Dataset Information

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

ABSTRACT: Nitrosomonas europaea has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of amoA were obtained, and they were found to differ by one nucleotide. To determine if both copies of amoA were functional, insertional mutagenesis was performed to inactivate either copy of amoA alone. A DNA cassette containing the lacZ and kan genes inserted into amoA was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of amoA. Mutations were obtained in both copies of amoA. Either copy of amoA was sufficient to support growth when the other copy was disrupted. However, inactivation of one copy of amoA, but not the other, resulted in slower growth. Measurements of ammonia-dependent O2 consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [14C]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type. Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.

SUBMITTER: Hommes NG

PROVIDER: S-EPMC107289 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

Hommes N G NG Sayavedra-Soto L A LA Arp D J DJ

Journal of bacteriology 19980701 13

Nitrosomonas europaea has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of amoA were obtained, and they were found to differ by one nucleotide. To determine if both copies of amoA were functional, insertional mutagenesis was performed to inactivate either copy of amoA alone. A DNA cassette containing the lacZ and kan genes inserted into amoA was constructed. Mutagenesis was done by using transformation and homologous recombination to mo ...[more]

PMID: 9642187

Similar Datasets

Project description:Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea's sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria.IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Project description:Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.

Dataset Information

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

Publications

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets