Date: 13 May, 2024

Dataset Title: Nitrogen stable isotope patterns in amino acids of a metabolically versatile anoxygenic phototrophic bacterium, Rhodopseudomonas palustris 

Dataset Creators: Dr. Jenan Kharbush, jenanjk@umich.edu; Raisha Rahman, rraisha@umich.edu; Dr. Ann Pearson, apearson@eps.harvard.edu

Dataset Contact: Dr. Jenan Kharbush, jenanjk@umich.edu


Research Overview:
Amino acids are the primary nitrogen-containing compounds in cells, participating in various metabolic processes such as protein synthesis and degradation into other nitrogenous compounds. Compound-specific nitrogen isotope analysis of amino acids in environmental samples can provide insight into nitrogen sources, cycling, and tropic modifications in ecosystems. However, we lack comprehensive understanding of N utilization patterns in microorganisms, which are the site of nitrogen assimilation in biological systems, due to the varied and complex mechanisms by which microbes can acquire and metabolize nitrogen-containing compounds. ?15N measurements for free intracellular amino acids and protein-bound amino acids for environmentally-relevant microbial groups may aid in untangling the biochemical processes involved in amino acid metabolism performed by these microbial communities. Here, we report intracellular amino acid ?15N values for R. palustris, a metabolically versatile anoxygenic phototrophic bacterium.


Methodology:
The dataset contains bulk and intracellular amino acid nitrogen isotope data for two strains of the metabolically versatile anoxygenic phototrophic bacterium, Rhodopseudomonas palustris. R. palustris CGA009 was grown micro-aerobically on minimal PM media, modified from Ritchie (2013), in capped 1L culture bottles with minimal headspace. The components were as follows (in mM): (NH4)2SO4, 10; KH2PO4, 12.5; Na2HPO4, 12.5; MgSO4, 0.238; CaCl2, 0.0454; Sodium pyruvate, 10; Sodium thiosulfate, 0.1; p-aminobenzoic acid, 0.0146; Sodium bicarbonate, 10. The medium additionally contained 10 mL of the ATCC MD-VS Vitamin Supplement and 1 mL of a trace elements mix. The trace elements mix contained the following components (in mM): Citric acid H2O, 105; FeSO4 7H2O, 2.511; ZnSO4 7H2O, 38.07; MnSO4 H2O, 9.11; CuSO4 5H2O, 1.570; Co(NO3)2 6H2O,0.859; Na2MoO4 2H2O, 0.02139; H3BO3, 1.856. R. palustris TIE1 was grown in completely anoxic conditions on minimal Freshwater media, modified from Ehrenreich and Widdel (1994), in capped 1L culture bottles. The components are as follows (in mM): NH4Cl, 10; KH2PO4, 1; MgCl2 6H2O, 1.97; CaCl2 2H2O, 1.36; Sodium thiosulfate, 5; Sodium pyruvate, 10; Sodium bicarbonate, 15. The medium additionally contained 1 mL of a vitamin supplement and 1 mL of a trace elements mix. Media preparation and culture inoculation were performed in an anaerobic glovebox. All cultures were incubated at 30¡C under 24-hour light while shaking at around 60 rpm. Growth rates were monitored by optical density (OD) at 600 nm on a UV-Vis spectrophotometer.
The R. palustris cultures were harvested via centrifugation at various points during the log and late-log phases of growth. The cell pellets were rinsed with nitrogen-free media between centrifugation steps. During cell collection, around 1 ml of spent media was collected, sterile filtered, and analyzed for NH4+ concentration using a Sigma-Aldrich Ammonia Assay Kit. 
Cell pellets for each culture were divided for bulk biomass, free intracellular amino acid, and protein-bound amino acid nitrogen isotope analysis. The bulk nitrogen isotopic composition of lyophilized cell samples were measured via elemental analysis with isotope ratio mass spectrometry (EA-IRMS), on a Thermo Scientific Flash IRMS Elemental Analyzer with EA Isolink, coupled to a Delta V Plus IRMS through a Conflo IV universal interface. Free intracellular amino acid subsamples were first lysed through a combination of bead-beating, freeze-thawing, and sonication. The subsamples were then purified with Dowex 50WX8 (200-400 mesh) cation exchange resin according to the method outlined by Owen et al. (1999), to isolate the individual amino acids from other cellular materials. The protein-bound amino acid subsamples were first treated with 6 M HCl at 110¡C for 20 hours to hydrolyze the protein peptide bonds, and then were also purified with the Dowex cation exchange resin. Prior to isotope analysis, the purified free and protein-bound amino acid subsamples were derivatized with MTBSTFA according to the procedure outlined in Molero et al. (2011). The derivatized free and protein-bound subsamples were then analyzed for 15N composition via  gas chromatography isotope ratio mass spectrometer (GC-IRMS), using a Thermo Scientific GC Isolink IRMS system with a Trace 1310 and TriPlus RSH Autosampler, coupled to a Delta V Plus IRMS though a Conflo IV universal interface (Agilent J&W DB-5ms GC Column; 60 m; GC program ramp: initial temperature of 90¡C, increased by 8.0¡C/min to 140¡C, then increased by 3.0¡C/min to 280¡C, and finally to 300¡C by 12¡C/min). Bulk, free amino acid, and protein-bound amino acid nitrogen isotope compositions were expressed in standard ? notation against atmospheric N2. ?15N values were corrected with in-house (amino acids) and authentic standards (USGS40, USGS41a, and AABA).

