Many enzymes depend upon protein networks that perform essential tasks such as cofactor distribution, membrane insertion, or covalent modification. These cellular networks are evolutionarily ancient and have diversified considerably through adaptation and coevolution with their client enzymes. As a consequence, cellular networks may be unable to recognize client enzymes from foreign species. Thus, many network-dependent enzymes often show limited or no activity when expressed within foreign species, and genes encoding such enzymes may be less likely to persist following horizontal transfer (Porse et al., 2018). Because the biosynthesis of many valuable natural products relies upon network-dependent enzymes, their compatibility with non-native cellular networks is an important question for bioengineering.

Iron-sulfur (Fe-S) enzymes depend upon cellular Fe-S biogenesis networks to acquire Fe-S cluster cofactors (Johnson et al., 2005). These networks assemble and distribute Fe-S clusters via Fe-S scaffold and carrier proteins (Figure 1A). Two Fe-S biogenesis pathways (ISC and SUF) have been identified in prokaryotes. Common to both ISC and SUF is the use of cysteine desulfurase enzymes (IscS or SufSE) to obtain sulfur from L-cysteine, and scaffold proteins (IscU or SufBCD) to assemble Fe-S clusters. Fe-S cluster carrier proteins of the A-type (IscA, SufA, ErpA), Nfu-type (NfuA), or ApbC-type (Mrp) deliver clusters to apo targets (Py and Barras, 2010; Roche et al., 2013). Fe-S biogenesis systems exemplify the diversification of essential cellular networks (Roche et al., 2013). It has been proposed that the diversification of Fe-S enzymes and networks is driven by adaptations to molecular oxygen, which reduces the environmental availability of iron and is destructive to Fe-S clusters (Andreini et al., 2017; Boyd et al., 2014; Imlay, 2006). Comparisons between SUF pathways, which appear to be more widespread, reveal further diversification: while core SUF components (SufB and SufC) are conserved, SUF systems differ in the number of proteins used to assist Fe-S cluster assembly, delivery, and maturation (e.g. SufD, SufE, SufS, SufU, SufT) (Boyd et al., 2014; Garcia et al., 2019).

Figure 1

Download asset Open asset

Fe-S enzymes often show no activity when expressed within foreign hosts (Lanz et al., 2012; Nakai et al., 2015; Nakamura et al., 1999). As with any heterologous enzyme, inactivity in vivo may arise from poor expression, i.e., inefficient transcription or translation. In a bioengineering context, these barriers are routinely overcome using tools such as strong promoters and codon optimization. Unfortunately, even if heterologous enzymes are expressed, few tools exist for ensuring activity is retained. Particularly for Fe-S enzymes, incompatibilities with foreign Fe-S biogenesis networks that arise from diversification may also limit activity. Here, we combine bioinformatics and phylogeny with an in vivo platform to systematically survey the ability of two types of Fe-S enzymes to retain activity within a foreign host. In doing so, we assessed how environmental adaptations and the dependencies of Fe-S enzymes upon cellular networks each contribute to limiting heterologous activity in prokaryotic hosts. We show that the activity of heterologous Fe-S enzymes can be recovered by broadening compatibility of host networks and by modifying growth conditions. This survey leads us to further investigate these barriers and enhance in vivo activity of a cobalamin-dependent radical S-adenosyl methionine (rSAM) methyltransferase enzyme involved in antibiotic biosynthesis.

Fe-S clusters are ancient metal cofactors present in all domains of life and genes encoding Fe-S containing proteins are likely to be transferred between organisms. Moreover, a wide array of Fe-S enzymes have biotechnological importance. However, inefficient maturation in heterologous hosts may impede activity of Fe-S proteins acquired via lateral transfer. By systematically mapping the transferability of Fe-S enzyme activities between prokaryote species, we have identified several factors affecting heterologous Fe-S enzyme activity: oxygen sensitivity, incompatibility with the host Fe-S biogenesis network, and incompatibility with host electron transfer proteins. Many orthologs are likely to be limited by multiple factors, e.g., oxygen sensitivity and Fe-S network incompatibility. The adaptations required for Fe-S enzymes to maintain activity within aerobic organisms are reflected by the existence of separate aerotolerant and oxygen-sensitive clades in NadA phylogeny. This in turn leads to a remarkable correlation between the likelihood of complementation by NadA orthologs and phylogenetic distance from E. coli NadA. Structural studies, as have been performed for the Fe-S enzyme IspH (Rao and Oldfield, 2016), could further explore the structural mechanisms underlying the oxygen tolerance of NadA orthologs from Clade 1.

Many NadA orthologs within the aerotolerant clade required a heterologous Fe-S biogenesis pathway, indicating that incompatibility with E. coli Fe-S biogenesis pathways can impede heterologous activity. Interestingly, NadA orthologs reactivated by SUF coexpression were obtained entirely from organisms that featured only an SUF pathway, suggesting that some orthologs require specific Fe-S scaffold or carrier proteins. Nevertheless, the native E. coli Fe-S cluster transfer and maturation pathways proved impressively versatile with heterologous Fe-S enzymes, as more than one-third of NadA orthologs tested – and by extension from our phylogenetically representative set, the same fraction of all known prokaryotic NadA orthologs – obtain Fe-S clusters when expressed within E. coli. Our results with IspG orthologs also reflect the versatility of the E. coli Fe-S network, as many IspG orthologs were reactivated using electron transfer proteins alone. For such orthologs, IspG activity is not limited by incompatibilities with any step facilitated by host Fe-S biogenesis pathways. Although Fe-S pathways must be versatile to provide Fe-S clusters to many structurally and functionally diverse Fe-S enzymes within a single species (e.g. >150 Fe-S enzymes in E. coli), it is nevertheless remarkable that this versatility also extends to Fe-S enzymes from distantly related species.

Unlike NadA, IspG orthologs at even a low patristic distance from E. coli IspG were inactive if compatible electron transfer proteins were not provided. The electron transfer proteins we identified may contribute essential roles during Fe-S cluster maturation, enzymatic catalysis, or cluster reduction following adventitious oxidation. The finding that electron transfer specificity prevents functional expression to such a severe degree was unexpected as many redox-dependent enzymes are compatible with multiple electron transfer proteins and vice versa. For instance, the E. coli flavodoxin FldA delivers electrons to E. coli BioB, IspG, and IspH (Wolff et al., 2003). Conversely, three B. subtilis electron transfer proteins (including YkuN and YkuP) support activity of B. subtilis acyl lipid desaturase (Chazarreta-Cifre et al., 2011) and five T. maritima ferredoxins are compatible with the T. maritima rSAM enzyme MiaB (Arcinas et al., 2019). Despite this promiscuity, prokaryotic genomes retain multiple electron transfer proteins whose structures and reduction potentials vary considerably. E. coli IspG requires low-potential reductants (E′ <–300 mV) (Xiao et al., 2009), suggesting that FldA reduces IspG as a hydroquinone (E′ = –433 mV) (McIver et al., 1998). However, the FldA potential is well below the corresponding potentials of B. subtilis YkuP and YkuN (–377 and –382 mV, respectively) as well as the reduction potential of cyanobacterial ferredoxins similar to Synechocystis PetF (approximately –380 mV) (Lawson et al., 2004; Motomura et al., 2019). Assuming that IspG orthologs exhibit reduction potentials similar to E. coli IspG, the selectivity we observe is likely determined by structural complementarity between electron transfer proteins and IspG orthologs. The presence of multiple electron transfer proteins enables specialization (Kaçar and Gaucher, 2013), and the retention of multiple electron transfer proteins reflects the nonredundant roles they play in their host. While this specialization may extend biosynthetic capabilities, it may also constrain the compatibility of electron transfer-dependent enzymes with heterologous hosts.

