Sinorhizobium melilotiGlutathione Reductase Is Required for both Redox Homeostasis and Symbiosis

ABSTRACTGlutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants inSinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways inS. meliloti. The first pathway is thede novosynthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether theS. melilotiGR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of theS. melilotigorgene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. Thegormutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2and sodium nitroprusside. Interestingly, the expression ofgshA,gshB1,katA, andkatBwas induced in the mutant. These findings reveal that the recycling of glutathione is important forS. melilotito maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCEThe antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacteriumS. meliloti, thede novosynthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection inS. meliloticells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

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Genome Analysis Suggests that the Soil Oligotrophic Bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) Is a Nitrogen-Fixing Symbiont of Aeschynomene indica

Applied and Environmental Microbiology ◽

10.1128/aem.00009-13 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2542-2551 ◽

Cited By ~ 32

Author(s):

Takashi Okubo ◽

Shohei Fukushima ◽

Manabu Itakura ◽

Kenshiro Oshima ◽

Aphakorn Longtonglang ◽

...

Keyword(s):

Nitrogen Fixation ◽

Phylogenetic Analyses ◽

Gc Content ◽

Gene Clusters ◽

Nodule Development ◽

Nitrogen Fixing ◽

Circular Chromosome ◽

Nod Factor ◽

Content Type ◽

Oligotrophic Bacterium

ABSTRACTAgromonas oligotrophica(Bradyrhizobium oligotrophicum) S58Tis a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments. Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165 bp with an average GC content of 65.1% lackingnodABCgenes and the typical symbiosis island. The genome showed a high level of similarity to the genomes ofBradyrhizobiumsp. ORS278 andBradyrhizobiumsp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant,Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nifandfix) withA. indicawere well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with fiveA. oligotrophicastrains (S58, S42, S55, S72, and S80). Surprisingly, all five strains ofA. oligotrophicaformed effective nitrogen-fixing nodules on the roots and/or stems ofA. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwellingA. oligotrophicagenerally possesses the ability to establish symbiosis withA. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis withA. indicais a common trait ofnodABC- and symbiosis island-lacking strains within the members of the photosyntheticBradyrhizobiumclade, includingA. oligotrophica.

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Complete Genome Sequence of Sinorhizobium Phage ΦM6, the First Terrestrial Phage of a Marine Phage Group

Microbiology Resource Announcements ◽

10.1128/mra.01143-18 ◽

2018 ◽

Vol 7 (13) ◽

Cited By ~ 2

Author(s):

Tess E. Brewer ◽

Brian K. Washburn ◽

Jason S. Lynn ◽

Kathryn M. Jones

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Sinorhizobium Meliloti ◽

Nitrogen Fixing ◽

Phage Group ◽

Content Type ◽

Marine Phage

Sinorhizobium phage ΦM6 infects the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. ΦM6 most closely resembles marine phages, such as Puniceispirillum phage HMO-2011, rather than previously sequenced rhizobial phages.

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Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

Applied and Environmental Microbiology ◽

10.1128/aem.01561-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 10

Author(s):

Michael J. Mitsch ◽

George C. diCenzo ◽

Alison Cowie ◽

Turlough M. Finan

Keyword(s):

Nitrogen Fixation ◽

Carbon Source ◽

Sinorhizobium Meliloti ◽

Rhizobium Leguminosarum ◽

Symbiotic Nitrogen Fixation ◽

Sequencing Analysis ◽

Wild Type ◽

Content Type ◽

Transcriptional Changes ◽

Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

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Nutrient-ColimitedTrichodesmiumas a Nitrogen Source or Sink in a Future Ocean

Applied and Environmental Microbiology ◽

10.1128/aem.02137-17 ◽

2017 ◽

Vol 84 (3) ◽

Cited By ~ 10

Author(s):

Nathan G. Walworth ◽

Fei-Xue Fu ◽

Michael D. Lee ◽

Xiaoni Cai ◽

Mak A. Saito ◽

...

