Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria.

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase.

Cyclic di-GMP inhibits nitrate assimilation by impairing the antitermination function of NasT in Pseudomonas putida.

The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.

Phyloecology of nitrate ammonifiers and their importance relative to denitrifiers in global terrestrial biomes.

Nitrate ammonification is important for soil nitrogen retention. However, the ecology of ammonifiers and their prevalence compared with denitrifiers, being competitors for nitrate, are overlooked. Here, we screen 1 million genomes for nrfA and onr, encoding ammonifier nitrite reductases. About 40% of ammonifier assemblies carry at least one denitrification gene and show higher potential for nitrous oxide production than consumption. We then use a phylogeny-based approach to recruit gene fragments of nrfA, onr and denitrification nitrite reductase genes (nirK, nirS) in 1861 global terrestrial metagenomes. nrfA outnumbers the nearly negligible onr counts in all biomes, but denitrification genes dominate, except in tundra. Random forest modelling teases apart the influence of the soil C/N on nrfA-ammonifier vs denitrifier abundance, showing an effect of nitrate rather than carbon content. This study demonstrates the multiple roles nitrate ammonifiers play in nitrogen cycling and identifies factors ultimately controlling the fate of soil nitrate.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

An unusual active site architecture in cytochrome c nitrite reductase NrfA-1 from Geobacter metallireducens.

Dissimilatory nitrate reduction to ammonia (DNRA) is a central pathway in the biogeochemical nitrogen cycle, allowing for the utilization of nitrate or nitrite as terminal electron acceptors. In contrast to the competing denitrification to N2, a major part of the essential nutrient nitrogen in DNRA is retained within the ecosystem and made available as ammonium to serve as a nitrogen source for other organisms. The second step of DNRA is mediated by the pentahaem cytochrome c nitrite reductase NrfA that catalyzes the six-electron reduction of nitrite to ammonium and is widely distributed among bacteria. A recent crystal structure of an NrfA ortholog from Geobacter lovleyi was the first characterized representative of a novel subclass of NrfA enzymes that lacked the canonical Ca2+ ion close to the active site haem 1. Here, we report the structural and functional characterization of NrfA from the closely related G. metallireducens. We established the recombinant production of catalytically active NrfA with its unique, lysine-coordinated active site haem heterologously in Escherichia coli and determined its three-dimensional structure by X-ray crystallography to 1.9 Å resolution. The structure confirmed GmNrfA as a further calcium-independent NrfA protein, and it also shows an altered active site that contained an unprecedented aspartate residue, D80, close to the substrate-binding site. This residue formed part of a loop that also caused a changed arrangement of the conserved substrate/product channel relative to other NrfA proteins and rendered the protein insensitive to the inhibitor sulphate. To elucidate the relevance of D80, we produced and studied the variants D80A and D80N that showed significantly reduced catalytic activity.

"Candidatus Subterrananammoxibiaceae," a New Anammox Bacterial Family in Globally Distributed Marine and Terrestrial Subsurfaces.

