As all previously characterized flavinylation systems contain genes that encode for an ApbE enzyme and a substrate that contains an FMN-binding domain, we reasoned that these features are indicative of flavinylation-mediated electron transfer. To identify flavinylation-mediated electron transfer systems, we searched for genes with ApbE (Pfam accession PF02424) or FMN-binding domains (Pfam accession PF04205) within a collection of 31,910 genomes that are representative of the genetic diversity of the prokaryotes (Parks et al., 2018; Parks et al., 2020). We found that 18,965 of bacterial genomes and 238 of archaeal genomes encode an FMN-binding domain-containing protein and/or an ApbE enzyme. To focus the search on extracytosolic electron transfer, we eliminated genes that lacked a computationally predicted signal peptide or lipidation site. This analysis provides evidence that ~50% of the bacterial (15,095) and ~4% of archaeal (63) genomes possess extracytosolic flavinylation components (Supplementary file 1). A phylogenetic analysis of the resulting dataset reveals that extracytosolic flavinylation components are broadly distributed across bacterial life – though strikingly underrepresented in Cyanobacteria and the candidate phyla radiation (Figure 2).

We next took advantage of the colocalization of genes for multi-subunit complexes on the genome to determine the prevalence and phylogenetic distribution of previously characterized flavinylated systems. We devised operational definitions in which the close proximity of a key gene in each system to an FMN-binding- or ApbE-containing gene was used to assign clusters to characterized systems (Supplementary file 2). These analyses revealed characterized systems in 8928 genomes broadly distributed across bacterial life (Figure 2).

In addition to assigning extracytosolic electron transfer functions, our analyses identified 6230 genomes that contained evidence of extracytosolic flavinylation but lacked a characterized extracytosolic electron transfer system. We also found that many genomes have multiple ApbE and/or FMN-binding genes and thus likely possess multiple extracytosolic electron transfer functions (Figure 1—figure supplement 1). As these observations suggested that a significant proportion of extracytosolic electron transfer systems remain uncharacterized, we next turned to the identification of uncategorized flavinylation-associated gene clusters.

Our initial approach only considered FMN-binding domains as potential flavinylation substrates. However, preliminary analyses identified 17% of genomes (2571 total) within our dataset that encoded an ApbE but no FMN-binding domain-containing protein – implying that some ApbE substrates lack an FMN-binding domain (Figure 3—figure supplement 1). To identify novel ApbE protein substrates, we examined the genomic context of these ‘orphan’ apbE genes, looking for gene cluster patterns conserved across multiple genomes (Supplementary file 1).

We observed that a subset of orphan apbE genes are associated with DUF2271 – an ~135 amino acid domain of unknown function. In some genomes a single gene encodes a protein with both ApbE and DUF2271 domains (e.g., NCBI accession RUL87804.1), but more commonly a separate DUF2271 gene is part of a gene cluster that includes an apbE. Consistent with DUF2271 serving as a flavinylation substrate, we identified a conserved [S/T]GA[S/T] motif within ApbE-associated DUF2271s (Figure 3A). We expressed the Amantichitinum ursilacus DUF2271 protein in Escherichia coli and found that it was flavinylated in the presence of its cognate ApbE (Figure 3B). Consistent with the conserved [S/T]GA[S/T] motif representing the sole ApbE target, we found that replacing the threonine at the predicted flavinylation site with an alanine abrogated flavinylation (Figure 3B).

We observed another subset of orphan apbE genes that are part of gene clusters that contain a gene annotated as DUF3570 – an ~420 amino acid domain of unknown function. Consistent with DUF3570 serving as a flavinylation substrate, we identified two conserved [S/T]GA[S/T] motifs within ApbE-associated DUF3570s (Figure 3C). Using the coexpression approach described above, we confirmed that the Chlorobium luteolum DUF3570 was flavinylated in the presence of its cognate ApbE (Figure 3D). Consistent with both of the identified [S/T]GA[S/T] motifs being modified, we found that replacing serines with alanines at both predicted flavinylation sites was required to abrogate flavinylation (Figure 3D).

