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K RE UZ E R. 1999. Diets containing either coconut oil or rumen-protected fat (54 g kg −1 dry matter each) were supplied to Rumen Simulation Technique fermenters filled with faunated and defaunated rumen fluid in a 2 × 2 factorial design. Defaunation immediately reduced methane formation by about 40% with each diet. With coconut oil, methane gradually declined in faunated and defaunated rumen fluid. Finally, the extent of methane suppression was similar, both with coconut oil and with defaunation. Independently of the status of protozoa, the population of methanogens in rumen fluid was significantly reduced by coconut oil. The results suggest that defaunation and coconut oil independently and additively suppress rumen methanogenesis.

Increasing quantities of man-made organic chemicals are released each year into the biosphere. Some of these compounds are both toxic and relatively resistant to physical, chemical, or biological degradation, and they thus constitute an environmental burden of considerable magnitude. Genetic manipulation of microbial catabolic pathways offers a powerful means by which to accelerate evolution of biodegradative routes through which such compounds might be eliminated from the environment. In the experiments described here, a catabolic pathway for alkylbenzoates specified by the TOL plasmid of Pseudomonas was restructured to produce a pathway capable of processing a new substrate, 4-ethylbenzoate. Analysis of critical steps in the TOL pathway that prevent metabolism of 4-ethylbenzoate revealed that this compound fails to induce synthesis of the catabolic enzymes and that one of its metabolic intermediates inactivates catechol 2,3-dioxygenase (C23O), the enzyme that cleaves the aromatic ring. Consequently, the pathway was sequentially modified by recruitment of genes from mutant bacteria selected for their production of either an altered pathway operon regulator that is activated by 4-ethylbenzoate or an altered C23O that is less sensitive to metabolite inactivation. The redesigned pathway was stably expressed and enabled host bacteria to degrade 4-ethylbenzoate in addition to the normal substrates of the TOL pathway.

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SummaryThe methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the doublestranded DNA phage ⌿M2. The complete phage genome sequence of 26 111 bp was established. Thirty-one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site-specific recombinases. Analysis of the N-terminal amino acid sequences of three phageencoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage ⌿M2 DNA sequence with parts of the sequence of the wild-type phage ⌿M1 suggests that ⌿M2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.

A family of flavoproteins, called A-type flavoproteins, is described. It consists of 14 protein sequences of 385Ϫ597 amino acids in length, 7 from methanogens (domain: Archaea), 5 from phototrophic prokaryotes, one from Escherichia coli, and a partial sequence from the sulfate reducer Desulfovibrio gigas (domain : Bacteria). No similar sequence could be found in the domain Eucarya. All sequences show significant similarity over a 385Ϫ400 amino acid portion overlapping a recognizable flavodoxin signature starting at positions 245Ϫ285 of the common core sequence. Cofactor analysis and, to some extent, analysis of the primary structure of six A-type flavoproteins, three of which are structurally characterized here, support the existence of four sub-families: Here we have aligned 14 sequences and FAD, which enables them to catalyze a variety of oneelectron and two-electron transfer reactions [1]. A number of and characterized the R. capsulatus protein and 2 additional members of this group of proteins as simple or complex flavoflavoproteins are known that contain additional redox centers like a disulfide bond, a heme group, a metal, or iron-sulfur cen-proteins. As a result, we propose to group them into a heterogeneous family, the A-type flavoproteins. ters [2]. Flavoproteins known to bind several cofactors, like cytochromes P-450, have distinct binding sites often located on separate protein domains [3Ϫ5].EXPERIMENTAL PROCEDURES We have recently purified an FMN-containing flavoprotein from cells of Methanobacterium thermoautotrophicum, flavoMaterials, Chemicals and enzymes. Restriction enzymes, protein A, which, like flavodoxins, is up-regulated under iron T4 DNA ligase, and Taq polymerase were purchased from and limitation and possesses a perfectly conserved flavodoxin signa-used as recommended by Fermentas. Oligonucleotide primers ture [6]. However, it differs from typical flavodoxins in being were synthesized at Microsynth. Q Sepharose Fast Flow, Sephmuch larger (a dimer of a 404-amino-acid polypeptide) and in acryl S-200 and S-300 HR were from Pharmacia, and Fractogel having a relatively high redox potential of Ϫ230 mV for the EMD-TMAE(650)-S from Merck (Darmstadt, Germany). Other quinone/hydroquinone couple [7]; so far, its function remains chemicals were obtained from Fluka (Buchs, Switzerland), and hypothetical [6Ϫ7]. At the time of its discovery, there was only were from the highest grade available. one flavoprotein clearly homologous to M. thermoautotroSequence analysis. Sequence analyses were carried out with phicum flavoprotein A, namely the product of an open reading the University of Wisconsin's Genetics Computer Group packframe in an operon involved in nitrogen fixation in Rhodobacter age, versions 8 and 9 and at the National Center for Biotechnolcapsulatus [8Ϫ9]. Since then, the completion of several se-ogy Information via the BLAST network service. quencing projects has permitted the discovery of ten predicted Amplification, cloning, and expression of E. coli orf479, polypeptide sequences unambiguou...

F. DOHME, A. MACHMÜLLER, A. WASSERFALLEN AND M. KREUZER. 2001. The objective of the present study was to investigate the effects of seven different pure fatty acids on rumen fermentation using the rumen simulation technique (RUSITEC). The fatty acids were supplied to a complete ruminant diet at a proportion of 50 g kg−1 dietary dry matter and compared with an unsupplemented control. Methane release and methanogenic counts were suppressed by the fatty acids C12 : 0, C14 : 0 and C18 : 2 whereas C8 : 0, C10 : 0, C16 : 0 and C18 : 0 showed no corresponding effects. Apart from C12 : 0 and C18 : 2, C8 : 0 and C10 : 0 also adversely affected ciliate protozoa suggesting independence from the methane‐suppressing effect of medium‐chain fatty acids (MCFA). Although MCFA but not C18 : 2 reduced ruminal fibre degradation, the influence on other fermentation traits remained low. In conclusion, the supply of certain fatty acids to ruminant diets seems to have the potential to reduce methane release.

The objective of the present study was to investigate the effects of seven different pure fatty acids on rumen fermentation using the rumen simulation technique (RUSITEC). The fatty acids were supplied to a complete ruminant diet at a proportion of 50 g x kg(-1) dietary dry matter and compared with an unsupplemented control. Methane release and methanogenic counts were suppressed by the fatty acids C12 : 0, C14 : 0 and C18 : 2 whereas C8 : 0, C10 : 0, C16 : 0 and C18 : 0 showed no corresponding effects. Apart from C12 : 0 and C18 : 2, C8 : 0 and C10 : 0 also adversely affected ciliate protozoa suggesting independence from the methane-suppressing effect of medium-chain fatty acids (MCFA). Although MCFA but not C18 : 2 reduced ruminal fibre degradation, the influence on other fermentation traits remained low. In conclusion, the supply of certain fatty acids to ruminant diets seems to have the potential to reduce methane release.