13C isotopic tracer experiments.
13C-labeled sodium bicarbonate was rapidly introduced (within 1 min) into the bioreactor containing N. moscoviensis cells growing under steady-state conditions to a final concentration of approximately 30 mM. Following 13C-label introduction, samples were rapidly withdrawn from the reactor at time points 0, 1.5, 3, 5, 8, 11, 15, 20, 30, 45, 60, 90, and 120 min. Samples were immediately filtered (Millipore 0.45-μm hydrophilic nylon filter HNWPO4700) using a vacuum pump to remove extracellular medium, and filters were placed face down in 1.5 ml of −80°C extraction solvent (40:40:20 acetonitrile-methanol-water) for cell quenching and metabolite extraction. Samples were then centrifuged (10,000 rpm, 4°C, 5 min), and 1 ml of cell-free supernatant was collected and stored at −80°C for metabolomic analysis. The time zero sample corresponded to the period directly before 13C-label addition. The ratio of 13C to 12C dissolved inorganic carbon remained constant during the course of the 2-h experimental period as determined by gas chromatography-mass spectrometry (GC-MS) analysis as described below.
Formate batch experiments.
N. moscoviensis cells were harvested from the membrane bioreactor. The biomass was centrifuged at 8,000 × g for 15 min at 25°C and washed by resuspending the cells in fresh mineral NOB medium. This was repeated until no nitrite or nitrate was detectable via nitrite/nitrate test strips (Nitrite test strips MQuant; Merck) in the culture. Subsequently, the cells were transferred to sterile 120-ml serum bottles (triplicate bottles per time point, 6 time points total) containing 50 ml NOB mineral salts medium with 0.5 mM sodium formate and no nitrite but 0.187 mM NH4Cl as the nitrogen source. Bottles were crimp sealed with a rubber stopper to allow monitoring of the gas headspace and were incubated at 39°C in the dark. Following 24 h of acclimation, 1 mM 13C-labeled sodium formate (Cambridge Isotopes Laboratories, MA, USA) was added to all incubations. At each time point (before addition and after 15, 30, 60, 180, and 300 min), the isotopic composition of the gas headspace was measured using GC-MS (see below), and subsequently, all 3 bottles corresponding to a given time point were sacrificed for metabolomics analysis. Bottle contents were filtered immediately using a vacuum pump, and metabolites were extracted using −80°C extraction solvent (40:40:20 acetonitrile-methanol-water) as described above.
For growth experiments, N. moscoviensis cells from the membrane bioreactor were harvested and washed until no nitrite or nitrate was detectable via nitrite/nitrate test strips (Nitrite test strips MQuant; Merck) (see above). The cells were transferred to sterile 120-ml serum bottles with 50 ml NOB mineral medium containing 0.187 mM NH4Cl as the nitrogen source and a pH adjusted to 6.6, 7, or 7.7 with 1 M KHCO3 and were sealed with rubber stoppers. The experiment was performed in duplicates (per pH value and substrate), and 5 mM sodium nitrite or 5 mM sodium formate was added to the incubations as the sole energy source. Growth was monitored for 7 days using optical density measurements at a wavelength of 600 nm.
Large-scale discovery proteomics of whole-cell and membrane fractions.