Keyword(s):

Nitrogen Fixation ◽

Nitrogen Source ◽

Nitrogen Metabolism ◽

Organic Nitrogen ◽

Open Ocean ◽

Nitrogen Fixing ◽

Content Type ◽

Nitrogen Inputs ◽

Ocean Conditions ◽

Nitrogen Scavenging

ABSTRACTNitrogen-fixing (N2) cyanobacteria provide bioavailable nitrogen to vast ocean regions but are in turn limited by iron (Fe) and/or phosphorus (P), which may force them to employ alternative nitrogen acquisition strategies. The adaptive responses of nitrogen fixers to global-change drivers under nutrient-limited conditions could profoundly alter the current ocean nitrogen and carbon cycles. Here, we show that the globally important N2fixerTrichodesmiumfundamentally shifts nitrogen metabolism toward organic-nitrogen scavenging following long-term high-CO2adaptation under iron and/or phosphorus (co)limitation. Global shifts in transcripts and proteins under high-CO2/Fe-limited and/or P-limited conditions include decreases in the N2-fixing nitrogenase enzyme, coupled with major increases in enzymes that oxidize trimethylamine (TMA). TMA is an abundant, biogeochemically important organic nitrogen compound that supports rapidTrichodesmiumgrowth while inhibiting N2fixation. In a future high-CO2ocean, this whole-cell energetic reallocation toward organic nitrogen scavenging and away from N2fixation may reduce new-nitrogen inputs byTrichodesmiumwhile simultaneously depleting the scarce fixed-nitrogen supplies of nitrogen-limited open-ocean ecosystems.IMPORTANCETrichodesmiumis among the most biogeochemically significant microorganisms in the ocean, since it supplies up to 50% of the new nitrogen supporting open-ocean food webs. We usedTrichodesmiumcultures adapted to high-CO2conditions for 7 years, followed by additional exposure to iron and/or phosphorus (co)limitation. We show that “future ocean” conditions of high CO2and concurrent nutrient limitation(s) fundamentally shift nitrogen metabolism away from nitrogen fixation and instead toward upregulation of organic nitrogen-scavenging pathways. We show that the responses ofTrichodesmiumto projected future ocean conditions include decreases in the nitrogen-fixing nitrogenase enzymes coupled with major increases in enzymes that oxidize the abundant organic nitrogen source trimethylamine (TMA). Such a shift toward organic nitrogen uptake and away from nitrogen fixation may substantially reduce new-nitrogen inputs byTrichodesmiumto the rest of the microbial community in the future high-CO2ocean, with potential global implications for ocean carbon and nitrogen cycling.

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Sinorhizobium meliloti Phage ΦM9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

Journal of Virology ◽

10.1128/jvi.01353-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 10945-10958 ◽

Cited By ~ 11

Author(s):

Matthew C. Johnson ◽

Kelsey B. Tatum ◽

Jason S. Lynn ◽

Tess E. Brewer ◽

Stephen Lu ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Open Reading Frames ◽

Host Cells ◽

Close Relative ◽

Tail Fiber ◽

Nitrogen Fixing ◽

Related Factors ◽

Content Type ◽

Fiber Assembly ◽

Host Infection

ABSTRACTRelatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of theSinorhizobium meliloti-infecting T4 superfamily phage ΦM9. This phage and its close relativeRhizobiumphage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-likeS. melilotiphages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 andS. melilotiphage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide.IMPORTANCEDespite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study ofS. meliloti-infecting T4 superfamily phage ΦM9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ΦM9 andS. meliloti-infecting T4 superfamily phage ΦM12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors.

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Transcriptional Activities of the Microbial Consortium Living with the Marine Nitrogen-Fixing Cyanobacterium Trichodesmium Reveal Potential Roles in Community-Level Nitrogen Cycling

Applied and Environmental Microbiology ◽

10.1128/aem.02026-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 6

Author(s):

Michael D. Lee ◽

Eric A. Webb ◽

Nathan G. Walworth ◽

Fei-Xue Fu ◽

Noelle A. Held ◽

...

