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A flavin-based extracellular electron transfer mechanism in diverse Gram-

positive bacteria

Article  in  Nature · September 2018

DOI: 10.1038/s41586-018-0498-z

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Letter https://doi.org/10.1038/s41586-018-0498-z

A flavin-based extracellular electron transfer

mechanism in diverse Gram-positive bacteria
­Samuel H. Light1, Lin Su2,3, Rafael Rivera-Lugo1, Jose A. Cornejo2, Alexander Louie1, Anthony T. Iavarone4,
Caroline M. Ajo-Franklin2 & Daniel A. Portnoy1,5*

Extracellular electron transfer (EET) describes microbial colorimetric change following an Fe2+-indicator overlay were visually
bioelectrochemical processes in which electrons are transferred identified and the location of their transposon insertion was mapped to
from the cytosol to the exterior of the cell1. Mineral-respiring the genome (Fig. 1b). From this screen, thirty-four independent trans-
bacteria use elaborate haem-based electron transfer mechanisms2–4 poson insertions that localized to a largely uncharacterized 8.5-kilobase
but the existence and mechanistic basis of other EETs remain largely locus were identified—with at least one insertion disrupting each of the
unknown. Here we show that the food-borne pathogen Listeria eight genes in this region (Fig. 1c). Genes in the locus were assigned
monocytogenes uses a distinctive flavin-based EET mechanism names on the basis of putative functions of their protein products
to deliver electrons to iron or an electrode. By performing a (see ‘Gene name assignment’ in Methods for a more detailed expla-
forward genetic screen to identify L. monocytogenes mutants with nation). The only transposon insertions outside the identified locus
diminished extracellular ferric iron reductase activity, we identified disrupt ribU, the substrate-binding subunit of a riboflavin transporter10
an eight-gene locus that is responsible for EET. This locus encodes
a specialized NADH dehydrogenase that segregates EET from a 25 b
aerobic respiration by channelling electrons to a discrete membrane-
20 S. o.
localized quinone pool. Other proteins facilitate the assembly of an
Current (μA cm–2)

abundant extracellular flavoprotein that, in conjunction with free- 15

L. m. (+)
molecule flavin shuttles, mediates electron transfer to extracellular 10
acceptors. This system thus establishes a simple electron conduit 5 5 mm
that is compatible with the single-membrane structure of the Gram- L. m. (–)
positive cell. Activation of EET supports growth on non-fermentable 0
0 4 8 12 16
carbon sources, and an EET mutant exhibited a competitive c
Time (h)
defect within the mouse gastrointestinal tract. Orthologues of ribU fmnA dmkA fmnB pplA ndh2 eetA eetB dmkB
the genes responsible for EET are present in hundreds of species
across the Firmicutes phylum, including multiple pathogens and d e
commensal members of the intestinal microbiota, and correlate 30
25
Maximum current

with EET activity in assayed strains. These findings suggest a 20

Fe2+ (μM min–1)

(μA cm–2)

greater prevalence of EET-based growth capabilities and establish 20 15

a previously underappreciated relevance for electrogenic bacteria *** 10
across diverse environments, including host-associated microbial 10 *** ***
5 ***
communities and infectious disease. *** *** *** *** *** *** *** 0
*** ***
0
L. monocytogenes is a fermentative Gram-positive bacterium that is n
n

n
pe

n
tA n
k tn
rib ype

kB tn
n
nB n
pp ::tn
nd ::tn
n n

ee ::tn

::t
::t

::t
:t
ee ::t

::t
fm A::t
fm ::t
dm A::

ty

frequently associated with decaying plant matter in the environment,

dm B::