(i) Whole-cell fraction sample preparation and proteolytic digestion. Biomass was harvested by centrifugation, washed once with Tris-EDTA buffer, pH 7.7, and frozen using liquid N2. Cell pellets were stored at −80°C until further processing. For protein extraction, cell pellets (445 mg wet weight) were resuspended in 5 ml 50 mM triethylammonium bicarbonate (TEAB) buffer, 1% sodium deoxycholate, pH 8, and mixed 1:1 with B-PER reagent (Thermo Fisher Scientific, Waltham, MA, USA). Cells were lysed by two rounds of sonification (Sonifier B-12 with microtip; Branson Sonic Power Company, Danbury, CT, USA) at setting 6 for 30 s, intermitted by cooling on ice. Cell debris and unopened cells were separated from the sample by centrifugation (14,000 × g, room temperature [RT], 10 min). Proteins were precipitated by addition of 250 μl 100% (wt/vol) trichloroacetic acid (TCA) to 1 ml of sample and incubation for 20 min on ice. Proteins were collected by centrifugation (14,000 rpm, RT, 5 min) and washed twice with ice-cold acetone. Next, the protein pellet was resuspended in 400 μl 50 mM TEAB buffer aided by a heating step at 40°C for 1 h with intermittent vortexing. One hundred microliters of the protein extract was diluted 5 times with 6 M urea, 200 mM ammonium bicarbonate (ABC) buffer followed by vortexing and a heating step for 20 min at 45°C at 1,450 rpm and 25 min of sonification. Acetonitrile (ACN) was added to a final concentration of 50%, followed by another heating step. A 200-μl aliquot of the sample was reduced by the addition of dithiothreitol (DTT) to a final concentration of 2.3 mM followed by incubation at 37°C for 1 h. Next, the sample was alkylated by the addition of iodoacetamide (IAM) to a final concentration of 3.75 mM followed by incubation in the dark at room temperature for 30 min. The sample was diluted with 200 mM ABC buffer to a concentration of urea of <1 M. Lastly, a 150-μl aliquot of the protein sample was digested with 5 μg of sequencing-grade trypsin (Promega, Madison, WI, USA) overnight at 37°C.
(ii) Membrane fraction sample preparation and proteolytic digestion. Biomass was harvested by centrifugation, washed once with Tris-EDTA (TE) buffer, and frozen using liquid N2. Cell pellets were stored at −80°C until further processing. For protein extraction, cell pellets (1.95 g wet weight) were resuspended in 13 ml 50 mM TEAB buffer, 1% sodium deoxycholate, pH 8, and mixed 1:1 with B-PER reagent (Thermo Fisher Scientific). Cells were lysed by three passages through a French press at 138 MPa pressure. Cell debris and unopened cells were pelleted from the sample by centrifugation (5,000 × g, 4°C, 15 min). The membrane fraction was prepared using ultracentrifugation (45,000 rpm, 4°C, 1 h on an Optima-90 ultracentrifuge; Beckman-Coulter, Brea, CA, USA) and washed once in 200 mM ABC buffer, followed by another ultracentrifugation step to collect the membrane pellet. Finally, the membrane pellet was resuspended in 400 μl 200 mM ABC, 1% N-dodecyl β-d-maltoside (DDM), and membrane proteins were solubilized overnight in an orbital shaker at 4°C. The sample was clarified by centrifugation (20,000 × g, RT, 15 min). Urea was added to the sample to a final concentration of 6 M. The sample was reduced by the addition of DTT to a final concentration of 2.3 mM followed by incubation at 37°C for 1 h. Next, the sample was alkylated by the addition of IAM to a final concentration of 3.75 mM, followed by incubation in the dark at RT for 30 min. The sample was diluted to a concentration of 1 mg/ml with 200 mM ABC, 1% DDM, and 6 M urea. Five aliquots of 100 μg protein each were digested or doubly digested using 2 μg of each selected proteinase; all digestion steps were performed at 37°C. The following different proteinases and combinations were tested: (i) LysC (Pierce), 4 h; (ii) trypsin (Promega), 4 h; (iii) chymotrypsin (Pierce), overnight; (iv) LysC, 4 h followed by trypsin, overnight; and (v) trypsin, 4 h followed by chymotrypsin, overnight. In digests with trypsin and chymotrypsin, ACN was added to a final concentration of 20%, and the concentration of urea in the sample was diluted to ≤1 M by the addition of ABC buffer.
(a) Solid phase extraction for membrane protein digest. Proteolytic digests were desalted using an Oasis HLB 96-well plate (Waters, Milford, MA, USA) according to the manufacturer’s protocol. The purified peptide eluate was further dried using a speed-vacuum concentrator.
(b) Large-scale shotgun proteomics.
