Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

Significance Nitrogen is a constituent of many essential biomolecules and plentiful on earth as inert N2 gas. For its assimilation by eukaryotes, N2 must be converted to a metabolically tractable form such as ammonium. Such conversion is catalyzed by nitrogenase, an enzyme produced by a select group of microorganisms called diazotrophs. Crop yields necessary to feed the world's population have critically depended on applying nitrogenous fertilizers. Incorporation of prokaryotic determinates required to produce active nitrogenase into crop plants would have enormous economic and environmental benefits. The active-site cofactors of all nitrogenases have a common metallocluster precursor synthesized by NifB. Here, we identify the genetic determinants for NifB function in mitochondria of Saccharomyces cerevisiae, thereby advancing prospects to generate N2-fixing crops.


Supplementary Materials and Methods
Generation of plasmids for galactose-induced yeast expression. E. coli DH5α was used for storage and amplification of yeast expression pESC vectors (Agilent Technologies). E .coli was grown at 37°C in Luria-Bertani (LB) medium supplemented with appropriate antibiotics. pN2GLT4 for yeast codon-optimized expression of mitochondria targeted A. vinelandii NifU (NifUAv) and NifS (NifSAv) has been previously described (1).
Sequence coding for su9-sam1p was created by overlapping PCR reactions as specified below.
Primers 5´-AAAGCGGCCGCATGGCCTCCACTCGTGTCCT-3´ and 5´-TTTACTAGTGCGAACTTCAAAGTCTTAGGCTTTTCCCAT-3´ were used for the overlapping PCR. su9-sam1p was cloned into pESC-LEU using NotI/SpeI, creating su9-sam1pflag. The sequence coding for su9-twinstrep-tev-nifX was created by overlapping PCR reactions as specified below. The sequence encoding su9-twinstrep was amplified from pN2XJ155 using 5´-AGTCGGATCCATGGCCTCCACTCGTGTCC-3´ and 5´-AGGTTCTCGCCGCCTCCTTCGAGTTTTTCAAATTGT-3´. The sequence encoding tev-nifX at 4ºC. The resulting supernatant containing soluble proteins was analyzed by SDS-PAGE and immunoblot analysis. Total yeast protein extracts of strain SB17Y were used to analyze temperature-dependent solubility of NifB as previously described (1). Mitochondria isolations were performed as previously described (4). Enrichment was verified using tubulin (cytoplasmic) and HSP60 (mitochondria) marker proteins. Similar loading on SDS-PAGE experiments was obtained by preparing samples according to optical density, and was confirmed by using either Commassie staining of polyacrylamide gels or Ponceau staining of nitrocellulose membranes. Additionally, immunoblotting with antibodies against tubulin or HSP60 were used as control of gel loading and sample precipitation.
Cultures for yeast expressed NifB purifications were grown in a 4 l fermenter (BIO-STAT) as previously reported (1), with the following modifications. For strains SB30Y-SB32Y (procedure 1), cultures were grown at 30ºC in selective SD-medium for 16 h, followed by 8 h in rich medium (0.25% yeast extract, 0.25% bactopeptone, 0.25% bactotryptone, 2.5% sucrose), supplemented with 25 mg/l ammonium iron(III) citrate, 0.225 mM iron (II) sulfate, 1.25 mM magnesium sulfate, 1.5 mM calcium chloride and trace element solution (5). Finally, protein expression was induced for 16 h by addition of 2.25% galactose, 0.25% bactopeptone, 0.25% bactotryptone and vitamin solution (5). The pH was automatically maintained around 5 using 0.8 M ammonium hydroxide. Air flow was maintained at 2.5 l air/min per 4 L culture, at 250 rpm. Dissolved oxygen dropped to zero (as measured by oxygen sensor, Mettler Toledo) before addition of galactose, and remained at zero during the rest of the process. For strains SB222Y-SB227Y (procedure 2), cultures were grown as for SB30Y-SB32Y, except that a 300 ml pre-inoculum grown in selective SD-medium was used to start a 4 L fermenter, with rich medium and galactose from the beginning, for 20 h. For constitutive Nif expression in strain SB187Y (procedure 3), a 4 L culture was grown as previously reported (6), but with glucose instead of galactose.
Growth of SU9-Samp1-FLAG-transformed sam5 and control cells was measured in YP media containing 3% glycerol as non-fermentable carbon source (7). S. cerevisiae wild-type BY4741, and BY4741 with deleted SAM5 (YNL003c, strain Y05331) were provided by EUROSCARF. GAL regulatory elements were induced using 0.1 M -estradiol in cells containing pGEV-His (2). Each strain was cultured in triplicates with a starting optical density (OD 600 ) of 0.10, and grown under aerobic conditions at 30ºC. Induction and protein expression was verified in total yeast protein extracts collected 75 h after the start of the experiment.
NifB pulldowns and activity screening assay. S. cerevisiae strains for Strep-tactin pulldowns and screening of NifB activity were flask-cultured in standard YPD medium, at 30ºC under aerobic conditions. Soluble yeast protein extracts to analyze NifB activity were prepared in anaerobic YeastBuster Protein Extraction Reagent (Novagen). Shortly, yeast cells corresponding to 25 ml culture at OD=8 were centrifuged at 3.000 g for 10 min. Pellets were washed in 1 ml milli-Q water, centrifuged, and then frozen in liquid N2 before introduced into an anaerobic glovebox (MBraun). Pellets were washed once in anaerobic buffer (100 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 2 mM DTH, 5 mM -ME), and then lysed in 1 ml anaerobic YeastBuster Protein Extraction Reagent (supplemented with 1xTHP, 2 mM DTH, 5 mM -ME, 1 mM PMSF, 1 μg/ml leupeptin, and 5 μg/ml DNAse I). Following incubation for 20 min at room temperature and 1,000 rpm, samples were centrifuged for 5 min at maximum speed using benchtop centrifuge. The resulting supernatants containing soluble proteins were analyzed by SDS-PAGE and immunoblot analysis. 700 l of the supernatant was added to 1.3 ml of the above described buffer containing Strep-tactin resin (IBA Lifesciences), and then incubated for 2 h with gentle shaking inside the glovebox. Strep-tactin beads were washed once with 1 ml buffer, and finally eluted in 180 l buffer containing 50 mM biotin (IBA Lifesciences). NifB enrichment in the supernatant was tested by SDS-PAGE and immunoblot analysis, and 75 ul (in duplicates) was used for FeMo-co synthesis and apo-NifDK reconstitution using UW140 cellfree extracts.
UV-visible spectroscopy, N-terminal sequencing, Western blotting and protein methods. Asisolated NifB preparations were used for colorimetric Fe determination (8), in vitro FeMo-co synthesis and nitrogenase activity assays (see sections below), and UV-visible spectroscopy. UVvisible absorption spectra were recorded under anaerobic conditions in septum-sealed cuvettes using a Shimadzu UV-2600 spectrophotometer. When indicated, anaerobic samples were exposed to air during 5 min. UV-visible absorption spectra were recorded against buffer C as baseline. Absorbance at 800 nm was subtracted and spectra were then normalized to 279 nm. The N-terminal amino acid sequence of purified NifB was determined by Edman degradation (Proteome Factory AG). Western blot signals were detected using X-ray films (AGFA), or using an iBright FL1000 Imaging System (ThermoFisher Scientific) with auto-contrast setting. Protein concentrations were measured using the BCA protein assay (PIERCE). Samples were pre-treated with iodoacetamide before performing the BCA assay to eliminate the interfering effect of DTH (9).

