Denitrification by bradyrhizobia under feast and famine and the role of the bc1 complex in securing electrons for N2O reduction.

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Metatranscriptomic Analyses of Plankton Communities Inhabiting Surface and Subpycnocline Waters of the Chesapeake Bay during Oxic-Anoxic-Oxic Transitions

We used metatranscriptomics to study the gene transcription patterns of microbial plankton (0.2 to 64 μm) at a mesohaline station in the Chesapeake Bay under transitions from oxic to anoxic waters in spring and from anoxic to oxic waters in autumn. Samples were collected from surface (i.e., above pycnocline) waters (3 m) and from waters beneath the pycnocline (16 to 22 m) in both 2010 and 2011. Metatranscriptome profiles based on function and potential phylogeny were different between 2010 and 2011 and strongly variable in 2011. This difference in variability corresponded with a highly variable ratio of eukaryotic to bacterial sequences (0.3 to 5.5), reflecting transient algal blooms in 2011 that were absent in 2010. The similarity between metatranscriptomes changed at a lower rate during the transition from oxic to anoxic waters than after the return to oxic conditions. Transcripts related to photosynthesis and low-affinity cytochrome oxidases were significantly higher in shallow than in deep waters, while in deep water genes involved in anaerobic metabolism, particularly sulfate reduction, succinyl coenzyme A (succinyl-CoA)-to-propionyl-CoA conversion, and menaquinone synthesis, were enriched relative to in shallow waters. Expected transitions in metabolism between oxic and anoxic deep waters were reflected in elevated levels of anaerobic respiratory reductases and utilization of propenediol and acetoin. The percentage of archaeal transcripts increased in both years in late summer (from 0.1 to 4.4% of all transcripts in 2010 and from 0.1 to 6.2% in 2011). Denitrification-related genes were expressed in a predicted pattern during the oxic-anoxic transition. Overall, our data suggest that Chesapeake Bay microbial assemblages express gene suites differently in shallow and deep waters and that differences in deep waters reflect variable redox states

Metagenomic Evidence for a Methylocystis Species Capable of Bioremediation of Diverse Heavy Metals

Heavy metal pollution has become an increasingly serious problem worldwide. Co-contamination with toxic mercury (Hg) and arsenic (As) presents a particularly difficult bioremediation trouble. By oxidizing the greenhouse gas methane, methanotrophs have been demonstrated to have high denitrification activity in eutrophic waters, indicating their possible potential for use in bioremediation of Hg(II) and As(V) in polluted water. Using metagenomics, a novel Methylocystis species (HL18), which was one of the most prevalent bacteria (9.9% of the relative abundance) in a CH4-based bio-reactor, is described here. The metagenomic-assembled genome (MAG) HL18 had gene products whose average amino acid identity against other known Methylocystis species varied from 69 to 85%, higher than the genus threshold but lower than the species boundary. Genomic analysis indicated that HL18 possessed all the genes necessary for the reduction of Hg(II) and As(V). Phylogenetic investigation of mercuric reductase (MerA) found that the HL18 protein was most closely affiliated with proteins from two Hg(II)-reducing bacteria, Bradyrhizobium sp. strain CCH5-F6 and Paracoccus halophilus. The genomic organization and phylogeny of the genes in the As(V)-reducing operon (arsRCCB) had significant identity with those from a As(V)-reducing bacterium belonging to the Rhodopseudomonas genus, indicating their reduction capability of As(V). Further analysis found that at least eight genera of methanotrophs possess both Hg(II) and As(V) reductases, illustrating the generally overlooked metabolic potential of methanotrophs. These results suggest that methanotrophs have greater bioremediation potential in heavy metal contaminated water than has been appreciated and could play an important role in the mitigation of heavy metal toxicity of contaminated wastewater