Fe-S enzymes catalyze many reactions that are relevant to biotechnology such as nitrogen reduction, dehydration, sulfur insertion, and methyl transfer (Johnson et al., 2005). The full potential of Fe-S enzymes cannot be harnessed until we are able to routinely express them within heterologous species. By identifying the separate contributions of oxygen sensitivity, Fe-S biogenesis network compatibility, and electron transfer protein specificity to impeding Fe-S enzyme function, our complementation and recovery assays suggest guidelines for improving the compatibility of Fe-S enzymes with engineered organisms. First, it should be explored whether identifying distinct phylogenetic clades within Fe-S enzyme families may reveal orthologs found predominantly in aerobic organisms, as we observed for NadA. Such efforts could guide selection of heterologous enzymes compatible with aerobic conditions. As we found with NadA, enzyme phylogeny may be more predictive of heterologous activity in aerobic conditions than the aerotolerance of the host organism. Second, the common approach of overexpressing host Fe-S pathways may not prove sufficient for some Fe-S enzymes. Many Fe-S enzymes may instead require heterologous Fe-S pathways (Corless et al., 2020; Nakamura et al., 1999). Our results with B. subtilis SUF suggest that the heterologous pathway used should match that of the original host. Finally, many heterologous Fe-S enzymes are often inactive not due to incompatibilities with Fe-S networks, but rather a lack of other essential compatible proteins such as electron transfer proteins, the importance of which may be often overlooked. As we demonstrated with IspG, suitable cofactors from the genome of the original host can often be identified by coexpression with the heterologous Fe-S enzyme. Likewise, in the case of TsrM, the low reducing potential of cobalamin informed the selection of [4Fe-4S]-type ferredoxins for screening. Additional studies evaluating these guidelines with a wider array of enzyme families could help formulate ‘design rules’ enabling the routine use of Fe-S enzymes in biosynthetic pathways, thus unlocking the chemical versatility of Fe-S clusters.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2 and 3. Data for phylogenetic analysis (containing protein database of the 248 prokaryotes, NadA/ IspG alignments and phylogenies and python script used to select sequences by clustering approach) is available on figshare.

1. 1. Garcia P

   (2021) figshare

   Dangelo_et_al_Supplementary_data.zip.

   https://doi.org/10.6084/m9.figshare.13664927.v1

1. 1. Altschul SF
   2. Madden TL
   3. Schäffer AA
   4. Zhang J
   5. Zhang Z
   6. Miller W
   7. Lipman DJ

   (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

   Nucleic Acids Research 25:3389–3402.

   https://doi.org/10.1093/nar/25.17.3389

   • PubMed
   • Google Scholar
2. 1. Andreini C
   2. Rosato A
   3. Banci L

   (2017) The Relationship between Environmental Dioxygen and Iron-Sulfur Proteins Explored at the Genome Level

   PLOS ONE 12:e0171279.

   https://doi.org/10.1371/journal.pone.0171279

   • PubMed
   • Google Scholar
3. 1. Arcinas AJ
   2. Maiocco SJ
   3. Elliott SJ
   4. Silakov A
   5. Booker SJ

   (2019) Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase

   Protein Science 28:267–282.

   https://doi.org/10.1002/pro.3548

   • PubMed
   • Google Scholar
4. 1. Balaban M
   2. Moshiri N
   3. Mai U
   4. Jia X
   5. Mirarab S

   (2019) TreeCluster: Clustering biological sequences using phylogenetic trees

   PLOS ONE 14:e0221068.

   https://doi.org/10.1371/journal.pone.0221068

   • PubMed
   • Google Scholar
5. 1. Beinert H

   (1983) Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins

   Analytical Biochemistry 131:373–378.

   https://doi.org/10.1016/0003-2697(83)90186-0

   • PubMed
   • Google Scholar
6. 1. Blaszczyk AJ
   2. Silakov A
   3. Zhang B
   4. Maiocco SJ
   5. Lanz ND
   6. Kelly WL
   7. Elliott SJ
   8. Krebs C
   9. Booker SJ

   (2016) Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase

   Journal of the American Chemical Society 138:3416–3426.

   https://doi.org/10.1021/jacs.5b12592

   • PubMed
   • Google Scholar
7. 1. Blaszczyk AJ
   2. Knox HL
   3. Booker SJ

   (2019) Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase

   Journal of Biological Inorganic Chemistry 24:831–839.

   https://doi.org/10.1007/s00775-019-01689-8

   • PubMed
   • Google Scholar
8. 1. Boyd ES
   2. Thomas KM
   3. Dai YY
   4. Boyd JM
   5. Outten FW

   (2014) Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway

   Biochemistry 53:5834–5847.

   https://doi.org/10.1021/bi500488r

   • PubMed
   • Google Scholar
9. 1. Broderick JB
   2. Duffus BR
   3. Duschene KS
   4. Shepard EM

   (2014) Radical S-adenosylmethionine enzymes

   Chemical Reviews 114:4229–4317.

   https://doi.org/10.1021/cr4004709

   • PubMed
   • Google Scholar
10. 1. Cammack R

    (1992) Iron—sulfur clusters in enzymes: Themes and variations

    Advances in Inorganic Chemistry 38:281–322.

    https://doi.org/10.1016/S0898-8838(08)60066-5

    • Google Scholar
11. 1. Chazarreta-Cifre L
    2. Martiarena L
    3. de Mendoza D
    4. Altabe SG

    (2011) Role of ferredoxin and flavodoxins in Bacillus subtilis fatty acid desaturation

    Journal of Bacteriology 193:4043–4048.

    https://doi.org/10.1128/JB.05103-11

    • PubMed
    • Google Scholar
12. 1. Corless EI
    2. Mettert EL
    3. Kiley PJ
    4. Antony E

    (2020) Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

    Journal of Bacteriology 202:e00496-19.

    https://doi.org/10.1128/JB.00496-19

    • PubMed
    • Google Scholar
13. 1. Criscuolo A
    2. Gribaldo S

    (2010) BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments

    BMC Evolutionary Biology 10:210.

    https://doi.org/10.1186/1471-2148-10-210

    • PubMed
    • Google Scholar
14. 1. Datsenko KA
    2. Wanner BL

    (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

    PNAS 97:6640–6645.

    https://doi.org/10.1073/pnas.120163297

    • PubMed
    • Google Scholar
15. 1. Elliott JI
    2. Brewer JM

    (1978) The inactivation of yeast enolase by 2,3-butanedione

    Archives of Biochemistry and Biophysics 190:351–357.

    https://doi.org/10.1016/0003-9861(78)90285-0

    • PubMed
    • Google Scholar
16. 1. Fish WW

    (1988) Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples

    Methods in Enzymology 158:357–364.

    https://doi.org/10.1016/0076-6879(88)58067-9

    • PubMed
    • Google Scholar
17. 1. Fourmond V
    2. Hoke K
    3. Heering HA
    4. Baffert C
    5. Leroux F
    6. Bertrand P
    7. Léger C

    (2009) SOAS: a free program to analyze electrochemical data and other one-dimensional signals

    Bioelectrochemistry (Amsterdam, Netherlands) 76:141–147.

    https://doi.org/10.1016/j.bioelechem.2009.02.010

    • PubMed
    • Google Scholar
18. 1. Garcia PS
    2. Gribaldo S
    3. Py B
    4. Barras F

    (2019) The SUF system: an ABC ATPase-dependent protein complex with a role in Fe-S cluster biogenesis

    Research in Microbiology 170:426–434.

    https://doi.org/10.1016/j.resmic.2019.08.001

    • PubMed
    • Google Scholar
19. 1. Gaudu P
    2. Weiss B

    (2000) Flavodoxin mutants of Escherichia coli K-12

    Journal of Bacteriology 182:1788–1793.