Bacteria specialized in anaerobic ammonium oxidation (anammox) are widespread in many anoxic habitats and form an important functional guild in the global nitrogen cycle by consuming bio-available nitrogen for energy rather than biomass production. Due to their slow growth rates, cultivation-independent approaches have been used to decipher their diversity across environments. However, their full diversity has not been well recognized. Here, we report a new family of putative anammox bacteria, "Candidatus Subterrananammoxibiaceae," existing in the globally distributed terrestrial and marine subsurface (groundwater and sediments of estuary, deep-sea, and hadal trenches). We recovered a high-quality metagenome-assembled genome of this family, tentatively named "Candidatus Subterrananammoxibius californiae," from a California groundwater site. The "Ca. Subterrananammoxibius californiae" genome not only contains genes for all essential components of anammox metabolism (e.g., hydrazine synthase, hydrazine oxidoreductase, nitrite reductase, and nitrite oxidoreductase) but also has the capacity for urea hydrolysis. In an Arctic ridge sediment core where redox zonation is well resolved, "Ca. Subterrananammoxibiaceae" is confined within the nitrate-ammonium transition zone where the anammox rate maximum occurs, providing environmental proof of the anammox activity of this new family. Phylogenetic analysis of nitrite oxidoreductase suggests that a horizontal transfer facilitated the spreading of the nitrite oxidation capacity between anammox bacteria (in the Planctomycetota phylum) and nitrite-oxidizing bacteria from Nitrospirota and Nitrospinota. By recognizing this new anammox family, we propose that all lineages within the "Ca. Brocadiales" order have anammox capacity. IMPORTANCE Microorganisms called anammox bacteria are efficient in removing bioavailable nitrogen from many natural and human-made environments. They exist in almost every anoxic habitat where both ammonium and nitrate/nitrite are present. However, only a few anammox bacteria have been cultured in laboratory settings, and their full phylogenetic diversity has not been recognized. Here, we present a new bacterial family whose members are present across both the terrestrial and marine subsurface. By reconstructing a high-quality genome from the groundwater environment, we demonstrate that this family has all critical enzymes of anammox metabolism and, notably, also urea utilization. This bacterium family in marine sediments is also preferably present in the niche where the anammox process occurs. These findings suggest that this novel family, named "Candidatus Subterrananammoxibiaceae," is an overlooked group of anammox bacteria, which should have impacts on nitrogen cycling in a range of environments.

Identification and examination of nitrogen metabolic genes in Lelliottia amnigena PTJIIT1005 for their ability to perform nitrate remediation.

Lelliottia amnigena PTJIIT1005 is a bacterium that utilizes nitrate as the sole nitrogen source and can remediate nitrate from media. The annotation was done related to nitrogen metabolic genes using the PATRIC, RAST tools, and PGAP from the genome sequence of this bacterium. Multiple sequence alignments and phylogenetic analysis of respiratory nitrate reductase, assimilatory nitrate reductase, nitrite reductase, glutamine synthetase, hydroxylamine reductase, nitric oxide reductase genes from PTJIIT1005 were done to find out sequence identities with the most similar species. The identification of operon arrangement in bacteria was also identified. The PATRIC KEGG feature mapped the N-metabolic pathway to identify the chemical process, and the 3D structure of representative enzymes was also elucidated. The putative protein 3D structure was analyzed using I-TASSER software. It gave good quality protein models of all nitrogen metabolism genes and showed good sequence identity with reference templates, approximately 81-99%, except for two genes; assimilatory nitrate reductase and nitrite reductase. This study suggested that PTJIIT1005 can remove N-nitrate from water because of having N-assimilation and denitrification genes.

Characteristics and comparisons of the aerobic and anaerobic denitrification of a Klebsiella oxytoca strain: Performance, electron transfer pathway, and mechanism.

The performance and electron (e-) transfer mechanisms of anaerobic and aerobic denitrification by strain Klebsiella were investigated in this study. The RT-PCR results demonstrated that the membrane bound nitrate reductase gene (narG) and Cu-nitrite reductase gene (nirK) were responsible for both aerobic and anerobic denitrification. The extreme low gene relative abundance of nirK might be responsible for the severe accumulation of NO2--N (nitrogen in the form of NO2- ion) under anaerobic condition. Moreover, the nitrite reductase (Nir) activity was 0.31 µg NO2--N min-1 mg-1 protein under anaerobic conditions, which was lower than that under aerobic conditions (0.38 µg NO2--N min-1 mg-1 protein). By using respiration chain inhibitors, the e- transfer pathways of anaerobic and aerobic denitrification of Klebsiella strain were constructed. Fe-S protein and Complex III were the core components under anaerobic conditions, while Coenzyme Q (CoQ), Complexes I and III played a key role in aerobic denitrification. Nitrogen assimilation was found to be the main way to generate NH4+-N (nitrogen in the form of NH4+ ion) during anaerobic denitrification, and also served as the primary nitrogen removal way under aerobic condition. The results of this study may help to improve the understanding of the core components of strain Klebsiella during aerobic and anaerobic denitrifications, and may suggest potential applications of the strain for nitrogen-containing wastewater.

A novel strategy to improving Rhodobacter azotoformans denitrification efficiency: Insight into the role of a two-component system NtrX/Y in denitrification regulation.

