-N/L.     

The adenylate kinases from a mesophilic and three thermophilic methanogenic members of the Archaea.

Adenylate kinase has been isolated from four related methanogenic members of the Archaea. For each, the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 degrees C for Methanococcus voltae, 70 degrees C for Methanococcus thermolithotrophicus, 80 degrees C for Methanococcus igneus, and 80 to 90 degrees C for Methanococcus jannaschii. The enzymes were sensitive to the adenylate kinase inhibitor P1, P5-di(adenosine-5')pentaphosphate, a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography. The enzymes had an estimated molecular mass (approximately 23 to 25 kDa) in the range common for adenylate kinases. Each of the enzymes had a region of amino acid sequence close to its N terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases. However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases, including an enzyme isolated from the archaeon Sulfolobus acidocaldarius. If verified as a nucleotide-binding domain, the methanogen sequence would represent a novel nucleotide-binding motif. There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.