Comparative Genomic and Genetic Evidence on a Role for the OarX Protein in Thiamin Salvage

Abstract

Salvage pathways for thiamin and its thiazole and pyrimidine moieties are poorly characterized compared to synthesis pathways. A candidate salvage gene is oarX, which encodes a short-chain dehydrogenase/reductase. In diverse bacteria, oarX clusters on the chromosome with genes of thiamin synthesis, salvage, or transport and is preceded by a thiamin pyrophosphate riboswitch. Thiamin and its moieties can undergo oxidations that convert a side-chain hydroxymethyl group to a carboxyl group, or the thiazole ring to a thiazolone, causing a loss of biological activity. To test if OarX participates in salvage of the carboxyl or thiazolone products, we used a genetic approach in Corynebacterium glutamicum ATCC 14067, which is auxotrophic for thiamin’s pyrimidine moiety. This strain could not utilize the pyrimidine carboxyl derivative. This excluded a role in salvaging this product and narrowed the function search to metabolism of the carboxyl or thiazolone derivatives of thiamin or its thiazole moiety. However, a ΔthiG (thiazole auxotroph) strain was not rescued by any of these derivatives. Nor did deleting oarX affect rescue by the physiological pyrimidine and thiazole precursors of thiamin. These findings reinforce the genomic evidence that OarX has a function in thiamin metabolism and rule out five logical possibilities for what this function is.

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Comparative Genomic and Genetic Evidence on a Role for the OarX
Protein in Thiamin Salvage
Edmar R. Oliveira-Filho,*Dmitry A. Rodionov, and Andrew D. Hanson
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ABSTRACT: Salvage pathways for thiamin and its thiazole and
pyrimidine moieties are poorly characterized compared to synthesis
pathways. A candidate salvage gene is oarX, which encodes a short-
chain dehydrogenase/reductase. In diverse bacteria, oarX clusters
on the chromosome with genes of thiamin synthesis, salvage, or
transport and is preceded by a thiamin pyrophosphate riboswitch.
Thiamin and its moieties can undergo oxidations that convert a
side-chain hydroxymethyl group to a carboxyl group, or the
thiazole ring to a thiazolone, causing a loss of biological activity. To
test if OarX participates in salvage of the carboxyl or thiazolone products, we used a genetic approach in Corynebacterium glutamicum
ATCC 14067, which is auxotrophic for thiamin’s pyrimidine moiety. This strain could not utilize the pyrimidine carboxyl derivative.
This excluded a role in salvaging this product and narrowed the function search to metabolism of the carboxyl or thiazolone
derivatives of thiamin or its thiazole moiety. However, a ΔthiG (thiazole auxotroph) strain was not rescued by any of these
derivatives. Nor did deleting oarX aect rescue by the physiological pyrimidine and thiazole precursors of thiamin. These findings
reinforce the genomic evidence that OarX has a function in thiamin metabolism and rule out five logical possibilities for what this
function is.
1. INTRODUCTION
Thiamin (vitamin B1), in its active pyrophosphate form, is the
cofactor for various enzymes that make or break carbon−
carbon bonds. Thiamin is essential in all organisms, although
not all can synthesize it, which makes thiamin biosynthesis and
metabolism pathways attractive drug targets.
1−3
Bacterial
biosynthesis pathways for thiamin and its constituent 4-
amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 5-(2-
hydroxyethyl)-4-methylthiazole (HET) moieties are well-
known; the widely distributed canonical pathways have no
missing steps or enzymes (Figure 1).
4−6
Salvage pathways are
less well-known, in part because there are at least 14 potential
degradation products of thiamin itself and its moieties.
5,7−10
As
some of these products are toxic,
11−13
they may need
detoxification; even less is known about detoxification routes
than about salvage.
8,9,13
Because all of the genes of thiamin biosynthesis have been
identified in various organisms, genes of unknown function
that comparative genomics evidence associates with thiamin
are likely to participate in salvage or detoxification, not
biosynthesis. One such gene, oarX, encoding a short-chain
dehydrogenase/reductase broadly similar to the 3-oxoacyl-
(acyl-carrier protein) reductase FabG, has long been known to
cluster on the chromosome with thiamin synthesis and salvage
genes and to be downstream of a thiamin pyrophosphate
(TPP) riboswitch.
4,14,15
Binding of TPP to this riboswitch
represses transcription and translation of the downstream
coding sequence.