2:

kB
kA

lA
h2
lA
U
t

pp
ild
t

dm

dm
ild

nd
W
W

but which transforms into an intracellular pathogen on encountering

a mammalian host5. Despite lacking a lifecycle or genes convention- Fig. 1 | An uncharacterized genetic locus associated with EET activity.
a, Chronoamperometry results from L. monocytogenes (L. m.)- or
ally associated with EET, a 25-year-old observation that L. monocyto­
Shewanella oneidensis (S. o.)-inoculated electrochemical reactors. For
genes possessed extracellular ferric iron reductase activity6 led us L. monocytogenes experiments, an electron donor (glucose) was present
to wonder whether a novel EET strategy existed. Because electrons in (+) or absent from (−) the medium; lactate was used as an electron
transferred out of the cell can be captured by an electrode, electro- donor for S. oneidensis. Results are representative of three independent
chemical measurements provide a useful tool for assaying EET7. By experiments. b, Image of one of the thirty-six independent mutants
performing chronoamperometry experiments, we observed that identified from the ferric iron reduction screen, minutes after ferrozine
L. monocytogenes produces a robust electric current in the presence of agar overlay (arrow). c, Location of transposon insertions (triangles) in
growth substrate (Fig. 1a, Extended Data Fig. 1a). In addition, we found mutants identified as having decreased ferric iron reductase activity in the
that cyclic voltammetry experiments—which monitor electric current genetic screen. Arrows represent genes, with the darker grey signifying
while the electrochemical potential is systematically varied—revealed a inclusion in a multi-gene operon. Previously uncharacterized genes on
the locus have been assigned names based on the putative functions of
distinctive catalytic wave reminiscent of other electrochemically active the proteins they encode. d, Ferric iron reductase activity of transposon
bacteria8,9 (Extended Data Fig. 1b). These results thus provide strong mutants identified from the screen. Results are expressed as mean ±
evidence that L. monocytogenes possesses EET activity. s.e.m. from three independent experiments. e, Maximum electric current
To address the genetic basis of EET activity, approximately 50,000 achieved from chronoamperometry experiments with representative
colonies of a pooled L. monocytogenes himar1 transposon library EET mutants. Strains that statistically differ from the wild-type strain are
were grown on Fe3+-containing agar plates. Mutants with decreased indicated; ***P < 0.001, ANOVA with Dunnett’s post-test.
1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. 2Molecular Foundry, Molecular Biophysics and Integrated Bioimaging, and Synthetic Biology
Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 3State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing,
210018, China. 4QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, CA, USA. 5Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA.
*e-mail: [email protected]

1 4 0 | N A T U RE | V O L 5 6 2 | 4 O C T O B ER 2 0 1 8
© 2018 Springer Nature Limited. All rights reserved.
 Letter RESEARCH

a Ndh1 Ndh2
NDH II NDH II CTD
1 403 1 410 628
DHNA OH O
b
O DMK MK
MenA OH OH
H MenG H
OH
DmkA x x
IPP

HepT OH OH
H
OP P DmkB OP P
x x

c 107 d Aerobic respiration

106 MK O2
Ndh1 QoxAB
CFUs per ml

105 HepT
e– CydAB
104 MenA
MenG
103
NADH DHNA and IPP
102
101 DmkA
e– DmkB
100 Ndh2
DMK Fe3+
Δn xA
ΔqdAB

m ::tn

n
dm ::tn
nd ::tn
dm ::tn
Δc ype

he nA
Δm 1

::t
dh
o

kB

G
kA
pT
h2
t
y

EET
en
ild
W

Fig. 2 | A parallel electron transfer pathway segregates EET from differ between the two quinones). c, Colony-forming units (CFUs) after
aerobic respiration. a, Domain layout of the L. monocytogenes proteins 24 h in aerobic respiration medium. Results from three independent
Ndh1 and Ndh2. CTD, C-terminal domain; NDH II, type II NADH experiments are expressed as mean ± s.e.m. The ∆cydAB ∆qoxA mutant
dehydrogenase domain. Grey regions represent predicted transmembrane lacks terminal cytochrome oxidases and thus provides an aerobic-
helices. b, Predicted reactions catalysed by L. monocytogenes DmkA respiration-deficient control. d, Probable electron transfer pathways
and DmkB, the paralogous proteins MenA and HepT, and MenG inferred from mutants with EET (red) or aerobic respiration (blue)
(highlighted by blue arrow). DHNA, 1,4-dihydroxy-2-naphthoyl-CoA; phenotypes. Dashed arrows highlight the path of electron flow and solid
DMK, demethylmenaquinone; IPP, isopentenyl pyrophosphate; MK, lines track quinone synthesis.
menaquinone; x, an unknown number of isoprene repeats (which may