    https://doi.org/10.1128/JB.182.7.1788-1793.2000

    • PubMed
    • Google Scholar
20. 1. Guindon S
    2. Dufayard JF
    3. Lefort V
    4. Anisimova M
    5. Hordijk W
    6. Gascuel O

    (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0

    Systematic Biology 59:307–321.

    https://doi.org/10.1093/sysbio/syq010

    • PubMed
    • Google Scholar
21. 1. Imlay JA

    (2006) Iron-sulphur clusters and the problem with oxygen

    Molecular Microbiology 59:1073–1082.

    https://doi.org/10.1111/j.1365-2958.2006.05028.x

    • PubMed
    • Google Scholar
22. 1. Jarrett JT
    2. Choi CY
    3. Matthews RG

    (1997) Changes in protonation associated with substrate binding and Cob(I)alamin formation in cobalamin-dependent methionine synthase

    Biochemistry 36:15739–15748.

    https://doi.org/10.1021/bi971987t

    • PubMed
    • Google Scholar
23. 1. Johnson DC
    2. Dean DR
    3. Smith AD
    4. Johnson MK

    (2005) Structure, function, and formation of biological iron-sulfur clusters

    Annual Review of Biochemistry 74:247–281.

    https://doi.org/10.1146/annurev.biochem.74.082803.133518

    • PubMed
    • Google Scholar
24. 1. Kaçar B
    2. Gaucher EA

    (2013) Experimental evolution of protein-protein interaction networks

    The Biochemical Journal 453:311–319.

    https://doi.org/10.1042/BJ20130205

    • PubMed
    • Google Scholar
25. 1. Katoh K
    2. Standley DM

    (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability

    Molecular Biology and Evolution 30:772–780.

    https://doi.org/10.1093/molbev/mst010

    • PubMed
    • Google Scholar
26. 1. Kirby J
    2. Dietzel KL
    3. Wichmann G
    4. Chan R
    5. Antipov E
    6. Moss N
    7. Baidoo EEK
    8. Jackson P
    9. Gaucher SP
    10. Gottlieb S
    11. LaBarge J
    12. Mahatdejkul T
    13. Hawkins KM
    14. Muley S
    15. Newman JD
    16. Liu P
    17. Keasling JD
    18. Zhao L

    (2016) Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

    Metabolic Engineering 38:494–503.

    https://doi.org/10.1016/j.ymben.2016.10.017

    • PubMed
    • Google Scholar
27. 1. Lanz ND
    2. Grove TL
    3. Gogonea CB
    4. Lee KH
    5. Krebs C
    6. Booker SJ

    (2012) RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins

    Methods in Enzymology 516:125–152.

    https://doi.org/10.1016/B978-0-12-394291-3.00030-7

    • PubMed
    • Google Scholar
28. 1. Lanz ND
    2. Blaszczyk AJ
    3. McCarthy EL
    4. Wang B
    5. Wang RX
    6. Jones BS
    7. Booker SJ

    (2018) Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli

    Biochemistry 57:1475–1490.

    https://doi.org/10.1021/acs.biochem.7b01205

    • PubMed
    • Google Scholar
29. 1. Larsson A

    (2014) AliView: a fast and lightweight alignment viewer and editor for large datasets

    Bioinformatics (Oxford, England) 30:3276–3278.

    https://doi.org/10.1093/bioinformatics/btu531

    • PubMed
    • Google Scholar
30. 1. Lawson RJ
    2. von Wachenfeldt C
    3. Haq I
    4. Perkins J
    5. Munro AW

    (2004) Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: biophysical properties and interactions with cytochrome P450 BioI

    Biochemistry 43:12390–12409.

    https://doi.org/10.1021/bi049131t

    • PubMed
    • Google Scholar
31. 1. Lee TS
    2. Krupa RA
    3. Zhang F
    4. Hajimorad M
    5. Holtz WJ
    6. Prasad N
    7. Lee SK
    8. Keasling JD

    (2011) BglBrick vectors and datasheets: A synthetic biology platform for gene expression

    Journal of Biological Engineering 5:12.

    https://doi.org/10.1186/1754-1611-5-12

    • PubMed
    • Google Scholar
32. 1. Lemoine F
    2. Domelevo Entfellner JB
    3. Wilkinson E
    4. Correia D
    5. Dávila Felipe M
    6. De Oliveira T
    7. Gascuel O

    (2018) Renewing Felsenstein’s phylogenetic bootstrap in the era of big data

    Nature 556:452–456.

    https://doi.org/10.1038/s41586-018-0043-0

    • PubMed
    • Google Scholar
33. 1. Lexa D
    2. Saveant JM

    (2002) The electrochemistry of vitamin B12

    Accounts of Chemical Research 16:235–243.

    https://doi.org/10.1021/ar00091a001

    • Google Scholar
34. 1. Loiseau L
    2. Gerez C
    3. Bekker M
    4. Ollagnier-de Choudens S
    5. Py B
    6. Sanakis Y
    7. Teixeira de Mattos J
    8. Fontecave M
    9. Barras F

    (2007) ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli

    PNAS 104:13626–13631.

    https://doi.org/10.1073/pnas.0705829104

    • PubMed
    • Google Scholar
35. 1. Mahanta N
    2. Hudson GA
    3. Mitchell DA

    (2017) Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

    Biochemistry 56:5229–5244.

    https://doi.org/10.1021/acs.biochem.7b00771

    • PubMed
    • Google Scholar
36. Conference

    1. McClure MA
    2. Smith C
    3. Elton P

    (1996)

    Proceedings. International Conference on Intelligent Systems for Molecular Biology

    Parameterization studies for the SAM and HMMER methods of hidden Markov model generation. pp. 155–164.

    • Google Scholar
37. 1. McIver L
    2. Leadbeater C
    3. Campopiano DJ
    4. Baxter RL
    5. Daff SN
    6. Chapman SK
    7. Munro AW

    (1998) Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli

    European Journal of Biochemistry 257:577–585.

    https://doi.org/10.1046/j.1432-1327.1998.2570577.x

    • PubMed
    • Google Scholar
38. 1. Mehta AP
    2. Abdelwahed SH
    3. Mahanta N
    4. Fedoseyenko D
    5. Philmus B
    6. Cooper LE
    7. Liu Y
    8. Jhulki I
    9. Ealick SE
    10. Begley TP

    (2015) Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions

    The Journal of Biological Chemistry 290:3980–3986.

    https://doi.org/10.1074/jbc.R114.623793

    • PubMed
    • Google Scholar
39. 1. Motomura T
    2. Zuccarello L
    3. Sétif P
    4. Boussac A
    5. Umena Y
    6. Lemaire D
    7. Tripathy JN
    8. Sugiura M
    9. Hienerwadel R
    10. Shen JR
    11. Berthomieu C

    (2019) An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties

    Biochimica et Biophysica Acta. Bioenergetics 1860:148084.

    https://doi.org/10.1016/j.bbabio.2019.148084

    • PubMed
    • Google Scholar
40. 1. Nakai T
    2. Ito H
    3. Kobayashi K
    4. Takahashi Y
    5. Hori H
    6. Tsubaki M
    7. Tanizawa K
    8. Okajima T

    (2015) The Radical S-Adenosyl-L-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

    The Journal of Biological Chemistry 290:11144–11166.

    https://doi.org/10.1074/jbc.M115.638320

    • PubMed
    • Google Scholar
41. 1. Nakamura M
    2. Saeki K
    3. Takahashi Y

    (1999) Hyperproduction of recombinant ferredoxins in Escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster

    Journal of Biochemistry 126:10–18.