14
Also, the SEED comparative genomics
database
16
has long computationally flagged a predicted FMN-
dependent monooxygenase gene (henceforth: fmnO) as being
in an operonic arrangement with oarX.
Rodionov et al. suggested 20 years ago that the “data seem
to be suciently strong to warrant experimental analysis of the
functional role of the oarX gene product in thiamin
metabolism”.
4
As many more genomes have been sequenced
since then and an association with f mnO has emerged, we
undertook a bioinformatic reanalysis of the function of oarX
and a genetic analysis using Corynebacterium glutamicum as a
test organism. C. glutamicum was chosen because it has an
oarX-fmnO operon preceded by a TPP riboswitch (Figure 2)
and is genetically tractable. We tested for potential salvage or
detoxification activities based on the likelihood that some of
these remain to be discovered (see above), on the fact that�
like known salvage genes�oarX occurs in some organisms but
not others,
4
and on the evolutionary premium on salvaging
thiamin because it is so energetically expensive to synthesize de
novo.
5,10,17,18
Further, we focused on the main five “orphan”
degradation products that are known to be excreted in
Received: April 11, 2024
Revised: May 30, 2024
Accepted: June 10, 2024
Published: June 21, 2024
Article
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mammalian urine
19,20
and formed by microbes
7,21
but whose
metabolic fates are unknown (Figure 1). In three of these,
thiamin acetic acid, 4-methyl-5-thiazoleacetic acid (MTA), and
2-methyl-4-amino-5-pyrimidinecarboxylic acid (PCA), a side-
chain primary alcohol group, has been oxidized to a carboxyl
group. In the other two, oxo-thiamin (the oxo derivative of
thiamin) or oxo-HET (the oxo derivative of the thiazole
precursor HET), the thiazole ring has been oxidized to a
thiazolone. Each of these compounds could theoretically be
recycled to thiamin or its HET or HMP precursor; after
activation, a carboxyl group can be reduced via an aldehyde to
an alcohol,
22
and ring oxo groups can likewise be reduced.
23
Salvage pathways for these compounds are accordingly
predicted to include dedicated reductases; OarX is a plausible
candidate for such a role.
2. MATERIALS AND METHODS
2.1. Bioinformatics Tools and Databases. The analyzed
microbial genomes were downloaded from GenBank
24
and
from the Bacterial and Viral Bioinformatics Resource Center
(BV-BRC) (https://www.bv-brc.org/), formerly known as
PATRIC.
25
The gene neighborhood and distribution analysis,
functional gene assignments, and metabolic subsystem analysis
for thiamin metabolism genes were performed using the SEED
database and web tools.
16
The thiamin biosynthesis subsystem
in SEED was adapted from the previously developed
subsystem for 2228 microbial genomes representing the
human gut microbiome
26
and was further enriched by
additional genomes encoding homologues of the oarX and
fmnO genes from the BV-BRC genomic database. Orthologs
were identified as bidirectional best hits using protein BLAST.
The analyzed functional roles of known and predicted thiamin
Figure 1. Canonical bacterial thiamin synthesis and salvage pathways. Enzymes: ThiD, hydroxymethylpyrimidine/phosphomethylpyrimidine
kinase; ThiE, thiamin phosphate synthase; ThiG, thiazole synthase; ThiL, thiamin phosphate kinase; ThiN, thiamin pyrophosphokinase; ThiO,
glycine oxidase; ThMPase, thiamin monophosphatase. Dehydroglycine can also be derived from tyrosine via ThiH. Compounds: AIR, 5-
aminoimidazole ribonucleotide; HET, 5-(2-hydroxy-ethyl)-4-methylthiazole; HMP, 4-amino-5-hydroxymethyl-2-methylpyrimidine; HMP-P, HMP
monophosphate; HMP-PP, HMP pyrophosphate; MTA, 4-methyl-5-thiazoleacetic acid; oxo-HET, oxo derivative of HET; oxo-thiamin, oxo
derivative of thiamin; PCA, 2-methyl-4-amino-5-pyrimidinecarboxylic acid; ThiS-COSH, ThiS thiocarboxylate; HET-P, HET-phosphate. Damage
products of thiamin and its thiazole or pyrimidine precursors are shown in blue and tied to the corresponding physiological compound with dashed
blue lines.
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metabolism enzymes and uptake transporters are summarized
in Supplementary Table 1, which also includes amino acid
sequences of the identified OarX and FmnO proteins. The
taxonomic distribution of TPP riboswitches was taken from the
RegPrecise database (https://regprecise.lbl.gov/)
27
for the
RF00059 family as described in the comparative genomics
study of riboswitch regulons in bacterial genomes.