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Microbial Communities ◽

Nitrogen Cycling ◽

Carbon Fixation ◽

Community Respiration ◽

Ecological Niches ◽

Nitrogen Fixing ◽

Content Type ◽

Globally Distributed

ABSTRACTTrichodesmiumis a globally distributed cyanobacterium whose nitrogen-fixing capability fuels primary production in warm oligotrophic oceans. Like many photoautotrophs,Trichodesmiumserves as a host to various other microorganisms, yet little is known about how this associated community modulates fluxes of environmentally relevant chemical species into and out of the supraorganismal structure. Here, we utilized metatranscriptomics to examine gene expression activities of microbial communities associated withTrichodesmium erythraeum(strain IMS101) using laboratory-maintained enrichment cultures that have previously been shown to harbor microbial communities similar to those of natural populations. In enrichments maintained under two distinct CO2concentrations for ∼8 years, the community transcriptional profiles were found to be specific to the treatment, demonstrating a restructuring of overall gene expression had occurred. Some of this restructuring involved significant increases in community respiration-related transcripts under elevated CO2, potentially facilitating the corresponding measured increases in host nitrogen fixation rates. Particularly of note, in both treatments, community transcripts involved in the reduction of nitrate, nitrite, and nitrous oxide were detected, suggesting the associated organisms may play a role in colony-level nitrogen cycling. Lastly, a taxon-specific analysis revealed distinct ecological niches of consistently cooccurring major taxa that may enable, or even encourage, the stable cohabitation of a diverse community withinTrichodesmiumconsortia.IMPORTANCETrichodesmiumis a genus of globally distributed, nitrogen-fixing marine cyanobacteria. As a source of new nitrogen in otherwise nitrogen-deficient systems, these organisms help fuel carbon fixation carried out by other more abundant photoautotrophs and thereby have significant roles in global nitrogen and carbon cycling. Members of theTrichodesmiumgenus tend to form large macroscopic colonies that appear to perpetually host an association of diverse interacting microbes distinct from the surrounding seawater, potentially making the entire assemblage a unique miniature ecosystem. Since its first successful cultivation in the early 1990s, there have been questions about the potential interdependencies betweenTrichodesmiumand its associated microbial community and whether the host's seemingly enigmatic nitrogen fixation schema somehow involved or benefited from its epibionts. Here, we revisit these old questions with new technology and investigate gene expression activities of microbial communities living in association withTrichodesmium.

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Differing Courses of Genetic Evolution of Bradyrhizobium Inoculants as Revealed by Long-Term Molecular Tracing in Acacia mangium Plantations

Applied and Environmental Microbiology ◽

10.1128/aem.02007-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5709-5716 ◽

Cited By ~ 2

Author(s):

M. M. Perrineau ◽

C. Le Roux ◽

A. Galiana ◽

A. Faye ◽

R. Duponnois ◽

...

Keyword(s):

Nitrogen Fixation ◽

Growth Promotion ◽

Acacia Mangium ◽

Housekeeping Genes ◽

Nitrogen Fixing ◽

Microbial Inoculants ◽

Nitrogen Fixing Bacteria ◽

Content Type ◽

Significant Difference

ABSTRACTIntroducing nitrogen-fixing bacteria as an inoculum in association with legume crops is a common practice in agriculture. However, the question of the evolution of these introduced microorganisms remains crucial, both in terms of microbial ecology and agronomy. We explored this question by analyzing the genetic and symbiotic evolution of twoBradyrhizobiumstrains inoculated onAcacia mangiumin Malaysia and Senegal 15 and 5 years, respectively, after their introduction. Based on typing of several loci, we showed that these two strains, although closely related and originally sampled in Australia, evolved differently. One strain was recovered in soil with the same five loci as the original isolate, whereas the symbiotic cluster of the other strain was detected with no trace of the three housekeeping genes of the original inoculum. Moreover, the nitrogen fixation efficiency was variable among these isolates (either recombinant or not), with significantly high, low, or similar efficiencies compared to the two original strains and no significant difference between recombinant and nonrecombinant isolates. These data suggested that 15 years after their introduction, nitrogen-fixing bacteria remain in the soil but that closely related inoculant strains may not evolve in the same way, either genetically or symbiotically. In a context of increasing agronomical use of microbial inoculants (for biological control, nitrogen fixation, or plant growth promotion), this result feeds the debate on the consequences associated with such practices.

