Neeraja Vajrala

Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea

The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH3) oxidation to nitrite (NO2−) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH3 oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH2OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH2OH or an initial oxidation product other than NH2OH. We now show through combined physiological and stable isotope tracer analyses that NH2OH is both produced and consumed during the oxidation of NH3 to NO2− by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH2OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH3 by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH2OH, the archaea possibly catalyzing NH2OH oxidation using a novel enzyme complex.

Use of Aliphatic n-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria

ABSTRACT Ammonia (NH3)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH3 oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C8 and C9. In contrast, NH3 oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C8 (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH4 +-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH4 +-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH4 +-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.

Role of a Fur homolog in iron metabolism in Nitrosomonas europaea.

BackgroundIn response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family.ResultsOf the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 μM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.ConclusionsOur studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.

Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe3+-ABC transporter permease components from other organisms. The N.europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N.europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe3+-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe3+ bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe3+ or Fe2+ forms or Fe3+ associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.

Cycloheximide prevents the de novo polypeptide synthesis required to recover from acetylene inhibition in Nitrosopumilus maritimus

Developing methods to differentiate the relative contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to ammonia (NH3) oxidation has been challenging due to the lack of compounds that selectively inhibit AOA. In this study, we investigated the effects of specific bacteria- and eukaryote-selective protein synthesis inhibitors on the recovery of acetylene (C2H2)-inactivated NH3 oxidation in the marine AOA Nitrosopumilus maritimus and compared the results with recovery of the AOB Nitrosomonas europaea. C2 H2 irreversibly inhibited N. maritimus NH3 oxidation in a similar manner to what was observed previously with N. europaea. However, cycloheximide (CHX), a widely used eukaryotic protein synthesis inhibitor, but not bacteria-specific protein synthesis inhibitors (kanamycin and gentamycin), inhibited the recovery of NH3-oxidizing activity in N. maritimus. CHX prevented the incorporation of (14)CO2 -labeling into cellular proteins, providing further evidence that CHX acts as a protein synthesis inhibitor in N. maritimus. If the effect of CHX on protein synthesis can be confirmed among other isolates of AOA, the combination of C2H2 inactivation followed by recovery of NH3 oxidation either in the presence of bacteria-selective protein synthesis inhibitors or CHX might be used to estimate the relative contributions of AOB and AOA to NH3 oxidation in natural environments.

Global analysis of the Nitrosomonas europaea iron starvation stimulon.

The importance of iron to the metabolism of the ammonia-oxidizing bacterium Nitrosomonas europaea is well known. However, the mechanisms by which N. europaea acquires iron under iron limitation are less well known. To obtain insight into these mechanisms, transcriptional profiling of N. europaea was performed during growth under different iron availabilities. Of 2,355 N. europaea genes on DNA microarrays, transcripts for 247 genes were identified as differentially expressed when cells were grown under iron limitation compared to cells grown under iron-replete conditions. Genes with higher transcript levels in response to iron limitation included those with confirmed or assigned roles in iron acquisition. Genes with lower transcript levels included those encoding iron-containing proteins. Our analysis identified several potentially novel iron acquisition systems in N. europaea and provided support for the primary involvement of a TonB-dependent heme receptor gene in N. europaea iron homeostasis. We demonstrated that hemoglobin can act as an iron source under iron-depleted conditions for N. europaea. In addition, we identified a hypothetical protein carrying a lipocalin-like domain that may have the ability to chelate iron for growth in iron-limited media.

Dissecting iron uptake and homeostasis in Nitrosomonas europaea.

The chemolithoautotroph Nitrosomonas europaea oxidizes about 25 mol of NH(3) for each mole of CO(2) that is converted to biomass using an array of heme and nonheme Fe-containing proteins. Hence mechanisms of efficient iron (Fe) uptake and homeostasis are particularly important for this Betaproteobacterium. Among nitrifiers, N.europaea has been the most studied to date. Characteristics that make N.europaea a suitable model to study Fe uptake and homeostasis are as follows: (a) its sequenced genome, (b) its capability to grow relatively well in 0.2 μM Fe in the absence of heterologous siderophores, and (c) its amenability to mutagenesis. In this chapter, we describe the methodology we use in our laboratory to dissect Fe uptake and homeostasis in the ammonia oxidizer N. europaea.