(Fig. 1c). We confirmed that the mutants had diminished ferric iron We next sought to address the downstream steps responsible for elec-
reductase (Fig. 1d) and electrochemical activity (Fig. 1e, Extended Data tron transfer from the quinone pool to extracellular electron accep-
Fig. 1b) and then turned to study the molecular basis of EET. tors. FmnB is a predicted lipoprotein that is annotated as possessing
Type II NADH dehydrogenase—or Ndh1 in L. monocytogenes— FMN transferase activity. Homologous FMN transferases catalyse a
catalyses electron exchange from cytosolic NADH to a lipid-soluble post-translational modification in which an FMN moiety is covalently
quinone derivative, which is the first step in the respiratory electron linked to a threonine side chain of substrate proteins13,14 (Fig. 3a). To
transport chain11. Ndh2, which is encoded by one of the genes in the identify protein substrates of FmnB, wild-type and fmnB::tn cells were
EET locus, is a protein with an N-terminal type II NADH dehydroge- subjected to a comparative mass spectrometric analysis. Only two
nase domain and a unique transmembrane C-terminal domain that is L. monocytogenes peptides met the criteria of selective FMNylation in
absent from functionally characterized enzymes (Fig. 2a). Consistent the wild-type sample and both of these mapped to distinct regions in
with Ndh2 being a novel NADH dehydrogenase, we observed that EET the protein product of the neighbouring gene in the EET locus, PplA
activation correlated with cellular NAD+ levels (Extended Data Fig. 2). (Supplementary Table 1).
Furthermore, the proteins DmkA and DmkB—which are encoded by Similar to FmnB, PplA is a predicted lipoprotein and, consistent with
two other genes in the EET locus—are paralogues of the highly con- this prediction, a trypsin-shaving experimental approach, in which
served microbial enzymes MenA and HepT, which catalyse terminal extracellular-surface-associated proteins liberated through a partial
steps in the production of the quinone demethylmenaquinone (Fig. 2b). digestion of the cell wall are identified by mass spectro­metry, confirmed
In Escherichia coli, three different quinones—demethylmenaquinone, that PplA is associated with the surface of the cell (Supplementary
menaquinone and ubiquinone—are used to selectively channel elec- Table 2). The N-terminal lipidation site on PplA is followed by
trons to different electron acceptors12. By analogy, we reasoned that a approximately 30 amino acids that are predicted to be unstructured.
distinct quinone derivative and NADH dehydrogenase might function- N-terminal unstructured regions are a common feature of bacterial
ally segregate electron fluxes for EET and aerobic respiration. lipoproteins and are thought to provide a loose tether that allows the
To clarify the relationship between EET and aerobic respiration, we for- active portion of the protein to diffuse further from the membrane and
mulated an ‘aerobic respiration medium’ that contained non-fermentable to partially or fully penetrate the cell wall15. This property, coupled
glycerol as the sole carbon source. Despite exhibiting wild-type levels of with the covalently bound redox-active FMNs, is consistent with PplA
ferric iron reductase activity (Extended Data Fig. 3a), ∆cydAB ∆qoxA representing the extracellular component of the EET machinery that
(a positive control that lacks terminal cytochrome oxidases), ΔmenA, facilitates electron transfer—via its FMNs—to extracellular electron
hepT::tn and Δndh1 strains failed to grow on aerobic respiration medium acceptors.
(Fig. 2c). By contrast, EET mutants grew similarly to wild-type strains Following its unstructured N-terminal region, PplA has sequential
under these conditions (Fig. 2c). Moreover, menG—which encodes domains that share 59% sequence identity with each other. From the
the enzyme that converts demethylmenaquinone to menaquinone—is proteomic analysis, it is evident that the FMNylated threonines on PplA
contained on an operon with hepT and is essential for growth on aer- assume equivalent positions on each of these related domains (Fig. 3b).
obic respiration medium, but not ferric iron reductase activity (Fig. 2c, To further clarify the mechanism of FMNylation, we tested FmnB sub-
Extended Data Fig. 3). Collectively, these results support the conclusion strate specificity using recombinant FmnB and PplA. These assays con-
that a demethylmenaquinone derivative used by Ndh2 and a menaqui- firm that FmnB catalyses FMNylation of PplA and demonstrate that
none derivative used by Ndh1 are selective for downstream enzymes that the enzyme specifically uses flavin adenine dinucleotide (FAD) as a
function in EET and aerobic respiration, respectively (Fig. 2d). substrate (Fig. 3c, Extended Data Fig. 4).