    https://doi.org/10.1093/oxfordjournals.jbchem.a022409

    • PubMed
    • Google Scholar
42. 1. Neidhardt FC
    2. Bloch PL
    3. Smith DF

    (1974) Culture medium for enterobacteria

    Journal of Bacteriology 119:736–747.

    https://doi.org/10.1128/jb.119.3.736-747.1974

    • PubMed
    • Google Scholar
43. 1. Nguyen L-T
    2. Schmidt HA
    3. von Haeseler A
    4. Minh BQ

    (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies

    Molecular Biology and Evolution 32:268–274.

    https://doi.org/10.1093/molbev/msu300

    • PubMed
    • Google Scholar
44. 1. Okada K
    2. Hase T

    (2005) Cyanobacterial non-mevalonate pathway: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase interacts with ferredoxin in Thermosynechococcus elongatus BP-1

    The Journal of Biological Chemistry 280:20672–20679.

    https://doi.org/10.1074/jbc.M500865200

    • PubMed
    • Google Scholar
45. 1. Peralta-Yahya PP
    2. Ouellet M
    3. Chan R
    4. Mukhopadhyay A
    5. Keasling JD
    6. Lee TS

    (2011) Identification and microbial production of a terpene-based advanced biofuel

    Nature Communications 2:483.

    https://doi.org/10.1038/ncomms1494

    • PubMed
    • Google Scholar
46. 1. Porse A
    2. Schou TS
    3. Munck C
    4. Ellabaan MMH
    5. Sommer MOA

    (2018) Biochemical mechanisms determine the functional compatibility of heterologous genes

    Nature Communications 9:522.

    https://doi.org/10.1038/s41467-018-02944-3

    • PubMed
    • Google Scholar
47. 1. Puan KJ
    2. Wang H
    3. Dairi T
    4. Kuzuyama T
    5. Morita CT

    (2005) fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

    FEBS Letters 579:3802–3806.

    https://doi.org/10.1016/j.febslet.2005.05.047

    • PubMed
    • Google Scholar
48. 1. Py B
    2. Barras F

    (2010) Building Fe-S proteins: bacterial strategies

    Nature Reviews. Microbiology 8:436–446.

    https://doi.org/10.1038/nrmicro2356

    • PubMed
    • Google Scholar
49. 1. Rao G
    2. Oldfield E

    (2016) Structure and Function of Four Classes of the 4Fe-4S Protein, IspH

    Biochemistry 55:4119–4129.

    https://doi.org/10.1021/acs.biochem.6b00474

    • PubMed
    • Google Scholar
50. 1. Reimer LC
    2. Vetcininova A
    3. Carbasse JS
    4. Söhngen C
    5. Gleim D
    6. Ebeling C
    7. Overmann J

    (2019) BacDive in 2019: bacterial phenotypic data for High-throughput biodiversity analysis

    Nucleic Acids Research 47:D631–D636.

    https://doi.org/10.1093/nar/gky879

    • PubMed
    • Google Scholar
51. 1. Roche B
    2. Aussel L
    3. Ezraty B
    4. Mandin P
    5. Py B
    6. Barras F

    (2013) Reprint of: Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity

    Biochimica et Biophysica Acta 1827:923–937.

    https://doi.org/10.1016/j.bbabio.2013.05.001

    • PubMed
    • Google Scholar
52. 1. Salis HM
    2. Mirsky EA
    3. Voigt CA

    (2009) Automated design of synthetic ribosome binding sites to control protein expression

    Nature Biotechnology 27:946–950.

    https://doi.org/10.1038/nbt.1568

    • PubMed
    • Google Scholar
53. 1. Virtanen P
    2. Gommers R
    3. Oliphant TE
    4. Haberland M
    5. Reddy T
    6. Cournapeau D
    7. Burovski E
    8. Peterson P
    9. Weckesser W
    10. Bright J
    11. van der Walt SJ
    12. Brett M
    13. Wilson J
    14. Millman KJ
    15. Mayorov N
    16. Nelson ARJ
    17. Jones E
    18. Kern R
    19. Larson E
    20. Carey CJ
    21. Polat İ
    22. Feng Y
    23. Moore EW
    24. VanderPlas J
    25. Laxalde D
    26. Perktold J
    27. Cimrman R
    28. Henriksen I
    29. Quintero EA
    30. Harris CR
    31. Archibald AM
    32. Ribeiro AH
    33. Pedregosa F
    34. van Mulbregt P
    35. SciPy 1.0 Contributors

    (2020) SciPy 1.0: fundamental algorithms for scientific computing in Python

    Nature Methods 17:261–272.

    https://doi.org/10.1038/s41592-019-0686-2

    • PubMed
    • Google Scholar
54. 1. Wolff M
    2. Seemann M
    3. Tse Sum Bui B
    4. Frapart Y
    5. Tritsch D
    6. Garcia Estrabot A
    7. Rodríguez-Concepción M
    8. Boronat A
    9. Marquet A
    10. Rohmer M

    (2003) Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein

    FEBS Letters 541:115–120.

    https://doi.org/10.1016/s0014-5793(03)00317-x

    • PubMed
    • Google Scholar
55. 1. Xiao YL
    2. Zahariou G
    3. Sanakis Y
    4. Liu PH

    (2009) IspG enzyme activity in the deoxyxylulose phosphate pathway: roles of the iron-sulfur cluster

    Biochemistry 48:10483–10485.

    https://doi.org/10.1021/bi901519q

    • PubMed
    • Google Scholar
56. 1. Zhou K
    2. Zou RY
    3. Stephanopoulos G
    4. Too HP

    (2012) Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

    Microbial Cell Factories 11:148.

    https://doi.org/10.1186/1475-2859-11-148

    • PubMed
    • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "Resolving the barriers that impair iron-sulphur cluster enzyme activity in non-native prokaryotic hosts" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Dennis Dean as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Wayne Outten (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted the following comments to help you prepare a revised submission: Your manuscript has been reviewed by three individuals that have considerable experience in the general area of Fe-S protein maturation. Although there is general agreement that there are interesting aspects of the study there are several concerns that preclude publication of the work in its present form. Most of these are described in detail in the initial review that is included elsewhere in this communication. In the discussion among the reviewers, they were asked to make specific suggestions about aspects that could be addressed without a demand for additional experiments such that the work might be suitable for publication in eLife. Please note that the rather specific suggestions provided here do not represent "conditions" for publication but rather points that should be considered if you and coworkers should decide to submit a revised manuscript. Also please be aware that any revised manuscript will be evaluated by all the participating reviewers.

The title and abstract would benefit from refinement. In the opinion of the reviewers the work does not, in fact, "resolve the barriers that impair Fe-S cluster enzyme activity in non-native prokaryotic hosts". If such barriers were resolved by the study this would suggest that future studies are not required for the field, which is probably not correct. As noted in the reviews there are barriers that have not been considered. A more descriptive title that could be considered and seems more appropriate is something like: "Using cellular selections for different iron-sulfur proteins to evaluate activities of divergent homologs in a non-native prokaryotic host". Such a title that emphasizes the cellular assay aspect would then be more inclusive of the figure 3 data which is about electron carriers rather than enzymes, which seems disconnected to the enzyme narrative highlighted in the title and abstract. It would also be useful if you would consider adjusting the last sentence in the abstract to convey more precisely what can be gleaned from the study.

Some modest further computational analysis of the data set that is more deeply connected to heterologous expression rather than maturation is an aspect the reviewers believe is necessary for a more complete story. You might want to consider an additional figure that involves such an analysis. This would not require additional experimentation but could involve, for example, plotting the calculated translational initiation strength using the Salis model, making some bar graphs that consider source organism thermostability, oxygen conditions, types of biogenesis systems, and relating this to the assay conditions (temperature, oxygen, host Fe-S machinery). Such an analysis would go a long way towards addressing the major concern of the reviewers. Namely, whether other properties correlate with the accumulation of active Fe-S proteins when heterologously expressed other than those directly considered in the manuscript in its present form. Regardless of trends, additional deeper reflection on the data set could be informative for readers to think about with respect to selection pressure on the divergent proteins as they relate assay conditions.

