RESUMEN
Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.
Asunto(s)
Alcaligenes , Amoníaco , Hidroxilaminas , Oxidorreductasas , Alcaligenes/enzimología , Amoníaco/metabolismo , Proteínas Bacterianas/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Hidroxilaminas/metabolismo , NAD/metabolismo , Nitrógeno/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , OxígenoRESUMEN
Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3âNH2OHâN2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.
Asunto(s)
Alcaligenes faecalis , Aerobiosis , Alcaligenes faecalis/genética , Alcaligenes faecalis/metabolismo , Amoníaco/metabolismo , Desnitrificación , Ecosistema , Nitrificación , Nitritos/metabolismo , Nitrógeno/metabolismoRESUMEN
Multicellular magnetotactic prokaryotes (MMPs) exhibit peculiar coordination of swimming along geomagnetic field lines. Approximately 40-80 cells assemble, with a helical geometry or axisymmetry, into spherical or ellipsoidal MMPs respectively. To contribute to a comprehensive understanding of bacterial multicellularity here we took multiple microscopic approaches to study the diversity, assembly, reproduction and motility of ellipsoidal MMPs. Using correlative fluorescence in situ hybridization and scanning electron microscopy analysis, we found an unexpected diversity in populations of ellipsoidal MMPs in the Mediterranean Sea. The high-pressure freezing/freeze substitution fixation technique allowed us to show, for the first time, that cells adhere via juxtaposed membranes and are held together by a rimming lattice. Fluorescence confocal microscopy and ultrathin section images revealed not only the one-layer hollow three-dimensional architecture, but also periphery-core unilateral constriction of constituent cells and unidirectional binary fission of the ellipsoidal MMPs. This finding suggests the evolution toward MMPs multicellularity via the mechanism of incomplete separation of offspring. Remarkably, thousands of flagellar at the periphery surface of cells underpin the coordinated swimming of MMPs in response to mechanical, chemical, magnetic and optical stimuli, including a magnetotactic photokinesis behaviour. Together these results unveil the unique structure and function property of ellipsoidal MMPs.
Asunto(s)
Fenómenos Magnéticos , Células Procariotas/fisiología , Adhesión Celular , División Celular , Membrana Celular , Hibridación Fluorescente in Situ , Microscopía Electrónica de Rastreo , Células Procariotas/ultraestructuraRESUMEN
The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.
Asunto(s)
Flagelos/metabolismo , Flagelina/metabolismo , Glicosiltransferasas/genética , Magnetospirillum/metabolismo , Proteínas Bacterianas , Campylobacter jejuni/enzimología , Flagelos/genética , Glicosilación , Magnetospirillum/enzimología , Magnetospirillum/genética , Ácidos Siálicos/química , Azúcares Ácidos/metabolismoRESUMEN
In E. coli and related species, flagellar brake protein YcgR responds to the elevated intracellular c-di-GMP, decreases the flagellar rotation speed, causes a CCW rotation bias, and regulates bacterial swimming. Boehm et al. suggested that c-di-GMP-activated YcgR directly interacted with the motor protein MotA to curb flagellar motor output. Paul et al. proposed that YcgR disrupted the organization of the FliG C-terminal domain to bias the flagellar rotation. The target proteins are controversial, and the role of motor proteins remains unclear in flagellar rotation speed and direction regulation by YcgR. Here we assayed the motor proteins' affinity via a modified FRET biosensor and accessed the role of those key residue via bead assays. We found that YcgR could interact with both MotA and FliG, and the affinities could be enhanced upon c-di-GMP binding. Furthermore, residue D54 of YcgR-N was needed for FliG binding. The mutation of the FliG binding residue D54 or the MotA binding ones, F117 and E232, restored flagellar rotation speed in wild-type cells and cells lacking chemotaxis response regulator CheY that switched the flagellar rotation direction and decreased the CCW ratio in wild-type cells. We propose that c-di-GMP-activated YcgR regulated the flagellar rotation speed and direction via its interaction with motor proteins MotA and FliG. Our work suggest the role of YcgR-motor proteins interaction in bacterial swimming regulation.
RESUMEN
Magnetotactic bacteria (MTB) are a group of Gram-negative bacteria characterized by synthesizing magnetosomes and swimming along geomagnetic field lines. Phylogenetically, they belong to different taxonomic lineages including Proteobacteria, Nitrospirae, Omnitrophica, Latescibacteria and Planctomycetes phyla on the phylogenetic tree. To date, six Nitrospirae MTB phylotypes have been identified from freshwater or low-salinity environments and described in the literature. Here, we report the identification of two Nitrospirae MTB phylotypes collected, for the first time, from the marine environment. Both have a spherical morphology with a cell size of ~ 5 µM and similar motility but are different colours (black-brown and ivory-white) under the optic microscope. They synthesized bullet-shaped iron-oxide magnetosomes that were arranged in multiple bundles of chains. Moreover, the cytoplasm of the black-brown Nitrospirae MTB contained sulphur inclusions that conferred on cells a rough, granular appearance. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that they are two novel species and cluster with the previously reported MTB affiliated with the phylum Nitrospirae, thus extending the distribution of Nitrospirae MTB from freshwater to the marine environment.