28
Additional
TPP riboswitch sequences in target genomes were predicted
using Riboswitch Scanner.
29
Phylogenetic trees were con-
structed with MEGA X
30
and the Robust Phylogenetic
Analysis tool (https://www.phylogeny.fr/).
31
2.2. C. glutamicum Gene Knockouts. Corynebacterium
glutamicum ATCC 14067 (DSM 20411) was obtained from
DSMZ (Braunschweig, Germany). Knockouts were obtained
via gene displacement using pk18sB
32
obtained from Addgene.
Primers were designed based on the ATCC 14067 genome
sequence (GenBank Accession NZ_CP022614.1).
33
Molecular
manipulations followed standard protocols
34
or kit manufac-
turers’ instructions. Phusion High-Fidelity DNA Polymerase
(Thermo Fisher Scientific, Waltham, MA) was used to amplify
DNA sequences in a T100 Thermal Cycler (Bio-Rad
Laboratories, Hercules, CA). Primer sequences are listed in
Supplementary Table 2. PCR products were gel-purified using
GeneJET Gel Extraction Kits (Thermo Fisher Scientific,
Waltham, MA) and assembled using NEBuilder HiFi DNA
Assembly or KLD Enzyme Mix (New England Biolabs,
Ipswich, MA). Assembly products were inserted into
Escherichia coli Top10 via electroporation using an E. coli
Pulser apparatus (Bio-Rad Laboratories, Hercules, CA).
Candidate clones (kanamycin-resistant) were then screened
by PCR to select those with successful ligation. Recombinant
plasmids were purified using GeneJET Plasmid Miniprep Kits
(Thermo Fisher Scientific, Waltham, MA) and sequence-
verified. pK18sB harboring C. glutamicum ATCC 14067 thiG
or oarX-fmnO deletion cassettes containing thiG or oarX-fmnO
800-bp flanking sequences (homology arms) were transformed
into C. glutamicum as described.
35
Briefly, C. glutamicum was
grown overnight on BHI medium and transferred to modified
Epo medium, starting OD600 = 0.3. When the culture reached
OD600 = 1.0, it was cooled on ice for 15 min, then cells were
harvested by centrifugation (4 °C, 4000 ×g, 10 min), washed
four times with ice-cold 10% glycerol (w/v), and resuspended
in 0.4% of the initial volume of 10% glycerol. For electro-
poration, 80 μL of cell suspension were mixed with ∼2000 ng
of plasmid DNA and given a single pulse at 12.5 kV/cm for 3−
5 ms. Cells were resuspended in 915 μL of BHIS medium,
incubated at 46 °C for 6 min, recovered for 2 h at 30 °C with
shaking at 250 rpm, and plated on LBHIS medium containing
Figure 2. Examples of chromosomal clustering of genes encoding OarX, FmnO, and acyl-CoA synthetase (ACS) with a thiamin pyrophosphate
riboswitch (R) and thiamin synthesis, salvage, and transport genes. ThiD, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase;
ThiE, thiamin phosphate synthase; ThiF, ThiS adenylyltransferase; ThiG, thiazole synthase; ThiM, HET kinase; ThiO, glycine oxidase; ThiS, sulfur
carrier protein; ThiV, predicted thiamin precursor transporter; YkoCDE, ECF thiamin transporter components; TenA, thiamin pyrimidine moiety
salvage enzyme; TenI, thiazole tautomerase; and Tbp, thiamin-binding protein.
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10 μg/mL kanamycin. The plates were incubated at 30 °C
until colonies appeared (up to 10 days). Following sucrose
selection (four passages on BHI + 10% sucrose), single
colonies were analyzed by colony PCR to identify those with
successful recombination.
2.3. Chemicals. Thiamin-related compounds were ob-
tained from the following sources: thiamin, Sigma-Aldrich (St.
Louis, MO); HMP, Ark Pharm Inc. (Arlington Heights, IL);
HET, Sigma-Aldrich; PCA, AstaTech Inc. (Bristol, PA), MTA,
AstaTech Inc.; oxo-thiamin (Toronto Research Chemicals,
North York, ON, Canada); oxo-HET, Toronto Research
Chemicals; thiamin acetic acid (hydrobromide bromide form),
Enamine Ltd. (Kyiv, Ukraine). Tests confirmed that bromide
ions did not aect growth at the maximum concentration (2
μM) present in the medium in experiments with thiamin acetic
acid. Aqueous stock solutions of thiamin-related compounds
(10−100 μM) were filter-sterilized and added to media to give
the concentrations indicated in the text.