Final technical report for award NO. DE-FG02-95ER20206

ABSTRACT Initial work focused on the regulation of nitrite reductase, the defining reaction of denitrification as well as nitric oxide (NO) reductase. Expression of the genes encoding both proteins was controlled by NnrR. This regulator was shown to be responsive to NO. More recent work has shown NnrR function is also likely inhibited by oxygen. Therefore, it is this protein that sets the oxygen level at which nitrate respiration takes over from aerobic respiration. The gene encoding NO reductase appears to only require NnrR for expression. Expression of the gene encoding nitrite reductase is more complex. In addition to NnrR, a two component sensor regulator complex termed PrrA and PrrB is also required for expression. These proteins are global regulators and serve to link denitrification with other bioenergetic processes in the cell. They also provide an additional layer of oxygen dependent regulation. The sequencing of the R. sphaeroides 2.4.3 genome allowed us to identify several other genes regulated by NnrR. Surprisingly, most of the genes were not essential for denitrification. Their high level of conservation in related denitrifiers suggests they do provide a selectable benefit to the bacterium, however. We also examined the role of nitrate reductase in contributing to denitrification in R. sphaeroides. Strain 2.4.3 is unusual in having two distinct, but related clusters of genes encoding nitrate reductase. One of these genes clusters is expressed under high oxygen conditions but is repressed, likely by PrrB-PrrA, under low oxygen conditions. The other cluster is expressed only under low oxygen conditions. This cluster expresses the nitrate reductase used during denitrification. The high oxygen expressed cluster encodes a protein used for redox homeostasis. Surprisingly, both clusters are fully expressed even in the absence of nitrate. During the course of this work we found that the type strain of R. sphaeroides, 2.4.1, is a partial denitrifier because it has the nitrate and NO reductases but lacks nitrite reductase. Like 2.4.3 it uses NnrR to regulate NO reductase. This unexpected arrangement suggested that it may use NO reductase to detoxify NO produced in its environment. Using a green fluorescent protein based reporter system we were able to demonstrate that NO produced by a denitrifier such as 2.4.3 can induce expression of NO reductase in 2.4.1. We then went on to show that the NO produced by denitrifiers can induce a stress response in other non-denitrifying bacteria. This suggests that the NO produced during denitrification will have a significant impact on the non-denitrifiers present in the surrounding environment. We also expanded our studies to include the denitrifier Agrobacterium tumefaciens. We demonstrated that the expression of the nitrite and NO reductase genes in this bacterium follows the same general scheme as in R. sphaeroides. We also were able to show that this bacterium would induce NO reductase in response to the NO produced by plants. Importantly, we were able to demonstrate that A. tumefaciens had difficulty transitioning from aerobic respiration to denitrification if the transition was sudden. This difficulty manifested as an accumulation of NO. In some conditions cells were slowly able to switch modes of respiration but in other cases NO accumulations seemed to kill the cells. The difficulty in transition appears to be due to an inability to produce enough energy once the oxygen has been completely consumed

Data used in analyses associated with: "Salinity-aided selection of progressive onset denitrifiers as a means of providing nitrite for anammox"

This is the protein sequence dataset used for determining reads derived from denitrification genes in Illumina read samples. The protein types in the dataset are the active site containing subunits from the following proteins: Nap – periplasmic nitrate reductase; Nar – membrane associated nitrate reductase; NirK – copper containing nitrite reductase; NirS- heme-containing nitrite reductase; cNor – cytochrome c oxidizing nitrite reductase; qNor – quinol oxidizing nitric oxide reductase and Nos – nitrous oxide reductase. Sequences were initially obtained from the data available from the Integrated Microbial Genomes (IMG) system (https://img.jgi.doe.gov/cgi-bin/m/main.cgi). Related proteins were then aligned and proteins predicted to not be related or that were significantly shorter in length than group average were removed

Identification, Functional Studies, and Genomic Comparisons of New Members of the NnrR Regulon in Rhodobacter sphaeroides▿

Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain. nnrV, under control of a previously identified NnrR site, was also identified. paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase. paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome c2 together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions

Expression of Nitrite and Nitric Oxide Reductases in Free-Living and Plant-Associated Agrobacterium tumefaciens C58 Cells

A number of the bacteria that form associations with plants are denitrifiers. To learn more about how the association with plants affects expression of denitrification genes, the regulation of nitrite and nitric oxide reductases was investigated in Agrobacterium tumefaciens. Analysis of free-living cells revealed that expression of the genes encoding nitrite and nitric oxide reductases, nirK and nor, respectively, requires low-oxygen conditions, nitric oxide, and the transcriptional regulator NnrR. Expression of nor was monitored in plant-associated bacteria using nor-gfp fusion expression. In root association experiments, only a small percentage of the attached cells were fluorescent, even when they were incubated under a nitrogen atmosphere. Inactivation of nirK had no significant effect on the ability of A. tumefaciens to bind to plant roots regardless of the oxygen tension, but it did decrease the occurrence of root-associated fluorescent cells. When wild-type cells containing the gfp fusion were infiltrated into leaves, most cells eventually became fluorescent. The same result was obtained when a nirK mutant was used, suggesting that nitric oxide activated nor expression in the endophytic bacteria. Addition of a nitric oxide synthase inhibitor to block nitric oxide generation by the plant prevented gfp expression in infiltrated nitrite reductase mutants, demonstrating that plant-derived nitric oxide can activate nor expression in infiltrated cells

Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers

To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide

Agrobacterium tumefaciens C58 Uses ActR and FnrN To Control nirK and nor Expression▿

Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression