Marco Fischer

Enrichment of a dioxin-dehalogenating Dehalococcoides species in two-liquid phase cultures.

Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.

The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases

Streptomyces coelicolor A3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all three narGHJI operons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triple narGHJI mutant, the moaA mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in S. coelicolor are also molybdenum-dependent. Analysis of S. coelicolor growth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-type S. coelicolor spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the moaA and triple nar mutants were chlorate-resistant. Complementation of the triple nar mutant with the individual narGHJI operons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nar1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.

Terminal reduction reactions of nitrate and sulfate assimilation in Streptomyces coelicolor A3(2): identification of genes encoding nitrite and sulfite reductases.

The model actinobacterium Streptomyces coelicolor A3(2) uses nitrate and sulfate as nitrogen and sulfur sources, respectively. The final step prior to assimilation into amino acids is the 6-electron reduction of the nitrite and sulfite anions, catalyzed by siroheme-dependent nitrite (NirBD) and sulfite (SirA) reductases. There are two predicted nitrite/sulfite reductases annotated in the genome of S. coelicolor, but it is unclear which is responsible for nitrite and which for sulfite reduction. Here we demonstrate that a knock-out in the genes SCO2487 and SCO2488 encoding NirBD prevents use of nitrite as a nitrogen source, while a knock-out in SCO6102 encoding SirA prevents sulfate assimilation. Both mutations could be phenotypically complemented by supplementation of the growth medium with ammonium or casamino acids in the case of the nirBD mutants or sulfur-containing amino acids in the case of the sirA mutants. No functional redundancy between the genes was observed and we demonstrate that NirBD is exclusively required for assimilatory nitrite (it does not detoxify nitrite) and SirA exclusively for assimilatory sulfite reduction.

A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

The Gram‐positive aerobe Streptomyces coelicolor undergoes a complex life cycle including growth as vegetative hyphae and the production of aerial hyphae and spores. Little is known about how spores retain viability in the presence of oxygen; however, nothing is known about this process during anaerobiosis. Here, we demonstrate that one of the three respiratory nitrate reductases, Nar‐1, synthesized by S. coelicolor is functional exclusively in spores. A tight coupling between nitrite production and the activity of the cytoplasmically oriented Nar‐1 enzyme was demonstrated. No exogenous electron donor was required to drive nitrate reduction, which indicates that spore storage compounds are used as electron donors. Oxygen reversibly inhibited nitrate reduction by spores but not by spore extracts, suggesting that nitrate transport might be the target of oxygen inhibition. Nar‐1 activity required no de novo protein synthesis indicating that Nar‐1 is synthesized during sporulation and remains in a latently active state throughout the lifetime of the spore. Remarkably, the rates of oxygen and of nitrate reduction by wetted spores were comparable. Together, these findings suggest that S. coelicolor spores have the potential to maintain a membrane potential using nitrate as an alternative electron acceptor.

Oxygen-Dependent Control of Respiratory Nitrate Reduction in Mycelium of Streptomyces coelicolor A3(2)

Several members of the obligately aerobic genus Streptomyces are able to reduce nitrate, catalyzed by Nar-type respiratory nitrate reductases. A unique feature of Streptomyces coelicolor A3(2) compared with other streptomycetes is that it synthesizes three nonredundant Nar enzymes. In this study, we show that Nar2 is the main Nar enzyme active in mycelium and could characterize the conditions governing its synthesis. Nar2 was present at low levels in aerobically cultivated mycelium, but synthesis was induced when cultures were grown under oxygen limitation. Growth in the presence of high oxygen concentrations prevented the induction of Nar2 synthesis. Equally, an abrupt shift from aerobiosis to anaerobiosis did not result in the immediate induction of Nar2 synthesis. This suggests that the synthesis of Nar2 is induced during a hypoxic downshift, probably to allow maintenance of a proton gradient during the transition to anaerobiosis. Although no Nar2 could be detected in freshly harvested mature spores, synthesis of the enzyme could be induced after long-term (several days) incubation of these resting spores under anaerobic conditions. Induction of Nar2 synthesis in spores was linked to transcriptional control. Nar2 activity in whole mycelium was strictly dependent on the presence of a putative nitrate transporter, NarK2. The oxygen-dependent inhibition of nitrate reduction by Nar2 was mediated by NarK2-dependent nitrate:nitrite antiport. This antiport mechanism likely prevents the accumulation of toxic nitrite in the cytoplasm. A deletion of the narK2 gene had no effect on Nar1-dependent nitrate reduction in resting spores. Together, our results indicate redox-dependent transcriptional and posttranslational control of nitrate reduction by Nar2.

Oxygen and Nitrate Respiration in Streptomyces coelicolor A3(2).

Streptomyces species belong to the phylum Actinobacteria and can only grow with oxygen as a terminal electron acceptor. Like other members of this phylum, such as corynebacteria and mycobacteria, the aerobic respiratory chain lacks a soluble cytochrome c. It is therefore implicit that direct electron transfer between the cytochrome bc1 and the cytochrome aa3 oxidase complexes occurs. The complex developmental cycle of streptomycetes manifests itself in the production of spores, which germinate in the presence of oxygen into a substrate mycelium that greatly facilitates acquisition of nutrients necessary to support their saprophytic lifestyle in soils. Due to the highly variable oxygen levels in soils, streptomycetes have developed means of surviving long periods of hypoxia or even anaerobiosis but they fail to grow under these conditions. Little to nothing is understood about how they maintain viability under conditions of oxygen limitation. It is assumed that they can utilise a number of different electron acceptors to help them maintain a membrane potential, one of which is nitrate. The model streptomycete remains Streptomyces coelicolor A3(2), and it synthesises three nonredundant respiratory nitrate reductases (Nar). These Nar enzymes are synthesised during different phases of the developmental cycle and they are functional only under oxygen-limiting (<5% oxygen in air) conditions. Nevertheless, the regulation of their synthesis does not appear to be responsive to nitrate and in the case of Nar1, it appears to be developmentally regulated. This review highlights some of the novel aspects of our current, but somewhat limited, knowledge of respiration in these fascinating bacteria.