2.4. Growth Media and Culture Conditions. E. coli
cultures were grown at 37 °C with shaking at 250 rpm in LB
medium (composition in g/L: Tryptone, 10; yeast extract, 5;
NaCl, 10). C. glutamicum precultures were grown at 30 °C
with shaking at 250 rpm in BHI medium (BD, Franklin Lakes,
NJ). Competent cells were prepared using modified Epo
medium (composition in g/L: Tryptone, 10; yeast extract, 5;
NaCl, 10; glycine, 25; Tween 80, 0.1%).
36
Cells were
recovered after transformation in BHIS medium (BHI, 18.5
g/L, sorbitol, 91 g/L). Recombinants were selected on LBHIS
agar (composition in g/L: Tryptone, 5; NaCl, 5; yeast extract,
2.5; BHI, 18.5; sorbitol, 91; agar, 18; pH 7.2). Growth
experiments were conducted on CGXII medium, modified
from the literature,
36
with the following composition in g/L:
(NH4)2SO4, 20; urea, 5; KH2PO4, 1; K2HPO4, 1;
MgSO4.7H2O, 0.25; MOPS, 42; glucose, 40; CaCl2, 0.013;
biotin, 0.0002, FeSO4, 0.001; and trace elements solution.
37
3. RESULTS AND DISCUSSION
3.1. Sequence-Based Analyses of OarX and FmnO
Proteins. Sequence-based analyses reinforced the original
inference
4
that OarX proteins dier in function from FabG
since they occur in genomes that encode a canonical FabG,
share only ∼30% sequence identity and ∼50% similarity with
FabG proteins, and belong to a dierent phylogenetic clade
(Supplementary Figure 1). Nothing beyond membership of a
short-chain dehydrogenase/reductase subgroup that includes
FabG and other carbonyl reductases could be gleaned from
OarX amino acid sequences. Similarly for FmnO, sequence and
phylogenetic analysis of FmnO proteins and FMN-dependent
monooxygenases with known activities indicated only that
FmnOs share at most ∼45% sequence identity with other
FMN-dependent monooxygenase family members and that
they form a separate clade (Supplementary Figure 2).
3.2. Comparative Genomics Analysis of oarX and
fmnO Genes. We analyzed the distribution and genomic
neighborhood of thiamin metabolism genes in bacterial
genomes possessing orthologs of the oarX and/or fmnO
genes using the SEED and BV-BRC databases.
16,25
Addition-
ally, we identified TPP riboswitches in regulatory regions of
thiamin metabolism genes using the RegPrecise resource.
27
We
found oarX genes in 87 genomes representing the phyla
Firmicutes (46 genomes), Actinobacteria (22 genomes), and
Proteobacteria (19 genomes); we found fmnO genes only in a
subset of 22 bacteria from these phyla, always clustered on the
chromosome with oarX (Supplementary Table 1).
Genomics-based reconstruction of thiamin metabolic path-
ways showed that oarX genes are frequently preceded by a
TPP riboswitch and clustered with thiamin synthesis, salvage,
or transport genes (Figure 2 and Supplementary Table 1). In
particular, oarX is clustered with one or more de novo thiazole
synthesis genes (thiOSGF and tenI) in four actinobacterial
genomes, in the Firmicutes Planococcus halotolerans and
Kurthia sp. JC30, and in the Proteobacterium Marinobacter
sp. ELB17, with the thiamin/HMP transporter gene thiV
38
in
four proteobacterial genomes, and with ykoCDE thiamin
transporter genes
39
in two genomes. In addition, oarX genes
cluster with the thiazole salvage gene thiM in five genomes,
with the pyrimidine salvage gene tenA in one genome, and with
thiamin synthesis genes thiD and thiE in eight genomes. Lastly,
oarX genes occasionally cluster with genes encoding acyl-CoA
synthetase, e.g., in Acinetobacter puyangensis ANC 4466 and
Corynebacterium nuruki (Figure 2).
By analyzing the genomic distribution of thiamin synthesis
and transport genes, we assigned requirements for thiamin or
for its HET or HMP precursors (see “Auxotrophy” column in
Supplementary Table 1) and capacities to take up these
molecules. Of the 87 OarX-encoding genomes, all encode ThiE
and so can synthesize thiamin from its HET and HMP
moieties. Of the 87 genomes, 45 are predicted prototrophs that
require no supplied precursors, while the other 42 are
predicted auxotrophs that require HET and/or HMP. Most
of the genomes (77 out of 87) are predicted to encode
transporters of thiamin, HET, or HMP that may also transport
derivatives of these compounds.