These results suggest that DUF2271 and DUF3570 are novel ApbE substrates. Including DUF2271 and DUF3570 within our analyses significantly expanded the number and diversity of predicted ApbE substrates and decreased the number of orphan apbE genomes within the dataset from 17% to 4% (670 total) (Figure 3—figure supplement 1). Subsequent analyses thus likely account for a significant fraction of flavinylation substrates – though the fact that orphan apbE genomes remain suggests that less prevalent ApbE substrates remain unidentified.

We next sought to gain insight into novel roles of protein flavinylation. Characterized flavinylation-based electron transfer systems are minimally defined by a cytosolic electron donor, a transmembrane electron transfer apparatus, and the flavinylated extracytosolic electron acceptor – though, in principle, the direction of electron flow could be reversed. We reasoned that identification of cytosolic and membrane components was important for understanding the extracytosolic capability of uncharacterized flavinylated systems.

To clarify the role of flavinylation-associated gene clusters, we analyzed the genomic context of a subset of representative genes with DUF3570, DUF2271, FMN-binding, and ApbE domains that were not assigned to a characterized system in our initial analyses. Annotations for the five upstream and five downstream genes were manually reviewed. From these gene clusters, we identified five putative transmembrane electron transfer apparatuses that are present in 6183 genomes, including 3635 of the 6230 genomes that lack a characterized extracytosolic electron transfer system (Figure 4, Figure 4—figure supplement 1 and Supplementary file 2). The following subsections describe the organization and likely functions of these systems. Annotations of the gene clusters associated with the flavinylated systems are provided in Supplementary file 1.

We identified 2465 flavinylation gene clusters in 2153 genomes that encode an ‘NapH-like’ iron-sulfur cluster-binding protein (Pfam accession PF12801 or KEGG accession K19339) that contains several transmembrane helices (Figure 2 and Supplementary file 1). These proteins are homologous to NapH, the putative quinone-binding subunit of periplasmic nitrate reductase, and exhibit a broad phylogenetic distribution (Figure 4A; Brondijk et al., 2002). The majority of NapH-like proteins identified in our analysis contain an extracytosolic N-terminal FMN-binding domain (1926 gene clusters) whereas the remaining ones (539 gene clusters) are in a genomic locus with a second gene that encodes an FMN-binding domain-containing protein. These proteins likely receive electrons from a donor (probably a quinol in some cases) that are transferred via the iron-sulfur cluster across the membrane (Figure 4A).

Several lines of evidence suggest that many NapH-like proteins function with respiratory oxidoreductases. Previously characterized NapH-like proteins PceC and NosZ have been shown to be flavinylated and are part of gene clusters for organohalide and nitrous oxide reduction, respectively (Figure 1; Buttet et al., 2018; Zhang et al., 2017). These previously characterized NapH-like systems were identified within 1197 genomes within our dataset (Figure 2). We also identified NapH-like gene clusters with an extracytosolic nitrite reductase in 133 genomes or an ethanol oxidase in 172 genomes. Thus, while the reactions catalyzed by the majority of NapH-like systems remain unknown, this electron transfer apparatus seems to be modularly employed to facilitate electron transfer to reductases or from oxidases (Figure 4A). This modularity of NapH-oxidoreductase associations is further underscored by a phylogenetic analysis, which suggests that NapH-like associations with nirS and exaA evolved independently multiple times (Figure 4—figure supplement 2).