4 O C T O B ER 2 0 1 8 | V O L 5 6 2 | N A T U RE | 1 4 1
© 2018 Springer Nature Limited. All rights reserved.
 RESEARCH Letter

a O
H
N O
b
PplA

N US FMN1 FMN2
N OH OH 23 55 143 178 265 299
O
N O
P
O
OH O

FMN Thr

c d
Acceptor
FMN
+ + + – + FmnB
+ + + + – PplA PplA e–
+ + – + + FAD PpIA
– – + – – FMN e–
FmnB
+ – – – – EDTA Flavin

50 kDa FmnA
RibU
37 kDa

25 kDa
FAD
EetA

e–

DmkA DMK EetB

50 kDa
DmkB Ndh2
37 kDa e–

25 kDa DHNA + IPP

NADH NAD+

Fig. 3 | A surface-associated flavoprotein establishes the extracellular representative of three independent experiments. See Supplementary
component of the EET apparatus. a, Post-translational modification Fig. 1 for uncropped gel. d, Model of the molecular basis of EET. DmkA
catalysed by the FMN transferase family of enzymes, of which FmnB and DmkB synthesize a demethylmenaquinone derivative (bottom inset).
is a member13,14. b, Domain architecture of PplA. FMN1, FMNylated RibU and FmnA secrete FAD that is used by FmnB to post-translationally
domain 1; FMN2, FMNylated domain 2; US, unstructured. The lipidated modify PplA (top inset). EET is achieved by a series of electron transfers.
cysteine on the N terminus after signal peptidase processing is shown in Ndh2 transfers electrons from NAD to DMK. Electrons are transferred
red and FMNylated threonines are shown in yellow. c, Analysis of FmnB from DMK to FMN groups on PplA or free flavin shuttles—possibly with
substrate specificity. SDS–PAGE of recombinant PplA after incubation involvement from uncharacterized membrane proteins in the EET locus,
under specified conditions. Ultraviolet illumination of the gel (bottom) EetA and EetB—and ultimately to a terminal electron acceptor.
enables visualization of protein with covalently bound flavin. Results are