Transcription factors (TFs) can manage the coordinated expression of genes clusters or multiple genes. TF was used to improve bacterial denitrification ability in this study. During denitrification, the ntrY of R. azotoformans, which encodes the sensor of NtrX/Y system, was significantly upregulated in transcription. Denitrification of the mutant △ntrY was significantly inhibited, and it was recovered after replenishing this gene to the mutant, which indicates the NtrX/Y system plays an important role in regulating bacterial denitrification. According to additional research, the NtrX/Y system regulates bacterial denitrification by directly promoting the expression of the nitrite reductase. ntrY overexpression appears to accelerate bacterial denitrification, and the introduction of a strong promoter tac in conjunction with iron supply optimization increases the rate by 72% further. This study realizes bacterial denitrification enhancement from the perspective of global transcription regulation, which provides a novel strategy for improving microbial ability to degrade pollutants.

Endothelial alpha globin is a nitrite reductase.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

Preparation for Denitrification and Phenotypic Diversification at the Cusp of Anoxia: a Purpose for N2O Reductase Vis-à-Vis Multiple Roles of O2.

Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O2 repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed "bet-hedging," where all cells synthesize the N2O reductase (NosZ) but only a minority synthesize nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia but with the possibility of escape if supplied with O2 or N2O. To test this, cells were exposed to gradual O2 depletion or sudden anoxia and subsequent spikes of O2 and N2O. The synthesis of NirS in single cells was monitored by using an mCherry-nirS fusion replacing the native nirS, and their growth was detected as dilution of green, fluorescent fluorescein isothiocyanate (FITC) stain. We demonstrate anoxic entrapment due to e--acceptor deprivation and show that O2 spiking leads to bet-hedging, while N2O spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N2O spike had much lower respiration rates than those rescued by the O2 spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O2. Our results bring into relief a fitness advantage of pairing restrictive nirS expression with universal NosZ synthesis in energy-limited systems. IMPORTANCE Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their N2O emissions because all cells express NosZ, reducing N2O to N2, while a minority express NirS + Nor, reducing NO2- to N2O. We hypothesized that the cells without Nir were entrapped in anoxia, without energy to synthesize Nir, and that they could be rescued by short spikes of O2 or N2O. We confirm such entrapment and the rescue of all cells by an N2O spike but only a fraction by an O2 spike. The results shed light on the role of O2 repression in bet-hedging and generated a novel hypothesis regarding the autocatalytic nirS expression via NO production. Insight into the regulation of denitrification, including bet-hedging, holds a clue to understanding, and ultimately curbing, the escalating emissions of N2O, which contribute to anthropogenic climate forcing.

Comparative Genome Analysis of a Novel Alkaliphilic Actinobacterial Species Nesterenkonia haasae.

In the present study, a comparative genome analysis of the novel alkaliphilic actinobacterial Nesterenkonia haasae with other members of the genus Nesterenkonia was performed. The genome size of Nesterenkonia members ranged from 2,188,008 to 3,676,111 bp. N. haasae and Nesterenkonia members of the present study encode the essential glycolysis and pentose phosphate pathway genes. In addition, some Nesterenkonia members encode the crucial genes for Entner-Doudoroff pathways. Some Nesterenkonia members possess the genes responsible for sulfate/thiosulfate transport system permease protein/ ATP-binding protein and conversion of sulfate to sulfite. Nesterenkonia members also encode the genes for assimilatory nitrate reduction, nitrite reductase, and the urea cycle. All Nesterenkonia members have the genes to overcome environmental stress and produce secondary metabolites. The present study helps to understand N. haasae and Nesterenkonia members' environmental adaptation and niches specificity based on their specific metabolic properties. Further, based on genome analysis, we propose reclassifying Nesterenkonia jeotgali as a later heterotypic synonym of Nesterenkonia sandarakina.

Myoglobin mutant with enhanced nitrite reductase activity regulates intracellular oxidative stress in human breast cancer cells.

Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.

Nitrite is reduced by nitrite reductase NirB without small subunit NirD in Escherichia coli.