Files contained here:
r_palustris_nitrogen_isotope_data.csv
This file contains 35 rows and 17 columns. Rows 2 to 13 contain data on R. palustris samples grown in N-replete conditions. Rows 15 to 17 contain data on R. palustris samples grown in N-limited conditions. The columns contain the following information:
B. Sample identifier, with each sample designated by experiment date, strain, and a letter. Samples with the same year-month designation were part of the same culture experiment.
C. Initial NH4 concentration
D. Raw OD600 measurement at time of culture harvest
E. Normalized OD, calculated relative to highest OD600 measurement for each set of culture experiments.
F. Fraction of NH4 remaining in sample, calculated from NH4 concentration measurements on spent media and initial NH4 concentration, column (C). 
G. Normalized f, calculated by multiplying Normalized OD/column (E) by a factor corresponding to the maximum fraction of added NH4 that could possibly be consumed by batch cultures during growth (determined by measuring the fraction of NH4 remaining in media at stationary phase, column (F)). Growth differed between cultures with different concentrations of added NH4, so this normalization allowed us to compare relative incorporation of NH4 between experiments and cultures. This factor was approximately equal to the fraction of N used when cultures reached late log/stationary phase, which was lower in N-replete cultures that became limited by something else other than N, and higher in N-limited cultures. 
H. Fractional Yield (f), calculated as (1-Normalized f, column G))
I. Ln(f), or Ln(Fractional Yield)
J. Substrate ?15N, or nitrogen isotopic composition of NH4+ used in media preparation
K. Bulk Biomass ?15N
L-W. Free Intracellular Amino Acid ?15N values for the following amino acids, in order: glutamate/glutamine, aspartate/asparagine, alanine, isoleucine, leucine, valine, proline, glycine, serine, lysine, phenylalanine, tyrosine. Each column is labelled as "Free" followed by the three-letter code for the given amino acid.
X-AI. Protein-bound Amino Acid ?15N values for the following amino acids, in order: glutamate/glutamine, aspartate/asparagine, alanine, isoleucine, leucine, valine, proline, glycine, serine, lysine, phenylalanine, tyrosine. Each column is labelled as "Prot" followed by the three-letter code for the given amino acid. For the columns for glutamate/glutamine and aspartate/asparagine, Glx and Asx are used, respectively.
Use and Access:
This data set is made available under a Creative Commons Public Domain license (CC0 1.0).