Following this theme, the manuscript might benefit from a bit more set up and discussion on what can be challenging mechanistically for expression and maturation of iron-sulfur proteins in the introduction. The current focus is restricted to challenges involving Fe-S maturation, which seems narrow and somewhat misleading (other potential limitations should be considered such as promoter strength, translation initiation, folding, maturation, etc.). The manuscript might also benefit from reflecting in the discussion on what is needed to further understand the mechanisms responsible for all these trends and what studies are needed in the future to develop design rules for the two proteins studied and other Fe-S family members. This could reflect on the power of the cellular assays used in tandem with expressing different biogenesis systems, adjusting expression of all components and so forth. Please be assured that the reviewers are not trying to guide your work but rather are providing recommendations that we believe would provide a presentation of higher impact such that it is suitable for publication in eLife.

We thank you for submitting your manuscript for consideration by eLife and hope you will find the reviews and the comments provided useful.

Reviewer #1 (Recommendations for the authors):

This reviewer found the work to be thorough and convincing. The rather concise and clear presentation contributes to the value of the manuscript. The only technical question that I would ask is that it is not clear to me whether or not a fully assembled Fe-S protein containing its cognate Fe-S cluster can be heterologously produced but is inactive because the compatible intact electron transfer component is missing, or if the cognate Fe-S cluster is not inserted during heterologous expression because the electron transfer component is missing. I believe the authors touch on this aspect in the manuscript, but it was not entirely clear to me which way this works? Perhaps this issue could be clarified or, perhaps, emphasized?

Reviewer #2 (Recommendations for the authors):

The conclusions of the paper are supported by the data presented. This work also provides one of the few systematic analyses of factors that influence heterologous Fe-S protein expression. However, there were several experiments that could have improved the manuscript and helped tease more information out of the studies performed.

1. Analysis of the IspG heterologous expression seems incomplete, especially since (as the authors point out) there are multiple explanations for lack of growth in a genetic complementation assay. It would be helpful for the reader if the authors could provide a Venn diagram or other graphical representation of the IspG family members tested. Such a figure should show which IspG proteins are (a) insoluble or undetectable (b) detectable but not able to reactivated at all, (c) detectable and active in E. coli, (d) detectable and active in E. coli but only with co-expression of partner electron transfer protein, etc. For example, it was unclear if some of the IspG proteins that were undetectable when expressed alone could be detected when their electron transfer protein was co-expressed with them. It is possible some IspG enzymes are designed to form complexes with their electron donors and may be inherently unstable without them. It was hard to tease that kind of information out of the data as shown.

2. It would have strengthened the paper to have an experiment that directly tests if IspG is receiving the appropriate Fe-S cluster (especially in those cases where the protein was detectable but inactive). Whole-cell EPR or whole-cell Mossbauer seem like appropriate techniques to address this question. Immunoprecipation of IspG after cell labeling with radioactive iron may also be sufficient. The in vitro Fe-S cluster transfer experiment with ErpA on its own is not enough to demonstrate that there is not a problem with Fe-S maturation of IspG in E. coli.

3. The last section of the paper on TsrM is interesting and indirectly relevant for the studies on activation of IspG. It would have been perhaps more informative for the authors to pursue the question of redox coupling directly with IspG and partner electron transfer proteins. In addition, it is important to answer the question of whether the partner protein was assisting IspG simply by increasing IspG stability and/or Fe-S cluster loading.

4. For NadA maturation, did the authors attempt more sophisticated analysis to determine if NadA enzymes and their compatible Fe-S biosynthesis pathways may have sequence motifs, different components, or other unique features that would explain why they work better with each other than more distantly related pathways/proteins? That is really a key question. Why do some pathways work better than others when maturing a given Fe-S enzyme? What features of each pathway have specifically diverged during evolution that created barriers between enzymes/maturation pathways?

Reviewer #3 (Recommendations for the authors):

As one considers the different mechanisms that could give rise to the observed trends, this study would be strengthened by further analysis.

Some simple things to consider include calculating the translation initiation rate variation using the Salis RBS calculator to determine how this relates to cellular complementation. Additionally, providing insight on how the native host growth temperature (or oxygen conditions) relates to function would beneficial as well. These types of computational analysis would help understand if expression and thermostability might underlie some of the trends observed.

The effect of iron-sulfur cluster assembly systems on rescuing and divergence from the host would benefit from a side-by-side comparison with host systems expressed the same way. Many of these systems have feedback regulation in their native hosts, and it isn't clear if the regulation of synthesizing these systems underlies the complementation observed or the evolutionary distance trends.

In cases where complementation is not observed, it would be interesting to try at lower temperatures (especially if host grows at lower temperature) and under anaerobic conditions where iron-sulfur cluster biogenesis is less burdensome. Additionally, if RBS strength varies, then one can express all of the homologous iron-sulfur proteins as fusions to a N-terminal peptide or GFP to maintain the context of the RBS and ensure more consistent expression conditions when comparing function.

The title and abstract would benefit from refinement. In the opinion of the reviewers the work does not, in fact, "resolve the barriers that impair Fe-S cluster enzyme activity in non-native prokaryotic hosts". If such barriers were resolved by the study this would suggest that future studies are not required for the field, which is probably not correct. As noted in the reviews there are barriers that have not been considered. A more descriptive title that could be considered and seems more appropriate is something like: "Using cellular selections for different iron-sulfur proteins to evaluate activities of divergent homologs in a non-native prokaryotic host". Such a title that emphasizes the cellular assay aspect would then be more inclusive of the figure 3 data which is about electron carriers rather than enzymes, which seems disconnected to the enzyme narrative highlighted in the title and abstract. It would also be useful if you would consider adjusting the last sentence in the abstract to convey more precisely what can be gleaned from the study.

We did not intend to imply that the barriers were “resolved” in the sense that no further work was needed. As suggested, we changed the title to better describe our work:

“Cellular assays identify barriers impeding iron-sulphur enzyme activity in a non-native prokaryotic host.”

We have also modified the final sentence of the abstract:

“Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineered Fe-S enzyme-dependent pathways.”

Some modest further computational analysis of the data set that is more deeply connected to heterologous expression rather than maturation is an aspect the reviewers believe is necessary for a more complete story. You might want to consider an additional figure that involves such an analysis. This would not require additional experimentation but could involve, for example, plotting the calculated translational initiation strength using the Salis model, making some bar graphs that consider source organism thermostability, oxygen conditions, types of biogenesis systems, and relating this to the assay conditions (temperature, oxygen, host Fe-S machinery). Such an analysis would go a long way towards addressing the major concern of the reviewers. Namely, whether other properties correlate with the accumulation of active Fe-S proteins when heterologously expressed other than those directly considered in the manuscript in its present form. Regardless of trends, additional deeper reflection on the data set could be informative for readers to think about with respect to selection pressure on the divergent proteins as they relate assay conditions.

As requested, we now include results from additional experiments and analyses to determine whether other properties correlate with Fe-S enzyme activity.

1. We have performed further complementation experiments in anaerobic conditions to explore oxygen sensitivity as a barrier to heterologous activity (lines 158-169).

2. We applied the RBS Calculator to predict translation initiation rates for heterologous orthologs. These values are now included in Tables 1 and 2. The distributions are depicted and compared in Figure 2 —figure supplement 3 and described in lines 251-257. This analysis did not reveal any significant differences between RBS strength distributions for groups of complementing and non-complementing orthologs.