We identified 1797 flavinylation gene clusters in 1468 genomes that encode an ‘MsrQ-like’ (Pfam accession PF01794) protein (Supplementary file 1). These clusters are broadly conserved in Actinobacteria and are infrequently identified in other lineages (Figure 2 and Figure 4B). MsrQ-like proteins are predicted to have six transmembrane helices and two heme-binding sites. MsrQ-like proteins are homologous to MsrQ, the quinone-binding subunit of periplasmic methionine sulfoxide reductase (Gennaris et al., 2015). MsrQ-like proteins are also distantly related to eukaryotic proteins that function in transmembrane electron transfer, including NADPH oxidase and STEAP iron reductases (Zhang et al., 2013).

MsrQ-like gene clusters typically include apbE and an FMN-binding domain-containing gene. MsrQ-like proteins often contain a C-terminal NAD-binding domain (Pfam accession PF00175, 1437 gene clusters). In other cases, the MsrQ-like gene clusters include an NuoF-like protein (Pfam accession PF10589, 31 gene clusters), homologous to the NAD-binding subunit of complex I. At least a subset of MsrQ-like proteins thus likely use NAD(P)H as a cytosolic electron donor (Figure 4B). We also identified 153 MsrQ-like gene clusters that encode a protein homologous to the eukaryotic ferrous iron transporter VIT1 (Pfam accession PF01988). This association suggests that some MsrQ-like systems function as assimilatory iron reductases that facilitate iron uptake through VIT1 (Figure 4B).

We identified 3220 flavinylation gene clusters in 3040 genomes that encode a PepSY-like (Pfam accessions PF03929 and PF16357) protein (Supplementary file 1). PepSY-like proteins contain three transmembrane helices and are broadly distributed throughout Gram-negative bacteria (Figures 2 and 4C). PepSY-like gene clusters frequently encode an ApbE enzyme, the flavinylation substrate DUF2271 (Pfam accession PF10029, 2833 clusters), and a secreted DUF4198 (Pfam accession PF10670, 1500 clusters) protein. Little is known about the structure or function of PepSY-like proteins. The only characterized PepSY-like homolog, Vibrio cholerae VciB (which is not associated with identified flavinylation components), was reported to possess extracytosolic iron reductase activity (Peng and Payne, 2017) an — an activity consistent with transmembrane electron transfer activity. We further identified 1077 PepSY-like genes that have a NAD-binding domain (Pfam accession PF00175), suggesting that a subset of these proteins use cytosolic NADH as an electron donor (Figure 4C).

Several observations implicate a role for identified PepSY-like gene clusters in iron reduction and assimilation. First, a functional connection to MsrQ-like proteins is suggested by our observation that 108 gene clusters contain both PepSY-like and MsrQ-like genes. Second, 167 PepSY-like gene clusters contain a VIT1 (Pfam accession PF01988) ferrous iron transporter (Figure 4C). Finally, PepSY-like gene clusters have been shown to be repressed by Fur (the primary transcription regulator that responds to iron limitation) in Shewanella oneidensis and Caulobacter crescentus (da Silva Neto et al., 2013; Wan et al., 2004). Moreover, in some cases, PepSY-like proteins may be actively involved in the extraction of siderophore-bound iron, as periplasmic reduction has been shown to be important for the uptake of siderophore-bound iron and 231 PepSY-like gene clusters encode a TonB receptor (Pfam accession PF03544) related to well-characterized outer membrane siderophore transporters (Figure 4C; Liu et al., 2018a; Manck et al., 2020).

We identified 285 flavinylation gene clusters in 275 genomes that contain a DsbD protein (Pfam accession PF02683) (Figure 2 and Supplementary file 1). DsbD is part of a well-studied transmembrane protein family that uses cysteine pairs to transfer electrons across the membrane (Krupp et al., 2001; Missiakas et al., 1995). DsbD family proteins generally transfer electrons onto extracytosolic thioredoxin-like proteins, which in turn use a similar thiol-disulfide exchange chemistry to promote extracytosolic redox-dependent activities, such as oxidative protein folding (Cho and Collet, 2013).

