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Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms., Competing Interests: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C 2 -C 8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment., (© 2023 The Authors. Environmental Microbiology Reports published by Applied Microbiology International and John Wiley & Sons Ltd.)

Importance: Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans , for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail., Competing Interests: The authors declare no conflict of interest.

Wetland soils are the greatest source of nitrous oxide (N 2 O), a critical greenhouse gas and ozone depleter released by microbes. Yet, microbial players and processes underlying the N 2 O emissions from wetland soils are poorly understood. Using in situ N 2 O measurements and by determining the structure and potential functional of microbial communities in 645 wetland soil samples globally, we examined the potential role of archaea, bacteria, and fungi in nitrogen (N) cycling and N 2 O emissions. We show that N 2 O emissions are higher in drained and warm wetland soils, and are correlated with functional diversity of microbes. We further provide evidence that despite their much lower abundance compared to bacteria, nitrifying archaeal abundance is a key factor explaining N 2 O emissions from wetland soils globally. Our data suggest that ongoing global warming and intensifying environmental change may boost archaeal nitrifiers, collectively transforming wetland soils to a greater source of N 2 O., (© 2022. The Author(s).)

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that-like for AOB-ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox., (© 2021. The Author(s).)

Background: Marine Group I (MGI) Thaumarchaeota, which play key roles in the global biogeochemical cycling of nitrogen and carbon (ammonia oxidizers), thrive in the aphotic deep sea with massive populations. Recent studies have revealed that MGI Thaumarchaeota were present in the deepest part of oceans-the hadal zone (depth > 6000 m, consisting almost entirely of trenches), with the predominant phylotype being distinct from that in the "shallower" deep sea. However, little is known about the metabolism and distribution of these ammonia oxidizers in the hadal water., Results: In this study, metagenomic data were obtained from 0-10,500 m deep seawater samples from the Mariana Trench. The distribution patterns of Thaumarchaeota derived from metagenomics and 16S rRNA gene sequencing were in line with that reported in previous studies: abundance of Thaumarchaeota peaked in bathypelagic zone (depth 1000-4000 m) and the predominant clade shifted in the hadal zone. Several metagenome-assembled thaumarchaeotal genomes were recovered, including a near-complete one representing the dominant hadal phylotype of MGI. Using comparative genomics, we predict that unexpected genes involved in bioenergetics, including two distinct ATP synthase genes (predicted to be coupled with H + and Na + respectively), and genes horizontally transferred from other extremophiles, such as those encoding putative di-myo-inositol-phosphate (DIP) synthases, might significantly contribute to the success of this hadal clade under the extreme condition. We also found that hadal MGI have the genetic potential to import a far higher range of organic compounds than their shallower water counterparts. Despite this trait, hadal MDI ammonia oxidation and carbon fixation genes are highly transcribed providing evidence they are likely autotrophic, contributing to the primary production in the aphotic deep sea., Conclusions: Our study reveals potentially novel adaptation mechanisms of deep-sea thaumarchaeotal clades and suggests key functions of deep-sea Thaumarchaeota in carbon and nitrogen cycling. Video Abstract.

Mechanosensitive channels are ubiquitous and highly studied. However, the evolution of the bacterial channels remains enigmatic. It can be argued that mechanosensitivity might be a feature of all membrane proteins with some becoming progressively less sensitive to membrane tension over the course of evolution. Bacteria and archaea exhibit two main classes of channels, MscS and MscL. Present day channels suggest that the evolution of MscL may be highly constrained, whereas MscS has undergone elaboration via gene fusion (and potentially gene fission) events to generate a diversity of channel structures. Some of these channel variants are constrained to a small number of genera or species. Some are only found in higher organisms. Only exceptionally have these diverse channels been investigated in any detail. In this review we consider both the processes that might have led to the evolved complexity but also some of the methods exploiting the explosion of genome sequences to understand (and/or track) their distribution. The role of MscS-related channels in calcium-mediated cell biology events is considered., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)