3. We now include the properties of the source organisms of each tested ortholog (oxygen tolerance, growth temperature, Fe-S biogenesis operon) in Tables 1 and 2. The properties are linked to complementation results in new bar graphs shown in Figure 2E.

These experiments and analyses revealed striking trends in our data, which are now discussed:

1. We found a correlation between source organism aerotolerance and complementation in aerobic conditions for NadA orthologs. This further revealed the existence of two clades with in the NadA phylogenetic tree: an aerotolerant clade, and a clade whose members largely required anaerobic conditions for activity. This is described in lines 185-201, and depicted in Figure 2.

2. We found no clear correlation between complementation ability and either thermotolerance or Fe-S biogenesis operons of the source organisms in the initial complementation experiments using the phylogenetically-based sets of orthologs.

3. However, this additional analysis revealed that the NadA orthologs recovered by heterologous SUF expression were entirely obtained from organisms with exclusively SUF-type pathways. This is described in lines 182-184 and depicted in Figure 2E.

4. The remarkable illustration of oxygen sensitivity as a selection pressure driving diversification within the NadA tree is now elaborated further in the Discussion section (lines 350-356).

Following this theme, the manuscript might benefit from a bit more set up and discussion on what can be challenging mechanistically for expression and maturation of iron-sulfur proteins in the introduction. The current focus is restricted to challenges involving Fe-S maturation, which seems narrow and somewhat misleading (other potential limitations should be considered such as promoter strength, translation initiation, folding, maturation, etc.).

Challenges related to heterologous protein expression are certainly relevant to our work. However, we focus upon challenges particular to Fe-S enzymes, rather than the problems related to protein expression. We therefore sought to exclude effects arising from low promoter strength or inefficient translation as much as possible by using codon-optimized genes and expression from multi-copy plasmids, thus ensuring that all Fe-S enzymes are expressed. We furthermore confirmed expression of a selection of heterologous orthologs using SDS-PAGE and mass spectrometry. The additional analysis by the RBS calculator, which found no evidence that inactive orthologs suffer from inefficient translation, supports our approach. This allows us to identify barriers to activity that are particular to Fe-S enzymes, such as incompatibility with Fe-S biogenesis pathways, oxygen sensitivity, and a lack of suitable electron transfer proteins.

We have modified the text to more clearly acknowledge that problems with heterologous expression may also impede Fe-S enzyme activity:

1. We broadened the introduction to encompass the challenges of heterologous protein expression before emphasizing that we are narrowly focusing upon barriers to activity that are specific to Fe-S enzymes (lines 76-83).

2. We emphasize our use of codon optimization and multicopy plasmids to prevent poor expression from influencing our complementation assays in the Results section (lines 106-109)

3. As explained above, we describe using the RBS Calculator and untargeted proteomics assays to confirm expression of the orthologs found to be inactive (Figure 2 —figure supplement 3, lines 251-257).

The manuscript might also benefit from reflecting in the discussion on what is needed to further understand the mechanisms responsible for all these trends and what studies are needed in the future to develop design rules for the two proteins studied and other Fe-S family members. This could reflect on the power of the cellular assays used in tandem with expressing different biogenesis systems, adjusting expression of all components and so forth. Please be assured that the reviewers are not trying to guide your work but rather are providing recommendations that we believe would provide a presentation of higher impact such that it is suitable for publication in eLife.

We have expanded the discussion to reflect upon further experiments needed for identifying mechanisms. We further elaborate on the insight provided for pathway engineers, and on the additional studies required for formulating reliable “design rules” for Fe-S enzymes:

1. Based on our new findings revealing aerotolerance as a key factor in heterologous NadA activity, we propose experiments to explore the structural basis of aerotolerance in NadA (lines 354-356).

2. We also propose that identifying clades within Fe-S enzyme families (as we did for NadA) may reveal the existence of aerotolerant clades more compatible with aerobic growth (lines 405-410).

3. We also emphasize that some Fe-S enzymes may require heterologous Fe-S biogenesis pathways. Our results suggest using heterologous SUF pathways for enzymes obtained from organisms with SUF pathways (lines 410-414).

4. We explicitly link the need for additional studies that test our suggested guidelines to the eventual formulation of design rules for Fe-S enzymes (lines 420-422).

We thank you for submitting your manuscript for consideration by eLife and hope you will find the reviews and the comments provided useful.

Reviewer #1 (Recommendations for the authors):

This reviewer found the work to be thorough and convincing. The rather concise and clear presentation contributes to the value of the manuscript. The only technical question that I would ask is that it is not clear to me whether or not a fully assembled Fe-S protein containing its cognate Fe-S cluster can be heterologously produced but is inactive because the compatible intact electron transfer component is missing, or if the cognate Fe-S cluster is not inserted during heterologous expression because the electron transfer component is missing. I believe the authors touch on this aspect in the manuscript, but it was not entirely clear to me which way this works? Perhaps this issue could be clarified or, perhaps, emphasized?

We thank the reviewer for the positive feedback. To address the reviewer’s question about whether electron transfer proteins assist either Fe-S cluster transfer or enzyme activity, we tested whether an IspG ortholog unable to complement growth contained an Fe-S cluster when expressed by E. coli. IspG from S. thermophilum was expressed in aerobic cultures and purified under anaerobiosis. in vitro assays (UV-visible spectra, together with iron and sulfur quantification) revealed that a significant fraction of purified IspG contained an Fe-S cluster. This suggests that this particular IspG ortholog is inactive because its cognate electron transport protein is missing, instead of an inability to acquire an Fe-S cluster. The missing electron transfer protein may be required during the IspG catalytic cycle or for cluster maturation step.

We have modified our manuscript to incorporate these results and to clarify our Discussion:

1. Our new characterization of purified Fe-S enzymes are now described in the new version lines 278-291 and Figure 2 —figure supplement 4. Further experiments along these lines with NadA orthologs are described below in our response to a similar question from Reviewer 2.

2. We now acknowledge in our manuscript that electron transfer proteins may be required for IspG cluster maturation, activity, or reduction after adventitious oxidation (lines 374-376 in Discussion).

3. We have removed our assertion that inactive IspG orthologs possess Fe-S clusters. While our new data supports this assertion for S. thermophilum IspG, we have not tested additional inactive IspG orthologs.

4. We have clarified that our observation that an electron transfer protein is sufficient to reactivate many IspG orthologs indicates that activities of these orthologs are not limited by any step facilitated by SUF or ISC Fe-S biogenesis and maturation pathways (lines 366-369 in Discussion). This is true even if the missing electron transfer proteins are required for Fe-S cluster transfer or further maturation.

Reviewer #2 (Recommendations for the authors):

The conclusions of the paper are supported by the data presented. This work also provides one of the few systematic analyses of factors that influence heterologous Fe-S protein expression. However, there were several experiments that could have improved the manuscript and helped tease more information out of the studies performed.

1. Analysis of the IspG heterologous expression seems incomplete, especially since (as the authors point out) there are multiple explanations for lack of growth in a genetic complementation assay. It would be helpful for the reader if the authors could provide a Venn diagram or other graphical representation of the IspG family members tested. Such a figure should show which IspG proteins are (a) insoluble or undetectable (b) detectable but not able to reactivated at all, (c) detectable and active in E. coli, (d) detectable and active in E. coli but only with co-expression of partner electron transfer protein, etc.

To clarify analysis of the IspG orthologs, we made several modifications to the text and tables. We have also added new graphical representations to Figures:

1. We now include a pie chart in Figure 2E depicting the numbers of IspG orthologs active, recovered using electron carriers, recovered in anaerobic conditions, and not recovered.