The DsbD gene clusters identified in our analyses typically include genes for ApbE, the flavinylation substrate DUF3570 (Pfam accession PF12094, 227 clusters), a thioredoxin-like protein (Pfam accession PF13899, 183 clusters), and a DUF4266 protein (Pfam accession PF14086, 221 clusters). DUF4266 is a small secreted protein with a highly conserved C-terminal cysteine – (any amino acid) – cysteine (CXC) sequence motif that may undergo redox cycling.

We also detected 316 gene clusters that encode ApbE, DUF3570, thioredoxin-like, and DUF4266 but do not colocalize on the genome with DsbD – implying that this system may be more common than is revealed by the DsbD-dependent analysis presented in Figure 2. These observations suggest that a hybrid thioredoxin-like/flavinylation-based system receives electrons from DsbD in some bacteria (Figure 4D). While the function of DUF3570-based electron transfer remains uncertain, 25 gene clusters contain a VIT1 ferrous iron transporter and thus may play a role in iron assimilation (Supplementary file 1).

We identified 127 flavinylation gene clusters in 127 genomes, primarily from the Actinobacteria and candidate phyla radiation, with evidence of partial NQR or RNF systems (Figure 2). As shown in Figure 1, the NQR and RNF complexes contain a common core apparatus with two transmembrane electron transfer pathways that together achieve a semicircular electron flow (Juárez et al., 2010; Steuber et al., 2014). A first path takes electrons from the cytosol to the extracytosolic FMN-binding domain, while a second path takes electrons from the FMN-binding domain back to a cytosolic substrate. NQR/RNF-like gene clusters encode for components associated with a single electron transfer pathway and thus likely function for unidirectional electron flow (Figure 4E).

NQR/RNF-like gene clusters encode a protein with an N-terminal membrane domain that is homologous to NqrB/RnfD and a cytosolic C-terminal domain homologous to the NAD-binding domain NqrF (Pfam accession PF00175). NqrB/RnfD is flavinylated by ApbE and is thought to play a role in electron transfer across the membrane (Juárez et al., 2010). This system presumably uses NAD(P)H as a cytosolic electron donor for electron transfer to an extracytosolic FMN-binding domain. Six NQR/RNF-like gene clusters contain a VIT1 ferrous iron transporter, suggesting that some of these systems function in iron assimilation (Supplementary file 1).

We next asked about the function of flavinylation-associated gene clusters that lack a core transmembrane electron transfer system. This category of extracytosolic proteins presumably relies on proteins encoded elsewhere on the genome to link up with membrane electron pools. Inspection of these gene clusters led to the identification of two noteworthy examples that are described in the following subsections.

A gene cluster that includes the ferrous iron-binding protein P19 (Pfam accession PF10634) and the FTR1 (Pfam accession PF03239) iron transporter has previously been shown to encode a mechanistically uncharacterized system involved in iron assimilation (Chan et al., 2010). We identified 260 P19 gene clusters, mostly in Gram-positive bacteria, that contain a gene with an FMN-binding domain (Figure 5—figure supplement 1A and B and Supplementary file 1). Interestingly, many Gram-negative P19 gene clusters lack an FMN-binding gene but contain an additional thioredoxin-like gene (Liu et al., 2018b; Figure 5—figure supplement 1B). The role of the FMN-binding/thioredoxin-like protein in these systems has not been defined but, similar to other assimilatory iron reductases, could engage in redox chemistry to facilitate iron uptake. These observations thus establish another connection between flavinylation and microbial iron assimilation and highlight functional parallels between flavinylation and thioredoxin-like extracytosolic electron transfer.