Supplementary Text
DNA and amino acid sequences DNA sequences used for parental strains and NifB library:

TTGGGTGAGTTCGTCAAAGTTATCCCAGTCTCCGCTGCCGCACACGCCCAAATGGAAGTC L G E F V K V I P V S A A A H A Q M E V
TAA -

A F P K L V C F K K R I E A I P Q I D K
TACTTAAAATCTTCTAAATATATCGCTTGGCCTTTGCAAGGTTGGCAAGCTACTTTTGGT

G G S G G G S G G S A W S H P Q F E K
Yeast optimized DNA sequences, and their translated amino acid sequences, cloned in pESC expression vectors. N-terminus for NifB as deduced from N-terminal sequencing is highlighted in green (SSSAW).

GAAAGAGGTAGAAGAGCCGGTACTGAAAATGCTGCATCTATTATAGGTTTGGGTGTTGCC E R G R R A G T E N A A S I I G L G V A
GCTGAAAGAGCTTTACAATTCATGGAACATGAAAACACTGAAGTTAAGAGATTGCGTGAT

TTGTTGTTGAACAAAGTTGGTATAGCAGCCTCCAGTGGTTCTGCTTGTACATCTGGTTCA L L L N K V G I A A S S G S A C T S G S
TTGGAACCATCACATGTTATGAGAGCAATGGATATTCCTTATACAGCTGCACACGGTACT