The assimilatory nitrite reductase enzyme NirB and small subunit NirD genes encoded in nir operon in Escherichia coli were cloned into the pET28a vector, and the recombinant enzyme was characterized for the first time. Docking of NirB with NirD, NADH, NO2-, NO3-, and CHO2- was performed using docking modeling programs. Methyl viologen and sodium dithionite were used as electron couples, and the amount of reduced nitrite was measured to calculate enzyme activity. NirB is the main enzyme and shows high activity with or without NirD. However, the inclusion of NirD into the enzyme solution at a ratio of 1NirD:2NirB resulted in 10% higher nitrite reductase activity. The enzyme tends to aggregate in the absence of ß-mercaptoethanol, which causes the conversion of tetrameric NirB to monomeric form, and the NirB enzyme shows its highest activity in monomeric form. The optimum temperature for enzyme activity was 37 °C and the optimum pH was found to be 7.0. Km and Vmax values of NirB were calculated as 9833 µM and 416.67 µmol NO2- reduced min-1 mg-1. Enzyme activity decreased by 55% and 50% in the presence of 100 mM nitrate and formate, respectively. The presence of 25 mM Cd2+ protected the enzyme at room temperature and the enzyme showed 10% higher activity in the presence of cadmium.

Homology modeling and virtual characterization of cytochrome c nitrite reductase (NrfA) in three model bacteria responsible for short-circuit pathway, DNRA in the terrestrial nitrogen cycle.

NrfA is the molecular marker for dissimilatory nitrate reduction to ammonium (DNRA) activity, catalysing cytochrome c nitrite reductase enzyme. However, the limited study has been made so far to understand the structural homology modeling of NrfA protein in DNRA bacteria. Therefore, three model DNRA bacteria (Escherechia coli, Wolinella succinogenes and Shewanella oneidensis) were chosen in this study for in-silico protein modeling of NrfA which roughly consists of similar length of amino acids and molecular weight and they belong to two contrasting taxonomic families (γ-proteobacteria with nrfABCDEFG and ε-proteobacteria with nrfHAIJ operon). Multiple bioinformatic tools were used to examine the primary, secondary, and tertiary structure of NrfA protein using three distinct homology modeling pipelines viz., Phyre2, Swiss model and Modeller. The results indicated that NrfA protein in E. coli, W. succinogenes and S. oneidensis was mostly periplasmic and hydrophilic. Four conserved Cys-X1-X2-Cys-His motifs, one Cys-X1-X2-Cys-Lys haem-binding motif and Ca ligand were also identified in NrfA protein irrespective of three model bacteria. Moreover, 11 identical conserved amino acids sequence was observed for the first time between serine and proline in NrfA protein. Secondary structure of NrfA revealed that α-helices were observed in 77.9%, 73.4%, and 77.4% in E. coli, W. succinogenes and S. oneidensis, respectively. Ramachandran plot showed that number of residue in favored region in E. coli, W. succinogenes and S. oneidensis was 97.03%, 97.01% and 97.25%, respectively. Our findings also revealed that among three pipelines, Modeller was considered the best in-silico tool for prediction of NrfA protein. Overall, significant findings of this study may aid in the identification of future unexplored DNRA bacteria containing cytochrome c nitrite reductase. The NrfA system, which is linked to respiratory nitrite ammonification, provides an analogous target for monitoring less studied N-retention processes, particularly in agricultural ecosystems. Furthermore, one of the challenging research tasks for the future is to determine how the NrfA protein responds to redox status in the microbial cells.

A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism.

Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations. IMPORTANCE To understand bacteria in diverse contexts, we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes, including a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

Effect of Copper on Expression of Functional Genes and Proteins Associated with Bradyrhizobium diazoefficiens Denitrification.

Nitrous oxide (N2O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils.

Novel nitrite reductase domain structure suggests a chimeric denitrification repertoire in the phylum Chloroflexi.

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported NOR subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.

Distinct Interaction Mechanism of RNA Polymerase and ResD at Proximal and Distal Subsites for Transcription Activation of Nitrite Reductase in Bacillus subtilis.

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis. The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at -87 to -84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended -10 promoter, it lacks a reasonable -35 element. Genetic analysis and structural simulations predicted that the absence of the -35 element might be compensated by interactions between σA and αCTD and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified, and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors, including the ubiquitous response regulators of two-component regulatory systems, particularly in Gram-positive bacteria.