Fumarate reductase-like enzymes (members of the Pfam accession PF00890 enzyme superfamily) are a group of evolutionarily related proteins that catalyze a variety of redox reactions – though phylogenetic analyses suggest that substrates for many members of the superfamily remain unknown (Jardim-Messeder et al., 2017; Light et al., 2019). We observe that fumarate reductase-like enzymes often contain an FMN-binding domain (3070 proteins encoded by 2979 distinct gene clusters in 1236 genomes) or are part of gene clusters (±2 genes) that contain an FMN-binding domain (189 gene clusters in 171 genomes) (Supplementary file 1). Several characterized fumarate reductase-like enzymes (including fumarate, urocanate, and methacrylate reductases) are extracytosolic and function in respiration (Bogachev et al., 2012; Light et al., 2019; Mikoulinskaia et al., 1999). The Listeria monocytogenes fumarate reductase and the S. oneidensis urocanate reductase have been shown to be flavinylated (Bogachev et al., 2012; Light et al., 2019). In both cases, the flavinylation motif is thought to facilitate electron transfer from electron transport chain components encoded elsewhere on the genome to the enzyme active site (Kees et al., 2019; Light et al., 2019). These observations thus suggest that FMN-binding domains mediate electron transfer from membrane components to a prevalent class of extracytosolic reductases and highlight another connection between flavinylation and respiration.

A comparison of extracytosolic electron transfer systems identified in our analyses revealed multiple instances in which cytochrome and flavinylated electron transfer components appear to be performing similar electron transfer roles within related systems. For example, we identified a flavinylated NapH-like protein that is well situated to mediate electron transfer from the extracytosolic alcohol oxidase to the electron transport chain, where a cytochrome c protein has been shown to play this role in other microbes (Figure 5A; Schobert and Görisch, 1999).

Another example of this dynamic is provided by a comparison of fumarate reductases. The extracytosolic S. oneidensis fumarate reductase contains an N-terminal multi-heme cytochrome c domain (Pfam accession PF14537), whereas the related L. monocytogenes enzyme uses an FMN-binding domain to connect to the electron transport chain (Figure 5B; DiChristina and DeLong, 1994; Light et al., 2019). Different microbes thus seem to utilize flavinylation and cytochrome domains in a similar fashion to link respiratory enzymes to electron transport chains.

We further found the type of flavinylation/cytochrome substitution observed for fumarate reductase to be indicative of a broader pattern within fumarate reductase-like enzymes. We identified 147 gene clusters within 108 genomes that encode separate fumarate reductase-like and cytochrome proteins and 879 genes within 360 genomes that encode a single protein with both fumarate reductase-like and cytochrome domains (Supplementary file 3). Many genomes encode multiple fumarate reductase-like paralogs and 99 genomes encode both cytochrome- and flavinylation-associated enzymes (Supplementary file 3). This dynamic is exemplified by S. oneidensis, which in addition to the mentioned cytochrome-associated fumarate reductase contains a flavinylated urocanate reductase that also exhibits a respiratory function (Figure 5C; Bogachev et al., 2012). Broadly similar flavinylation- and cytochrome-based extracytosolic electron transfer mechanisms thus seemingly coexist within some microbes.

Multi-heme cytochromes are proteins that bind multiple hemes to achieve longer distance extracytosolic electron transfer (Blumberger, 2018). Among other functions, multi-heme proteins are important for transferring electrons across the cell envelope to insoluble electron acceptors that are inaccessible within the cytosolic membrane (El-Naggar et al., 2010; Wang et al., 2019). We have observed that extracytosolic proteins with multiple FMN-binding domains are also common (2081 proteins in 1530 genomes), particularly in Gram-positive bacteria, and contain as many as 13 predicted flavinylation sites (Figure 6A and B and Supplementary file 1). Multi-cofactor binding properties thus establish another parallel between cytochrome and flavinylation-based electron transfer.