10,40
This comparative genomic evidence points to four
deductions about the functions of OarX and FmnO. First,
the clustering and/or co-occurrence of oarX with thiamin,
thiazole, and pyrimidine transporter genes and with the core
thiamin synthesis gene thiE is consistent with a role for OarX
in salvaging a thiamin-related breakdown product that can be
taken up and reused for thiamin synthesis. Second, that oarX
clusters with genes for synthesis and salvage of the thiazole
moiety (thiOSGF,tenI, and thiM) and for transport of thiamin
(thiV,ykoCDE) suggests that OarX acts on a thiazole or
thiamin derivative. Third, the clustering of oarX with acyl-CoA
synthetase genes, although weaker than other associations, is
suggestive because CoA thioester formation is a common
activation step in carboxyl reduction pathways.
22
This
clustering thus suggests that OarX might catalyze reduction
of the CoA thioester derivative of thiamin acetic acid, MTA, or
PCA. Other members of the short-chain dehydrogenase-
reductase family carry out such a reduction on a fatty acyl-CoA
substrate.
41,42
Fourth, that oarX occurs in many genomes that
lack fmnO and�among the genomes analyzed�that fmnO is
always in an operonic arrangement with oarX implies that the
function of OarX does not depend on FmnO, although that of
FmnO could depend on OarX.
These deductions prompted genetic tests in C. glutamicum
for roles for OarX and FmnO in salvage of PCA, MTA, oxo-
HET, thiamin acetic acid, or oxo-thiamin. For these tests, the
oarX-fmnO operon was deleted as a unit for the sake of
eciency. We reasoned that if knocking out both genes does
not give a phenotype, this excludes a role for either gene and
that if it does give a phenotype, this would warrant making
single knockouts to dissect which gene is responsible. We
tested all compounds at the physiological concentrations
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appropriate for thiamin and its precursors (≤1μM);
10,38
concentrations higher than this are most unlikely to occur in
natural environments.
43
3.3. Evidence That OarX and FmnO Do Not Salvage
PCA. We first tested whether OarX and FmnO are needed to
salvage the pyrimidine carboxylic acid PCA, which seemed
relatively unlikely given the association of oarX and f mnO with
thiazole synthesis and salvage genes (see above). We used C.
glutamicum ATCC 14067, which has a full set of thiamin
synthesis genes except thiC
33
and consequently requires HMP
(or thiamin) for growth.
44
We simply compared the ability of
various PCA concentrations to support the growth of ATCC
14067 with that of HMP or thiamin (Figure 3A). No
concentration of PCA allowed growth, meaning that neither
OarX nor FmnO enabled its salvage. Nor are OarX and FmnO
likely to detoxify PCA, as there is no indication that PCA is
toxic (Supplementary Figure 3).
45,46
3.4. Evidence That OarX and FmnO Do Not Salvage
MTA, Oxo-HET, Oxo-thiamin, or Thiamin Acetic Acid. To
test for roles of OarX and FmnO in salvage of thiamin or
thiazole oxidation products, we deleted thiG in C. glutamicum
ATCC 14067 to create a thiazole auxotroph (i.e., to force
dependence on an external source of thiazole) and attempted
to rescue the deletant by supplying 100 nM or 1 μM MTA,
oxo-HET, oxo-thiamin, or thiamin acetic acid using HET and
thiamin as benchmarks. HET or thiamin supported growth as
expected, but the other compounds did not (Figure 3B−E);
the slight growth at the highest concentration of oxo-HET
could be due to trace contamination with HET. Tests with
wild-typeC. glutamicumATCC 14067 showed that, at 100 nM
or1μM, none of the thiamin degradation products inhibited
growth except for a minor transient eect of oxo-HET
(Supplementary Figure 3). These results exclude roles for
OarX and FnmO in salvaging MTA, oxo-HET, oxo-thiamin, or
thiamin acetic acid.
The above data left open the possibility that OarX and
FmnO have an unrecognized accessory (i.e., nonessential) role
in salvaging thiamin itself or its physiological precursors HMP
or HET, or in detoxifying an unidentified product of thiamin
breakdown. We therefore created a triple ΔthiG ΔoarX−fmnO
C. glutamicum ATCC 14067 knockout strain and compared its
responses to the normally used concentrations of thiamin,
HMP, and HET with those of the ΔthiG single knockout.