Because both FmnB and PplA are membrane-anchored lipoproteins, L. monocytogenes proliferates21,22. To determine whether flavins could be
a source of FAD substrate is required for FmnB to FMNylate PplA on used as electron shuttles, we tested the effect of exogenous riboflavin, FMN
the surface of the cell. The only transposon insertions identified outside and FAD on EET activity. Injection of FMN into an L. monocytogenes-
the EET locus disrupt ribU, which has previously been shown to encode inoculated electrochemical chamber resulted in a pronounced increase
the substrate-binding subunit of an energy-coupling factor (ECF) in electric current (Extended Data Fig. 6a). Moreover, while cells
transporter that functions in riboflavin uptake10. In addition to a sub- immersed in soluble ferric iron exhibited a high baseline level of reduc-
strate-binding subunit, ECF transporters contain a transmembrane tase activity that was unresponsive to flavins, flavins caused a marked
subunit and two distinct ATPase subunits, which drive the transport of concentration-dependent enhancement in the reduction of insoluble
substrate across the membrane10 (Extended Data Fig. 5a). FmnA in the ferric (hydr)oxide (Extended Data Fig. 6b). These data thus support
EET locus shares 50% sequence identity with the transmembrane subunit the conclusion that L. monocytogenes can use environmental flavins to
of the RibU–ECF riboflavin transporter (EcfT) and this led us to propose shuttle electrons to outlying acceptors.
that FmnA interacted with RibU to promote FAD secretion (Extended Integrating all of our insights into the roles of the components of
Data Fig. 5b). Consistent with this interpretation, proteomic analysis the EET apparatus, we propose a molecular model of electron travel
of ribU::tn and fmnA::tn strains revealed a marked decrease in PplA from intracellular NADH to membrane-confined quinone, then to
FMNylation (Supplementary Table 1). Furthermore, addition of FAD to extracellular flavoprotein (and/or other shuttles), and ultimately to a
the growth medium specifically restored ferric iron reductase activity to kinetically favourable terminal electron acceptor (Fig. 3d). To deter-
the ribU::tn and fmnA::tn strains (Extended Data Fig. 5c). On the basis of mine whether EET established a bona fide growth-supporting activity,
these findings, we propose that RibU and FmnA establish a transporter we next screened a library of common microbial growth substrates and
that secretes the FAD required for FmnB-catalysed FMNylation of PplA. found that the inclusion of ferric iron or an electrode was required for
The term ‘extracellular electron shuttle’ refers to redox-active anaerobic growth on the sugar alcohols xylitol and d-arabitol (Fig. 4a,
small molecules that are cyclically reduced by cells and oxidized by Extended Data Fig. 7). Genes for aerobic respiration but not EET
extracellular electron acceptors16,17. The relevance of shuttles for were essential for aerobic growth on xylitol, whereas this pattern was
EET is exemplified by Shewanella species, which use an efflux-type reversed under anaerobic conditions—that is, EET genes were essen-
transporter to secrete flavins that shuttle electrons to acceptors that tial and aerobic respiration genes dispensable (Fig. 4a, Extended Data
are not directly contacting the cell18–20. In contrast to Shewanella, Fig. 7). These data demonstrate that the distinct electron transport
L. monocytogenes is a flavin auxotroph and thus by definition envi- chains that segregate aerobic respiration and EET promote aerobic and
ronmental flavins must be present in its replicative niche. Indeed, anaerobic growth, respectively.
micromolar flavin concentrations are typical of nutrient-rich envi- We next asked whether EET has a role in host colonization.
ronments, such as the plant biomass and mammalian hosts in which Consistent with EET being dispensable for aerobic growth,

1 4 2 | N A T U RE | V O L 5 6 2 | 4 O C T O B ER 2 0 1 8
© 2018 Springer Nature Limited. All rights reserved.
 Letter RESEARCH

a 109 10

Current (μA cm–2)

108 Wild type

CFUs per ml
Wild type
6

4
107 ndh2::tn
2
Wild type (–) ndh2::tn
106 0
0 1 2 3 4 5 0 1 2 3 4 5
Time (days) Time (days)
b c *
* *
102
Control
100 * *

L. monocytogenes activity
ndh2::tn *

Percentage of wild-type
** ** *
CI in faeces

101
NS

50
100

10–1 0
1 3 5

L o n
al L. . lacua
ca m vie s
ss ati ae
E. lifla m

cc . f ec s
B. rol ciums

L. L ula s
pl . c ns

S. S. fino um
ll t s
E. oly ans
B. oli us
bt 2
ilis
ra ta ei
E du us
E. ro ar cti

sa E . fa ran
ha ae ali

rc u

ga mu su
n t

su K1
E. an as
e cu
in 2::

ci ytic

c tic
v

f r
Time (days)