Gene cluster analyses provide some insight into the basis of multi-flavinylated protein electron transfer. We find that multi-flavinylated proteins are often associated with established transmembrane electron transfer components and thus likely receive electrons through conventional mechanisms (Figure 6C). We observe that some clusters contain large unannotated proteins with putative cell wall-binding domains – such as SLH (Pfam accession PF00395), Rib (Pfam accession PF08428), FIVAR (Pfam accession PF07554), or LysM (Pfam accession PF01476) – and are thus likely involved in redox chemistry within the cell wall or at the cell surface (Figure 6D). We also identified 157 clusters that encode a multi-flavinylated protein and additional proteins with FMN-binding domains (Figure 6D and E). These clusters encode as many as five proteins with FMN-binding domains and frequently contain multiple multi-flavinylated proteins (Figure 6D). These observations suggest that some multi-flavinylated proteins are part of multi-step electron transfer pathways and may form elaborate multi-subunit complexes that span the cell wall.

The role of multi-flavinylated gene clusters is generally unclear, with only a minority of clusters providing limited functional clues. We identified a subset of clusters that encode proteins with multiple fumarate reductase-like domains that likely establish an unusual multi-functional reductase platform (Figure 6D and G). We also identified a number of RNF clusters that contain multi-flavinylated RnfGs (the extracytosolic flavinylated subunit in RNF complexes) (Figure 6C). These multi-flavinylated RNF complexes likely provide a second electron transfer pathway that facilitates transfer to alternative extracytosolic acceptors. This type of bifurcated electron transfer would be similar to a multi-heme cytochrome-based transfer mechanism recently suggested in studies of a methanogen RNF (Holmes et al., 2019). Interestingly, we also identified a subset of multi-flavinylated RnfGs in the family Christensenellales that contain a multi-heme cytochrome domain and thus may assume additional functions relevant for cytochrome-based electron transfer (Figure 6H). While much remains to be learned, these preliminary observations are consistent with multi-flavinylated proteins establishing elaborate and functionally diverse electron transfer pathways.

The 30,238 bacterial and 1672 archaeal genomes from the GTDB (release 05-RS95 of July 17, 2020) were downloaded with the taxonomy and the predicted protein sequences of the genomes (Parks et al., 2020).

Protein sequences were functionally annotated based on the accession of their best Hmmsearch match (version 3.3) (E-value cut-off 0.001) (Eddy, 1998) against the KOfam database (downloaded on February 18, 2020) (Aramaki et al., 2020). Domains were predicted using the same Hmmsearch procedure against the Pfam database (version 33.0) (Mistry et al., 2021). SIGNALP (version 5.0) was run to predict the putative cellular localization of the proteins using the parameters -org arch in archaeal genomes and -org gram +in bacterial genomes (Almagro Armenteros et al., 2019). Prediction of transmembrane helices in proteins was performed using TMHMM (version 2.0) (default parameters) (Krogh et al., 2001).

The five genes downstream and upstream of an ApbE, FMN-binding domain, DUF3570 or DUF2271 encoding genes were first collected. Only gene clusters with at least one signal peptide or lipidation site predicted in one of the four target genes were considered for further analysis and were referred to as "flavinylation-associated gene clusters." The flavinylation-associated gene clusters were then assigned to 1 of the 10 flavinylated systems based on the presence of key genes reported in Supplementary file 2. The RNF system was considered present if a Na⁺-translocating ferredoxin:NAD⁺ oxidoreductase subunit B (RnfB, KEGG accession K03616) was encoded within a flavinylation-associated gene cluster. The nitrous oxide reduction system (Nos) was considered present if a nitrous oxide reductase (NosZ, KEGG accession K00376) was encoded within a flavinylation-associated gene cluster. The organohalide respiration system was defined by the presence of a PceA enzyme (Pfam accession PF13486) encoded within a flavinylation-associated gene cluster. The extracellular electron transfer system was considered present if a NADH dehydrogenase (KEGG accession K03885) with a transmembrane helix was encoded within a flavinylation-associated gene cluster. The NQR was defined by the Na⁺-transporting NADH:ubiquinone oxidoreductase subunit F (NqrF, KEGG accession K00351) encoded within a flavinylation-associated gene cluster. The NapH-like was considered present if an ‘NapH-like’ iron-sulfur cluster-binding protein (Pfam accession PF12801 or KEGG accession K19339) was encoded within a flavinylation-associated gene cluster. NapH-like gene clusters that encoded a NirS (KEGG accession K15864) were identified as containing a nitrite reductase. NapH-like gene clusters that encoded an ExaA enzyme (KEGG accession K00114) were identified as containing an alcohol oxidase. The MsrQ-like system was defined by the presence of ‘MsrQ-like’ (Pfam accession PF01794) gene within a flavinylation-associated gene cluster. The PepSY-like system was defined by a PepSY-like (Pfam accessions PF03929 and PF16357) protein encoded within a flavinylation-associated gene cluster. The DsbD system was considered present if a DsbD protein (Pfam accession PF02683) was encoded within a flavinylation-associated gene cluster. Finally, the NQR/RNF-like system was considered present if a flavinylation-associated gene cluster encoded a protein with an N-terminal membrane domain that is homologous to NqrB/RnfD (Pfam accession PF03116) and a cytosolic C-terminal domain homologous to the NAD-binding domain NqrF (Pfam accession PF00175).