2. In Tables 1 and 2, we now indicate NadA and IspG orthologs analyzed and detected by MS.

3. In Table 2, we have consolidated data from electron transfer protein recovery experiments into a single column to more cleanly indicate the IspG orthologs that were recovered. Information on the specific electron carriers activating the IspG orthologs has been moved to Supplementary File 6.

4. We provide a pie chart in Figure 2 —figure supplement 3 that depicts the numbers of inactive IspG orthologs that were analyzed using MS, and which of these were successfully detected. This pie chart better clarifies that our MS detection focused upon the inactive IspG orthologs, as MS experiments were motivated by our need to confirm that inactive orthologs were successfully expressed. For orthologs that were detected and undetected, the characteristics of the native hosts are displayed.

5. We emphasize in the text that our MS analysis was focused primarily upon orthologs unable to complement in any condition tested (lines 260-266).

For example, it was unclear if some of the IspG proteins that were undetectable when expressed alone could be detected when their electron transfer protein was co-expressed with them. It is possible some IspG enzymes are designed to form complexes with their electron donors and may be inherently unstable without them. It was hard to tease that kind of information out of the data as shown.

We did not directly test whether expression of compatible electron transfer proteins stabilizes IspG orthologs. This is because our mass spectroscopy experiments were primarily motivated by our need to confirm that orthologs inactive in our complementation assays were expressed. We therefore focused upon orthologs that did not complement growth in any condition. Therefore, the only orthologs we submitted to mass spec analysis were those that did not show any evidence of expression, i.e. because they did not complement growth either alone in aerobic conditions or when co-expressed with a SUF pathway or an electron transfer protein. As stated above, we now clarify that MS was used primarily to detect inactive orthologs on lines 260-263.

Nevertheless, our prior mass spec results can partly address the reviewer’s question of whether inactive IspG can be detected in the absence of suitable electron donors. Aerobic cultures expressing 16 inactive IspG orthologs were analyzed by mass spec. As 10 of the 16 orthologs were detected by mass spec without co-expression of any compatible electron transfer protein, it can be concluded that these orthologs apparently do not require electron transfer proteins for protein stability. We cannot state whether the 6 orthologs analyzed but not detected by mass spec would be detected had we been able to identify and co-express compatible electron transfer proteins.

However, complementation experiments in anaerobic conditions (performed for the revised manuscript) revealed that one of the 6 IspG orthologs that could not be detected by MS (from Collinsella stercoris) recovered growth when expressed under anaerobic conditions. This implies that some IspG orthologs may indeed be unstable (or insoluble) in aerobic conditions, which would make them less detectable by MS. This observation is highlighted on lines 272-276.

2. It would have strengthened the paper to have an experiment that directly tests if IspG is receiving the appropriate Fe-S cluster (especially in those cases where the protein was detectable but inactive). Whole-cell EPR or whole-cell Mossbauer seem like appropriate techniques to address this question. Immunoprecipation of IspG after cell labeling with radioactive iron may also be sufficient. The in vitro Fe-S cluster transfer experiment with ErpA on its own is not enough to demonstrate that there is not a problem with Fe-S maturation of IspG in E. coli.

We agree, and we address a nearly identical question in our response to Reviewer 1. While equipment limitations related to the COVID pandemic precluded a timely whole-cell EPR analysis, we have performed an additional biochemical analysis on a subset of NadA and IspG proteins, which is now presented in the revised manuscript (lines 278-291 and Figure 2 —figure supplement 4).

We tested whether several NadA and IspG proteins that scored negative in the aerobic complementation test contained an Fe-S cluster within aerobic E. coli cultures. After anaerobic purification, UV-visible spectroscopy together with iron and sulfur quantification (by both colorimetric method and ICP-MS) indicated that at least a small fraction of each of the 4 enzymes purified contained an Fe-S cluster. This indicates that each of these 4 orthologs receive Fe-S clusters. In the case of D. thermophilum and D. indicum NadA orthologs and T. maritima IspG, each of which complement growth in anaerobic conditions, the lack of complementation during aerobic conditions leads us to speculate that the Fe-S clusters detected may be oxidized or otherwise inactivated. In the case of S. thermophilum IspG, which is inactive even in anaerobic conditions, the detection of an Fe-S cluster further suggests that this particular ortholog is inactive because its cognate electron transfer protein is missing, not because it cannot receive a cluster. These new data are now described in the new version lines 287-291 and in Figure 2 —figure supplement 4.

3. The last section of the paper on TsrM is interesting and indirectly relevant for the studies on activation of IspG. It would have been perhaps more informative for the authors to pursue the question of redox coupling directly with IspG and partner electron transfer proteins.

Examining redox coupling between IspG and electron transfer proteins would indeed be more relevant to IspG complementation. However, this would require additional experiments that may not reveal much that is not already known, as the potentials for several electron transfer proteins that we used to activate heterologous IspG have been measured and published. We have now included these published redox potentials in an expanded Discussion (lines 385-393) in which we compare the potentials to in vitro measurements of IspG activity. Given that the redox potentials of FldA and the electron transfer proteins used to activate heterologous IspG are very similar, it is likely that selectivity for specific IspG is driven by structural recognition between IspG and electron transfer proteins, rather than by redox potential.

In addition, it is important to answer the question of whether the partner protein was assisting IspG simply by increasing IspG stability and/or Fe-S cluster loading.

The reviewer is correct that we cannot distinguish for most IspG orthologs whether the electron transfer proteins recover IspG activity by providing electrons for the IspG catalytic cycle (as we originally stated), or whether recovery is enabled through Fe-S cluster loading, maturation, or stability. We have therefore clarified that electron transfer proteins may contribute to any of these steps in the discussion (lines 374-376).

While we cannot conclusively answer this question for all IspG orthologs tested, our experiment with S. thermophilum IspG, which remained inactive in our complementation assays and yet retained an Fe-S cluster following anaerobic purification from aerobic cultures of E. coli, suggests that an activating electron transfer protein is not absolutely required for Fe-S cluster loading (lines 277-281). Furthermore, as we state above, mass spectroscopy detection of 10 of 16 IspG orthologs that are inactive in aerobic conditions indicates that electron transfer proteins are not necessarily needed to stabilize the IspG protein itself. On the other hand, the Collinsella stercoris IspG ortholog was not detected in samples from an aerobic culture, but nevertheless recovered growth in an anaerobic culture. This may indicate that some oxygen-sensitive IspG orthologs may be unstable or insoluble during aerobic growth (lines 272-276).

4. For NadA maturation, did the authors attempt more sophisticated analysis to determine if NadA enzymes and their compatible Fe-S biosynthesis pathways may have sequence motifs, different components, or other unique features that would explain why they work better with each other than more distantly related pathways/proteins? That is really a key question. Why do some pathways work better than others when maturing a given Fe-S enzyme? What features of each pathway have specifically diverged during evolution that created barriers between enzymes/maturation pathways?

We agree, and we suggest a similar study that would investigate the sequence motifs and structures underlying the aerotolerance of NadA orthologs in the Discussion (lines 354-356). Concerning motifs that influence compatibility between Fe-S pathways and NadA orthologs, we are currently performing such an in-depth analysis that combines bioinformatics with structural and functional analysis. The data will be published in a separate publication.

Reviewer #3 (Recommendations for the authors):

As one considers the different mechanisms that could give rise to the observed trends, this study would be strengthened by further analysis.

Some simple things to consider include calculating the translation initiation rate variation using the Salis RBS calculator to determine how this relates to cellular complementation. Additionally, providing insight on how the native host growth temperature (or oxygen conditions) relates to function would beneficial as well. These types of computational analysis would help understand if expression and thermostability might underlie some of the trends observed.