There were no significant dierences between the single and
triple knockout strains (Supplementary Figure 4), which makes
a cryptic role in salvage or detoxification unlikely.
4. CONCLUSIONS
Our comparative genomic analysis, based on ∼600×more
genomes than were available in 2002
47
when the OarX-thiamin
association was first flagged,
4
greatly strengthens the case for
this association and for its being functional rather than
fortuitous. The fact that (i) OarX belongs to a subgroup of
short-chain dehydrogenase/reductases whose members typi-
cally mediate carbonyl reduction reactions and (ii) carboxylate
and thiazolone oxidation products of thiamin are prevalent
favored the simple hypothesis that OarX mediates a reductive
step in the salvage of one or more of these products. Our
genetic data invalidate this hypothesis with respect to five
known oxidation products. These negative results are
important to document because the products that we tested
are obvious ones that anyone probing the function of OarX
would be likely to test (fruitlessly, in fact). The hypothesis of a
salvage role for OarX, and possibly also for FmnO, nevertheless
still stands because the thiazole and pyrimidine moieties of
thiamin and thiamin itself can carry other modifications
besides carboxyl or oxo groups,
7
i.e., the substrate for OarX
could be a derivative of MTA, oxo-HET, thiamin acetic acid,
oxo-thiamin, or PCA, not one of these compounds themselves.
Future deep characterization of thiamin breakdown products
by modern mass spectrometric methods (as opposed to the
classical radiochemical methods used previously) could show
Figure 3. Growth responses of the C. glutamicum ATCC 14067 wild-
type strain or its ΔthiG mutant to oxidative degradation products of
thiamin. Growth was measured after 24 h. The corresponding
physiological precursor (HMP or HET) and thiamin served as
benchmarks. (a) Response of the wild-type strain to PCA. (b)
Response of the ΔthiG strain to MTA. (c) Response of the ΔthiG
strain to oxo-HET. (d) Response of the ΔthiG strain to oxo-thiamin.
(e) Response of the ΔthiG strain to thiamin acetic acid. Data are from
three independent cultures. Mean values are shown by horizontal bars
(±standard error of the mean).
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whether such derivatives are formed in sucient amounts to
make them plausible candidates for salvage by OarX.
■ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.4c03514.
Phylogenetic tree of representative OarX and FabG
proteins; phylogenetic tree of representative FmnO
proteins and FMN-dependent monooxygenase family
proteins with known enzymatic activities; growth
responses of C. glutamicum ATCC 14067 to HMP
only (no additions) or HMP + thiamin or thiazole
oxidation products; and growth responses of the C.
glutamicum ATCC 14067 ΔthiG and ΔthiG ΔoarX-
fmnO mutants to thiamin or its physiological precursors
(HMP + HET) (PDF)
Thiamin metabolism subsystem in OarX-encoding
bacterial genomes (XLSX)
Primer sequences used in this work (XLSX)
■AUTHOR INFORMATION
Corresponding Author
Edmar R. Oliveira-Filho −Horticultural Sciences
Department, University of Florida, Gainesville, Florida
32611, United States; orcid.org/0000-0002-1597-0830;
Email: ramosdeoli.edmar@ufl.edu
Authors
Dmitry A. Rodionov −Infectious and Inflammatory Diseases
Center, Sanford Burnham Prebys Medical Discovery Institute,
La Jolla, California 92037, United States; orcid.org/
0000-0002-0939-390X
Andrew D. Hanson −Horticultural Sciences Department,
University of Florida, Gainesville, Florida 32611, United
States; orcid.org/0000-0003-2585-9340
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.4c03514
Author Contributions
E.R.O.-F.: conceptualization, methodology, investigation,
visualization, and writing�original draft. D.A.R.: formal
analysis, data curation, and writing�review and editing.
A.D.H.: conceptualization, writing�original draft, funding
acquisition, and project administration.
Funding
This work was supported primarily by the U.S. Department of
Energy, Oce of Science, Basic Energy Sciences under Award
DE-SC0020153, and by USDA NIFA Hatch project FLA-
HOS-005796 and an Endowment from the C.V. Grin, Sr.
Foundation. D.R. was supported by NIH award DK030292-35.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
We thank Dr. Rosane Aparecida Moniz Piccoli, Eric Velasco,
and Dr. Henrique da Costa Oliveira for advice on C.
glutamicum cultivation and gene deletion procedures.
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