The ‘NapH-like’ iron-sulfur cluster-binding protein tree was built as follows. Sequences were aligned using MAFFT (version 7.390) (–auto option) (Katoh and Standley, 2016). The alignment was further trimmed using Trimal (version 1.4.22) (–gappyout option) (Capella-Gutierrez et al., 2009). Tree reconstruction was performed using IQ-TREE (version 1.6.12) (Nguyen et al., 2015), using ModelFinder to select the best model of evolution (Kalyaanamoorthy et al., 2017) and with 1000 ultrafast bootstrap (Hoang et al., 2018).

A maximum-likelihood tree was calculated based on the concatenation of 14 ribosomal proteins (L2, L3, L4, L5, L6, L14, L15, L18, L22, L24, S3, S8, S17, and S19). Homologous protein sequences were aligned using MAFFT (version 7.390) (--auto option) (Katoh and Standley, 2016) and alignments refined to remove gapped regions using Trimal (version 1.4.22) (--gappyout 570 option) (Capella-Gutierrez et al., 2009). The protein alignments were concatenated with a final alignment of 9152 genomes and 2850 positions. Tree reconstruction was performed using IQ-TREE (version 1.6.12) (Nguyen et al., 2015). A LG + I + G4 model of evolution was selected using ModelFinder (Kalyaanamoorthy et al., 2017) and 1000 ultrafast bootstraps were performed (Hoang et al., 2018).

Sequences of flavinylation-associated DUF3570 and DUF2271 proteins were aligned using EMBL-EBI Clustal Omega Multiple Sequence Alignment (Madeira et al., 2019). Sequence logos of the flavinylation sites shown in Figure 3 were generated in R using the ‘ggseqlogo’ package (Wagih, 2017).

A synthetic construct of the signal peptide-truncated A. ursilacus IGB-41 DUF2271 gene (NCBI accession WP_053936890.1) was subcloned into the pMCSG53 vector. A second construct contained a ribosome-binding site and the signal peptide-truncated cognate apbE (NCBI accession WP_053936888.1) just downstream of the DUF2271 gene. A similar cloning strategy was used for the C. luteolum DSM 273 DUF3570 (NCBI accession ABB24424.1) and its cognate apbE (NCBI accession ABB24423.1). Point mutations of the DUF3570- and DUF2271-encoding genes were generated using the NEB Q5 Site-Directed Mutagenesis Kit. Briefly, overlapping primers containing mutated sequences were used in a PCR using pMCSG53::DUF3570 or pMCSG53::DUF2771 as DNA template to generate expression vectors containing respective mutant sequences. Plasmids containing wild-type protein sequences were removed using digestion with the DpnI enzyme, which only acts on methylated DNA sequences.