We have performed additional experiments and analysis requested (as also described in our response to the Editor):

1. In order to explore how variations in translation initiation rate relate to the trends we observed, we used the RBS calculator (Salis et al.,) to compare the predicted translation initiation rates of complementing and non-complementing orthologs. No significant differences between RBS strength distributions of complementing and non-complementing orthologs could be found. These analyses are presented in Tables 1 and 2, depicted in Figure 2 —figure supplement 3, and described on lines 251-257.

2. As described in our response to the Editor, we have also analyzed whether growth temperature differences between E. coli and sources of the heterologous orthologs, the presence of oxygen, and presence of ISC or SUF pathways relate to function in E. coli. These results are depicted in Figure 2E, and the data provided in Table 2. We did not find any clear relationship between growth temperature and complementation.

3. Additional complementation results in anaerobic conditions revealed the existence of two clades within the NadA phylogeny – one of which is aerotolerant, the other likely oxygen-sensitive. Interestingly, membership of NadA orthologs within either clade better predicts complementation in aerobic conditions that does the oxygen tolerance of the original host organisms. This is more fully described in lines 158-169 and lines 185-201.

4. While we found no strong relationship between presence of ISC or SUF pathways in original hosts and activity within E. coli, we did find that re-activation of NadA orthologs by heterologous SUF pathways was only possible if the source organism also possessed a SUF pathway. This is described in lines 182-184 and in the Discussion (lines 412-414).

The effect of iron-sulfur cluster assembly systems on rescuing and divergence from the host would benefit from a side-by-side comparison with host systems expressed the same way. Many of these systems have feedback regulation in their native hosts, and it isn't clear if the regulation of synthesizing these systems underlies the complementation observed or the evolutionary distance trends.

Considering our NadA reactivation experiments with SUF operons, both E. coli and B. subtilis SUF operons were cloned into identical plasmid backbones (pBbA5a) and expressed using an IPTG-inducible promoter, thus precluding any differences in regulation from affecting our results.

For our initial complementation experiments, we acknowledge that the feedback-limited expression of the E. coli ISC pathway encoded in the genome, in contrast with the overexpression of the SUF pathways from multi-copy plasmids, may preclude a side-by-side comparison. This is why we do not compare the compatibility of the E. coli ISC pathway against the SUF pathways that we tested.

In cases where complementation is not observed, it would be interesting to try at lower temperatures (especially if host grows at lower temperature) and under anaerobic conditions where iron-sulfur cluster biogenesis is less burdensome. Additionally, if RBS strength varies, then one can express all of the homologous iron-sulfur proteins as fusions to a N-terminal peptide or GFP to maintain the context of the RBS and ensure more consistent expression conditions when comparing function.

As described in our responses to other reviewers, we repeated our experiments in anaerobic conditions. This revealed interesting relationships between oxygen sensitivity and activity, which are detailed in our manuscript.

We repeated our complementation assays at low temperature (28°C). This did not change the outcome (lines 145-146). We do not see a clear relationship between optimal growth temperature of each source organism and complementation results (Figure 2E).

Finally, we acknowledge that N-terminal fusions to our Fe-S enzymes together with a consistent RBS would likely ensure a more consistent expression level between orthologs; however, such fusions might affect enzyme activity and complicate our results. As we did not observe any differences in the RBS strength distributions obtained for active and inactive orthologs, we are confident that our results are not driven by differences in RBS strength.

Author details

1. Francesca D'Angelo

   Unit Stress Adaptation and Metabolism of Enterobacteria, Department of Microbiology, Université de Paris, UMR CNRS 2001, Institut Pasteur, Paris, France

   Contribution

   These authors are listed alphabetically, Data curation, Formal analysis, Investigation, Writing – original draft

   Contributed equally with

   Elena Fernández-Fueyo, Pierre Simon Garcia and Helena Shomar

   Competing interests

   No competing interests declared

   Additional information

   These authors are listed alphabetically

   "This ORCID iD identifies the author of this article:" 0000-0001-5880-1087

2. Elena Fernández-Fueyo

   Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands

   Contribution

   These authors are listed alphabetically, Conceptualization, Formal analysis, Investigation, Methodology

   Contributed equally with

   Francesca D'Angelo, Pierre Simon Garcia and Helena Shomar

   Competing interests

   No competing interests declared

   Additional information

   These authors are listed alphabetically

3. Pierre Simon Garcia

   1. Unit Stress Adaptation and Metabolism of Enterobacteria, Department of Microbiology, Université de Paris, UMR CNRS 2001, Institut Pasteur, Paris, France
   2. Institut Pasteur, Université de Paris, CNRS UMR6047, Evolutionary Biology of the Microbial Cell, Department of Microbiology, Paris, France

   Contribution

   These authors are listed alphabetically, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft

   Contributed equally with

   Francesca D'Angelo, Elena Fernández-Fueyo and Helena Shomar

   Competing interests

   No competing interests declared

   Additional information

   These authors are listed alphabetically

4. Helena Shomar

   Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands

   Contribution

   These authors are listed alphabetically, Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Francesca D'Angelo, Elena Fernández-Fueyo and Pierre Simon Garcia

   Competing interests

   No competing interests declared

   Additional information

   These authors are listed alphabetically

   "This ORCID iD identifies the author of this article:" 0000-0002-1090-2871

5. Martin Pelosse

   Univ. Grenoble Alpes, CNRS, CEA, IRIG, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

6. Rita Rebelo Manuel

   Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8068-2053

7. Ferhat Büke

   Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Siyi Liu

   Aix-Marseille Université-CNRS, Laboratoire de Chimie Bactérienne UMR 7283, Institut de Microbiologie de la Méditerranée, Institut Microbiologie Bioénergies Biotechnologie, Marseille, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Niels van den Broek

   Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

10. Nicolas Duraffourg

    Univ. Grenoble Alpes, CNRS, CEA, IRIG, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

11. Carol de Ram

    Department of Biotechnology, Delft University of Technology, Delft, Netherlands

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

12. Martin Pabst

    Department of Biotechnology, Delft University of Technology, Delft, Netherlands

    Contribution

    Investigation, Methodology, Resources, Supervision

    Competing interests

    No competing interests declared

13. Emmanuelle Bouveret

    Unit Stress Adaptation and Metabolism of Enterobacteria, Department of Microbiology, Université de Paris, UMR CNRS 2001, Institut Pasteur, Paris, France

    Contribution

    Investigation, Methodology, Resources

    Competing interests

    No competing interests declared

14. Simonetta Gribaldo

    Institut Pasteur, Université de Paris, CNRS UMR6047, Evolutionary Biology of the Microbial Cell, Department of Microbiology, Paris, France

    Contribution

    Conceptualization, Investigation, Methodology, Resources, Software, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7662-021X

15. Béatrice Py

    Aix-Marseille Université-CNRS, Laboratoire de Chimie Bactérienne UMR 7283, Institut de Microbiologie de la Méditerranée, Institut Microbiologie Bioénergies Biotechnologie, Marseille, France

    Contribution

    Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

16. Sandrine Ollagnier de Choudens

    Univ. Grenoble Alpes, CNRS, CEA, IRIG, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France

    Contribution

    Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-0080-6659

17. Frédéric Barras

    Unit Stress Adaptation and Metabolism of Enterobacteria, Department of Microbiology, Université de Paris, UMR CNRS 2001, Institut Pasteur, Paris, France

    Contribution

    Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing

    Contributed equally with

    Gregory Bokinsky

    For correspondence

    frederic.barras@pasteur.fr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3458-2574

18. Gregory Bokinsky

    Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands

    Contribution

    Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing

    Contributed equally with

    Frédéric Barras

    For correspondence

    g.e.bokinsky@tudelft.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7256-4492

• 1,398

  Page views

• 204

  Downloads

• 3

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.