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Novel Genes and Regulators That Influence Production of Cell Surface Exopolysaccharides inSinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.00501-17 ◽

2017 ◽

Vol 200 (3) ◽

Cited By ~ 5

Author(s):

Melanie J. Barnett ◽

Sharon R. Long

Keyword(s):

Cell Surface ◽

Biofilm Formation ◽

Sinorhizobium Meliloti ◽

Developmental Process ◽

Root Nodules ◽

Critical Role ◽

Nitrogen Fixing ◽

Symbiotic Interaction ◽

Content Type ◽

Mutant Phenotypes

ABSTRACTSinorhizobium melilotiis a soil-dwelling alphaproteobacterium that engages in a nitrogen-fixing root nodule symbiosis with leguminous plants. Cell surface polysaccharides are important both for adapting to stresses in the soil and for the development of an effective symbiotic interaction. Among the polysaccharides characterized to date, the acidic exopolysaccharides I (EPS-I; succinoglycan) and II (EPS-II; galactoglucan) are particularly important for protection from abiotic stresses, biofilm formation, root colonization, and infection of plant roots. Previous genetic screens discovered mutants with impaired EPS production, allowing the delineation of EPS biosynthetic pathways. Here we report on a genetic screen to isolate mutants with mucoid colonial morphologies that suggest EPS overproduction. Screening with Tn5-110, which allows the recovery of both null and upregulation mutants, yielded 47 mucoid mutants, most of which overproduce EPS-I; among the 30 unique genes and intergenic regions identified, 14 have not been associated with EPS production previously. We identified a new protein-coding gene,emmD, which may be involved in the regulation of EPS-I production as part of the EmmABC three-component regulatory circuit. We also identified a mutant defective in EPS-I production, motility, and symbiosis, where Tn5-110 was not responsible for the mutant phenotypes; these phenotypes result from a missense mutation inrpoAcorresponding to the domain of the RNA polymerase alpha subunit known to interact with transcription regulators.IMPORTANCEThe alphaproteobacteriumSinorhizobium meliloticonverts dinitrogen to ammonium while inhabiting specialized plant organs termed root nodules. The transformation ofS. melilotifrom a free-living soil bacterium to a nitrogen-fixing plant symbiont is a complex developmental process requiring close interaction between the two partners. As the interface between the bacterium and its environment, theS. meliloticell surface plays a critical role in adaptation to varied soil environments and in interaction with plant hosts. We isolated and characterizedS. melilotimutants with increased production of exopolysaccharides, key cell surface components. Our diverse set of mutants suggests roles for exopolysaccharide production in growth, metabolism, cell division, envelope homeostasis, biofilm formation, stress response, motility, and symbiosis.

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Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

Applied and Environmental Microbiology ◽

10.1128/aem.01276-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 8056-8061 ◽

Cited By ~ 32

Author(s):

Ji Xu ◽

Xiao-Lin Li ◽

Li Luo

Keyword(s):

Nitrogen Fixation ◽

Drought Stress ◽

Sinorhizobium Meliloti ◽

Nitrogenase Activity ◽

Root Nodules ◽

Severe Drought ◽

Control Strain ◽

Free Living ◽

Content Type ◽

Host Tolerance

ABSTRACTCytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineeredSinorhizobiumstrains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed theAgrobacterium iptgene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although theiptgene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineeredSinorhizobiumstrains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.

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Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

Applied and Environmental Microbiology ◽

10.1128/aem.03556-15 ◽

2016 ◽

Vol 82 (8) ◽

pp. 2270-2279 ◽

Cited By ~ 15

Author(s):

Kristen Bennett ◽

Natalie C. Sadler ◽

Aaron T. Wright ◽

Chris Yeager ◽

Michael R. Hyman

Keyword(s):

De Novo ◽

Click Reaction ◽

Nitrosomonas Europaea ◽

Amino Acid Sequences ◽

Protein Profiling ◽

Ammonia Monooxygenase ◽

Peptide Fragments ◽

Content Type ◽

Sds Page ◽

In Cells

ABSTRACTNitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2−) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.

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