The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, ␤-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfurcompound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfurcompound oxidation by ␤-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.Thiobacillus denitrificans, first isolated by Beijerinck over a century ago (4), was one of the first nonfilamentous bacteria ever described to be capable of growth on inorganic sulfur compounds as sole energy sources (47, 49). Characterized by its ability to conserve energy from the oxidation of inorganic sulfur compounds under either aerobic or denitrifying conditions, T. denitrificans is the best studied of the very few obligate chemolithoautotrophic species known to couple denitrification to sulfur-compound oxidation (Thiomicrospira denitrificans and Thioalkalivibrio thiocyanodenitrificans also have this ability [76,85]). Despite many years of work on the biochemistry of inorganic sulfur-compound oxidation by Thiobacillus thioparus and T. denitrificans, the mechanisms of oxidation and how they are coupled to energy conservation are still not well understood in these ␤-proteobacteria, relative to the advances made with facultatively chemolithotrophic ␣-proteobacterial genera, such as Paracoccus and Starkeya (28,39,45,50). The availability of the complete genome sequence should enable elucidation of the sulfur-oxidation pathway(s) and lead to specifically focused biochemical investigations to resolve these knowledge gaps.Rec...

The capacity of some bacteria to metabolize hydrocarbons in the absence of molecular oxygen was first recognized only about ten years ago. Since then, the number of hydrocarbon compounds shown to be catabolized anaerobically by pure bacterial cultures has been steadily increasing. This review summarises the current knowledge of the bacterial isolates capable of anaerobic mineralization of hydrocarbons, and of the biochemistry and molecular biology of enzymes involved in the catabolic pathways of some of these substrates. Several alkylbenzenes, alkanes or alkenes are anaerobically utilized as substrates by several species of denitrifying, ferric iron‐reducing and sulfate‐reducing bacteria. Another group of anaerobic hydrocarbon degrading bacteria are ‘proton reducers’ that depend on syntrophic associations with methanogens. For two alkylbenzenes, toluene and ethylbenzene, details of the biochemical pathways involved in anaerobic mineralization are known. These hydrocarbons are initially attacked by novel, formerly unknown reactions and oxidized further to benzoyl‐CoA, a common intermediate in anaerobic catabolism of many aromatic compounds. Toluene degradation is initiated by an unusual addition reaction of the toluene methyl group to the double bond of fumarate to form benzylsuccinate. The enzyme catalyzing this first step has been characterized at both the biochemical and molecular level. It is a unique type of glycyl‐radical enzyme, an enzyme family previously represented only by pyruvate‐formate lyases and anaerobic ribonucleotide reductases. Based on the nature of benzylsuccinate synthase as a radical enzyme, a hypothetical reaction mechanism for the addition of toluene to fumarate is proposed. The further catabolism of benzylsuccinate to benzoyl‐CoA and succinyl‐CoA appears to occur via reactions of a modified β‐oxidation pathway. Ethylbenzene is first oxidized at the methylene carbon to 1‐phenylethanol and subsequently to acetophenone, which is then carboxylated to 3‐oxophenylpropionate and converted to benzoyl‐CoA and acetyl‐CoA. Anaerobic mineralization of alkanes involves an oxygen‐independent oxidation to fatty acids, followed by β‐oxidation. In one strain of an alkane‐mineralizing sulfate‐reducing bacterium, the activation appears to proceed via a chain‐elongation, possibly by addition of a C1‐group at the terminal methyl group of the alkane. Finally, aspects concerned with the regulation and ecological significance of anaerobic hydrocarbon catabolic pathways are discussed.

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.Interest in the anaerobic metabolism of alkylbenzenes is increasing because of the subject's relevance to bioremediation of fuel-contaminated aquifers and because of novel aspects of the biochemical reactions involved. Of particular biochemical interest is how aromatic hydrocarbons can be activated in the absence of mono-and dioxygenase-catalyzed reactions. Toluene appears to be the most readily degraded of the alkylbenzenes under anaerobic conditions and has been the most intensively studied. Since 1989, pure cultures capable of anaerobic toluene degradation have been reported under denitrifying conditions (10,13,15,21,22), ferric iron-reducing conditions (17, 18), and sulfate-reducing conditions (5, 20); fermentative-methanogenic mixed enrichment cultures that degrade toluene have also been reported (11,16,24). Several anaerobic toluene mineralization pathways have been proposed, with varying degrees of experimental support. Initial reactions of toluene in these proposed pathways include (i) hydroxylation of the methyl group to form benzyl alcohol (1, 2), (ii) hydroxylation of the aromatic ring to form p-cresol (e.g., references 16 and 24), (iii) attack of the methyl carbon by acetyl coenzyme A (CoA) to form phenylpropionyl-CoA (12), and very recently (iv) addition of the methyl carbon to fumarate to form benzylsuccinate (7). To date,...

We have engineered Escherichia coli to overproduce saturated and monounsaturated aliphatic methyl ketones in the C 11 to C 15 (diesel) range; this group of methyl ketones includes 2-undecanone and 2-tridecanone, which are of importance to the flavor and fragrance industry and also have favorable cetane numbers (as we report here). We describe specific improvements that resulted in a 700-fold enhancement in methyl ketone titer relative to that of a fatty acid-overproducing E. coli strain, including the following: (i) overproduction of ␤-ketoacyl coenzyme A (CoA) thioesters achieved by modification of the ␤-oxidation pathway (specifically, overexpression of a heterologous acyl-CoA oxidase and native FadB and chromosomal deletion of fadA) and (ii) overexpression of a native thioesterase (FadM). FadM was previously associated with oleic acid degradation, not methyl ketone synthesis, but outperformed a recently identified methyl ketone synthase (Solanum habrochaites MKS2 [ShMKS2], a thioesterase from wild tomato) in ␤-ketoacyl-CoA-overproducing strains tested. Whole-genome transcriptional (microarray) studies led to the discovery that FadM is a valuable catalyst for enhancing methyl ketone production. The use of a two-phase system with decane enhanced methyl ketone production by 4-to 7-fold in addition to increases from genetic modifications.