Sequence verified plasmids were transformed in E. coli BL21 cells (Rosetta DE3, Novagen). A single colony of each expression strain was isolated on Luria-Bertani (LB) agar supplemented with carbenicillin (100 μg/mL) and inoculated into 15 mL of LB. Following overnight growth, cultures were diluted in 500 mL of brain heart infusion broth to a final OD600 of 0.1. When the OD600 reached 0.7–1 protein overexpression was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. The culture was then incubated overnight at 25°C with aeration and collected by centrifugation (7000 × g for 15 min). After removing the supernatant, cells were washed in 30 mL of lysis buffer (5:1 v/weight of pellet; 300 mM NaCl, 1 mM dithiothreitol, 10 mM imidazole, 1 mM ethylenediaminetetraacetic acid [EDTA], and 50 mM Tris-HCl pH = 7.5). Pelleted cells were stored at −80°C overnight, resuspended in lysis buffer, lysed by sonication (8 × 30 s pulses), and cleared by centrifugation (40,000 × g for 30 min).

For the purification of A. ursilacus DUF2271, cell lysate was collected and loaded onto a 5 mL HisTrapTM column (GE Healthcare) using the ÄKTA Pure FPLC. Protein was eluted using an imidazole concentration gradient with a maximal concentration of 500 mM. Protein concentrations of eluted fractions containing His₆-tagged DUF2271 were measured on a DeNovix DS-11 FX+Spectrophotometer based on protein molar mass and extinction coefficient and standardized to 0.4 mg/mL.

Initial observations revealed that the majority of expressed C. luteolum DUF3570 was present in the lysed cell pellet. To purify C. luteolum DUF3570, the cell pellet was washed with wash buffer (10:1 v/weight of pellet; 100 mM Tris-HCl pH = 7.5, 300 mM NaCl, 2 mM 2-mercaptoethanol, 1 M guanidine-HCl, 1 mM EDTA, and 2% w/v Triton X-100) and centrifuged at 40,000 × g for 30 min. The supernatant was then discarded and this washing step was repeated until the supernatant was clear. The cell pellet was then resuspended in wash buffer (10:1 v/weight of pellet) without guanidine-HCl and Triton X-100 and centrifuged (40,000 × g for 15 min) to remove guanidine-HCl and Triton X-100. After supernatant was discarded, the pellet was resuspended in extraction buffer (4:1 v/weight of pellet; 100 mM Tris-HCl pH = 7.5, 300 mM NaCl, 10 mM imidazole, 6 M guanidine-HCl, 2 mM 2-mercaptoethanol, and 1 mM EDTA) and protein was denatured by overnight rotator mixation. Supernatant containing denatured DUF3570 was collected by centrifugation (20,000 × g for 30 min).

Subsequent C. luteolum DUF3570 purification steps were conducted on a Ni-NTA column. Specifically, 4 mL of Ni-NTA slurry (Nuvia IMAC Resin, 25 mL) were added into a glass chromatography column (Econo-Column, 1.0 × 10 cm²). The column was prepared with 10 mL of column wash buffer and the samples containing denatured DUF3570 were then loaded. Bound protein was eluted using 5 mL of modified column wash buffer containing 50, 100, 200, or 500 mM of imidazole. To prevent guanidine-HCl from forming precipitates with SDS in following steps, eluted samples were mixed with 100% ethanol (9:1 v/v) and incubated at −20°C for 10 min. After centrifugation at 21,100 × g for 5 min and removal of the supernatant, the pelleted protein was washed once with 90% ethanol. The sample was centrifuged again and the pellet was resuspended in diH₂O. Protein concentrations of eluted fractions containing His₆-tagged DUF3570 were measured as described above and standardized to 1.2 mg/mL.

Purified and normalized DUF2271 and DUF3570 were loaded and separated on a 12% Bis-Tris gel. Prior to gel staining, flavinylated bands were visualized under UV due to the UV resonance of the flavin molecule. Visualizations of Coomassie blue stained protein were captured with an iBright 1500 gel imager.