All dried peptide fractions were resuspended in H2
O containing 3% acetonitrile and 0.1% formic acid using mild vortexing. An aliquot of every sample corresponding to approximately 100 to 200 ng protein digest was analyzed in duplicates using a one-dimensional shotgun proteomics approach (48
). Briefly, 1 μl of sample was injected to a nano-liquid chromatography system consisting of an EASY nano LC 1200, equipped with an Acclaim PepMap RSLC RP C18
separation column (50 μm by 150 mm, 2 μm, and 100 Å), and a QE plus Orbitrap mass spectrometer (Thermo). The flow rate was maintained at 300 nl/min over a linear gradient using H2
O containing 0.1% formic acid as solvent A, and 80% acetonitrile in H2
O and 0.1% formic acid as solvent B. The soluble protein extract fractions were analyzed using a gradient from 5% to 30% solvent B over 90 min, and finally to 75% B over 25 min. The soluble membrane protein fractions were analyzed using a shorter gradient from 4% to 30% B over 32.5 min followed by a second step to 65% B over 12.5 min, and data were acquired in total over 50 min. In either case, the Orbitrap was operated in data-dependent acquisition mode acquiring peptide signals from 350 to 1,400 m/z
at 70,000 resolution, where the top 10 signals were isolated from a window of 2.0 m/z
and fragmented using a normalized collision energy (NCE) of 30. The AGC target was set to 1E5, at a maximum IT of 54 ms and 17,500 resolution.
(c) Database search and data processing.
Raw shotgun proteomics data from membrane proteins and soluble protein extract fractions were analyzed and combined using PEAKS Studio 8.5 (Bioinformatics Solutions Inc., Waterloo, Canada). Database search was performed allowing 20-ppm parent ion and 0.02-Da fragment mass error tolerance. Search conditions further considered 3 missed cleavages for the respective enzymes used, carbamidomethylation as fixed and methionine oxidation and N/Q deamidation as variable modifications. Peptide spectra were matched against a N. moscoviensis
sequence database (UniProt TrEMBL, June 2018, taxon identifier [Tax ID] 42253), which was modified by filtering out duplicated entries. The database search included the GPM cRAP contaminant database (https://www.thegpm.org/crap/
) and a decoy fusion for determining false-discovery rates. Peptide spectrum matches were filtered against a 1% false-discovery rate (FDR), and protein identifications with 2 or more unique peptides were considered significant hits. For the prefractionated whole-cell proteome, proteins were counted if they were significantly identified in at least one of the subfractions.
To relate the proteome data to the previously published transcriptome of N. moscoviensis
under stable cultivation conditions grown on nitrite (13
), we reanalyzed the transcriptome data (GEO series accession number GSE123406
) using the N. moscoviensis
genome annotation version CP011801.1
as a reference, from which the UniProt protein sequences were derived (5
). The transcriptomic reanalysis was performed as described by Mundinger et al. (13
). In short, quality-filtered raw reads from IonTorrent PGM sequencing (minimum quality score of 0.05, maximum sequencing length of 300 bp, allowing two ambiguous nucleotides) were mapped to the N. moscoviensis
genome (NCBI accession number CP011801.1
) using the mapping tool BBMap v35.92 (https://sourceforge.net/projects/bbmap/
) and counted using featureCounts (50
) with the parameters minid-0.95 with fracOverlap-0.9 for a minimum alignment identity of 95% over 90% of the read length and ambig=random to assign reads with multiple top-scoring mapping locations randomly to a single location.
Since the genome includes several duplicated genes, the 4,790 protein coding sequences (CDSs) correspond to only 4,733 UniProt entries for nonidentical proteins. To directly compare transcription to proteome data, the transcriptome reads of those identical genes were thus summed up.
Expression levels of CDSs were compared by ranking them from high to low based on their reads per kilobase per million reads (RPKM) values. Additionally, the log2
fold to median was calculated for genome-wide visualizations of the gene expression levels in a Circos plot (51