Desulfitobacterium strains have the ability to dechlorinate halogenated compounds under anaerobic conditions by dehalorespiration. The complete genome of the tetrachloroethene (PCE)-dechlorinating strain Desulfitobacterium hafniense Y51 is a 5,727,534-bp circular chromosome harboring 5,060 predicted protein coding sequences. This genome contains only two reductive dehalogenase genes, a lower number than reported in most other dehalorespiring strains. More than 50 members of the dimethyl sulfoxide reductase superfamily and 30 paralogs of the flavoprotein subunit of the fumarate reductase are encoded as well. A remarkable feature of the genome is the large number of O-demethylase paralogs, which allow utilization of lignin-derived phenyl methyl ethers as electron donors. The large genome reveals a more versatile microorganism that can utilize a larger set of specialized electron donors and acceptors than previously thought. This is in sharp contrast to the PCE-dechlorinating strain Dehalococcoides ethenogenes 195, which has a relatively small genome with a narrow metabolic repertoire. A genomic comparison of these two very different strains allowed us to narrow down the potential candidates implicated in the dechlorination process. Our results provide further impetus to the use of desulfitobacteria as tools for bioremediation.Halogenated organic compounds are released into the environment from natural and anthropogenic sources. Many anthropogenic halogenated chemicals, like chlorinated haloalkenes (7, 10, 46), benzenes (1), and dioxins (5), are of particular concern due to their toxicity to humans and other forms of life. This toxicity is often paired with high recalcitrance to degradation, especially in anaerobic environments, leading to persistent contamination.Anaerobic environments are frequently characterized by limited availability of electron acceptors. Theoretical calculations have shown that coupling the reduction of many halogenated organic compounds to the oxidation of suitable substrates is a way to harness energy (46). As determined two decades ago, this source of energy is utilized by the microbial community. The oxidation of available electron donors coupled to the reduction of halogenated organic compounds while energy is conserved is called dehalorespiration (7,10,46). Dehalorespiring strains have been isolated independently from contaminated sites around the world. The two most prominent genera resulting from these isolation efforts are Dehalococcoides (29) and Desulfitobacterium (51), and various strains of these genera are used as model systems to study dehalorespiration (8,11,51).Dehalococcoides ethenogenes 195 is one of the few strains isolated to date which can dechlorinate tetrachloroethene (PCE) to ethene (29). D. ethenogenes 195 can use only hydrogen as an electron donor and chlorinated compounds as electron acceptors (29).Desulfitobacterium strains are also known to dechlorinate a wide variety of substrates, including halophenolic compounds and chloroalkenes (7,10,46). Although several s...

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.

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A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygenlimiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.Benzene, toluene, ethylbenzene, and xylenes (BTEX) are one of the most common groups of groundwater contaminants. Of these contaminants, toluene is degraded by many strains of aerobic bacteria, and five different aerobic toluene degradation pathways have been identified. Burkholderia cepacia G4, Ralstonia pickettii PKO1, and Pseudomonas mendocina KR1 first oxidize toluene using specific monooxygenases to form o-, m-, and p-cresol, respectively (34, 35, 52). The cresols formed by strains G4 and PKO1 undergo a second monooxygenation to form 3-methylcatechol, which is then degraded by a meta ring fission pathway (35,45). In strain KR1, the methyl group of p-cresol is oxidized, and the resulting 4-hydroxybenzoate is degraded by an ortho cleavage pathway (51). On the other hand, Pseudomonas putida mt-2 oxidizes the methyl group of toluene to form benzoic acid, which is further metabolized through a meta cleavage pathway via catechol (4). P. putida F1 carries the chromosomally encoded tod pathway. Toluene dioxygenase (TodC1C2BA) oxidizes toluene to cis-toluene dihydrodiol. Then, toluene dihydrodiol dehydrogenase (TodD) transforms the dihydrodiol to 3-methylcatechol, which is cleaved by the meta fission enzyme 3-methylcatechol 2,3-