GGTTCACTTACAGGAATCCCACCTGAAAGATTGGCTGAGGCAATGAGAGAAATACAGGCA G S L T G I P P E R L A E A M R E I Q A
AGGTAA R -

GAAATTTCTGCTTCAAAATGCACCGAGTGTGATGGAGACTATGCTGAAAAGCAATGCGCA E I S A S K C T E C D G D Y A E K Q C A
TCTATTTGTCCAGTTGAAGGTGCTATCTTGTTAGCAGACGGAACTCCTGCTAACCCACCT

GGTTCACTTACAGGAATCCCACCTGAAAGATTGGCTGAGGCAATGAGAGAAATACAGGCA G S L T G I P P E R L A E A M R E I Q A
AGGTATCCATATGATGTTCCAGATTATGCTTAA

AACGAAAAGGTTCATGATAAGTACGGTAGAGTTCATTTGCCAGTTGCTCCAAGATGTAAC N E K V H D K Y G R V H L P V A P R C N
ATTGCTTGTAAGTTCTGCAAGAGGTCCGTTTCTAAAGAATGTTGTGAACATAGACCAGGT

A Y V Q N I I P L I P Q Y K M K E L R A
CCAACTTGCGAAGAAATCAAAAAGGTCAGAAAAGAGTGCGAGAAGTACATCCCACAATTC

CCAACTTGCGAAGAAATCAAAAAGGTCAGAAAAGAGTGCGAGAAGTACATCCCACAATTC P T C E E I K K V R K E C E K Y I P Q F
AGAGCTTGTGGTCAATGTAGAGCTGATGCTGTTGGTCTGATCAAAGAAAAAGAGCTGTTG

GTTGACAGAATCAAGCAATTGAACATCGATCATGTTACCATCACCATCAACATGGTTGAT V D R I K Q L N I D H V T I T I N M V D
CCAGAAATTGGCACTAAGATCTATCCATGGGTTCACTACAGAAGAAAGAGATACAAAGGT

GAAGCCTACGATGTTATTGAAAAGGTTGCCTTGGAATTCTACGAGAAGTGGATTTTGGAA E A Y D V I E K V A L E F Y E K W I L E
GCCAGAGATTGA A R D -

GAAAGGTCCTTCAACAACTACGAAATTAGACCAGGTGTTTCTAGAACCGTTATTACTCCT E R S F N N Y E I R P G V S R T V I T P
GAAGAAGCTTTGGACGCTATTAGAAGGGCTTTAGAAGTTTGCCCAGATATTCATGTTGCT

A C G A S A V R Q L M A I G V Q P I K V
TCCGAGGGAGCAAGGATCGCGGAGCTCATTGAGGCCCTGCAGGTCGAGCTGAGGGAGGGA

CTAGTTGCCGCTGGTTTATGTAAGAGAGTTCAAGTTCAATTTTCTTATGCCATCGGTATT L V A A G L C K R V Q V Q F S Y A I G I
GCGGAACCATTGTCCTTGCACGTTGACACCTATGGTACTGCGACCAAGTCTGACGAAGAA

ACGACCGAGGACTTAAGAGCGCAACTAAAGTCCGAGATCATTGAAAAAGTCATCCCAAGA T T E D L R A Q L K S E I I E K V I P R
GACATGTTGGACGAAAACACCAAATACTTTATCCAACCTTCCGGTAGATTCGTCATCGGT      . Positive control reactions for the assay contained purified NifB-co (+ NifB-co). Negative control reactions for assay were performed in the absence of NifBco (ctrl). Error bars represent mean ± standard deviation (n=2). The soluble extracts for the assay were prepared using the YeastBuster extraction method.         Table S5 for details).   Table S5) (1), and subcomponents as in Fig. 3E (2 to 4). Experimental data is shown in black solid line, while overall spectral simulation (using AC1 cluster (10)) is shown in red dotted line. The g values of each species, spin concentration and cluster nomenclature (adapted from (10)) are indicated in the figure.        Origin of the 28 nifB genes selected for the library screening (euryarch., euryarchaeota; -bact., proteobacteria; chlor., chlorobi; cyano., cyanobacteria; -bact., -proteobacteria; firmi., firmicutes; -bact., -proteobacteria; chlorofl., chloroflexi). Multidomain NifB protein architecture (presence of the C-terminal NifX-like domain) is indicated as NifX. Expression and accumulation of soluble NifB, and yield when purified using GAL-regulated expression plasmids, are indicated.  Processed forms refer to molecular mass following SU9 cleavage (RAY-SSAW, SI Appendix, Fig.  S13B). For SU9-TS-TEV-NifX two values are reported in the "Processed" column where the smaller represent size after TEV protease treatment.