L. dh
n

t a g
.m

E
C
Fig. 4 | EET supports anaerobic growth, confers a competitive durans; n = 6 for Listeria innocua, E. faecalis and Streptococcus mutans;
advantage in the intestinal lumen, and is active in multiple Firmicutes. n = 5 for Carnobacterium maltaromaticum, Enterococcus casseliflavus,
a, L. monocytogenes CFUs (left) and electric current (right) from Streptococcus gallolyticus and Bacillus subtilis; n = 4 for Lactococcus lactis,
chronoamperometry experiments conducted with xylitol growth Enterococcus faecium, Enterococcus saccharolyticus, Bacillus circulans,
medium. (−), control condition without an electrode. Results from three Lactobacillus plantarum and Enterococcus raffinosus; n = 3 for Lactobacillus
independent experiments are expressed as mean ± s.e.m. b, Mice (n = 5) casei and E. coli K12) are expressed as mean ± s.e.m. Strains that
were fed bread inoculated with a 1:1 mixture of ∆hly and ∆hly ndh2::tn statistically differ from ndh2::tn are indicated; *P < 0.05, ANOVA with
L. monocytogenes strains. The competitive index (CI) at three time Dunnett’s post-test. Some members of Lactobacillales lack the ability to
points after infection is indicated. Median values and statistically synthesize DHNA, the precursor for demethylmenaquinone biosynthesis,
significant differences compared to a control that competed two ∆hly and require an exogenous source for quinone-dependent processes36.
strains are indicated; **P = 0.01, unpaired two-sided t-test. Results Organisms with genes in the EET locus and menC (which catalyses an
are representative of three independent experiments. c, Iron reductase essential step in DHNA biosynthesis) are coloured grey. L. casei,
activity in a panel of Firmicutes species, expressed as a percentage of wild- L. plantarum and E. raffinosus contain genes for EET, but not menC.
type L. monocytogenes activity. Results from at least three independent The remaining species lack genes in the EET locus.
experiments (n = 7 for ndh2::tn, Lactococcus garvieae and Enterococcus

EET-deficient mutants resembled wild-type L. monocytogenes in commercial applications in food fermentation or probiotics (Lactococcus
an intracellular macrophage growth assay and an intravenous spp., Lactobacillus spp., Oenococcus spp., Tetragenococcus spp. and so on)
infection model (Extended Data Fig. 8). Because anaerobic growth (Supplementary Table 3). The functionality of identified loci could
mechanisms are important for microbial proliferation within the explain previous reports of EET-like activity in a number of species25–35
intestinal lumen23,24, we proposed that the food-borne pathogen might and assays of ferric iron reductase activity of a panel of Firmicutes
use EET in this context. Consistent with the hypothesis, the faecal provided additional evidence that the presence of necessary genetic
burden of the ndh2::tn strain was decreased approximately sixfold components correlates with EET activity (Fig. 4c).
in a streptomycin-pretreated model of L. monocytogenes intestinal In conclusion, our study reveals a novel electron transport chain
colonization (Fig. 4b). These results thus suggest a role for EET within that supports growth on extracellular electron acceptors. This mech-
the dysbiotic gut and raise the possibility that EET constitutes a anism lacks an elaborate multi-haem apparatus and, partly by taking
generally important metabolic activity within the mammalian gastro- advantage of the single-membrane architecture of the Gram-positive
intestinal tract. cell, is characterized by considerably fewer electron transfer steps than
We next turned to the phylogenetic distribution of the genes respon- comparable systems in mineral-respiring Gram-negative bacteria1. The
sible for EET. BLAST searches revealed that homologues of these genes genes identified in the EET locus are present in a wide-ranging group
are widespread in hundreds of species that span the Firmicutes phylum of microorganisms that occupy a diverse array of ecological niches.
(Extended Data Fig. 9a, Supplementary Table 3). Many of these genes Defying conventional views of EET, this distinctive system is abun-
are likely to encode functional EET systems, as the identified locus dant in bacteria that prioritize fermentative metabolic strategies and
is typically conserved, though noteworthy distinctions are evident in reside in nutrient-rich environments, including the lactic acid bacteria.
some genomes (Extended Data Fig. 9b). Microorganisms that possess a Within this context, environmental flavins may represent a feature of
locus with EET genes adopt a wide range of different lifestyles, includ- the ecological landscape that can be exploited to promote EET activity.
ing within thermophilic (Caldanaerobius spp., Thermoanaerobacter These observations suggest that, rather than being a specialized process
spp. and so on) and halophilic (Halolactibacillus spp., Halothermothrix confined to mineral-respiring bacteria, the use of extracellular electron
spp. and so on) habitats. Orthologues of the identified genes in the acceptors represents a fundamental facet of microbial metabolism that
EET locus are found in a number of human pathogens (Clostridium is relevant across diverse environments. In addition to obvious bioen-
perfringens, Enterococcus faecalis, Streptococcus dysgalactiae and so on), ergetic applications, the characterization of a flavin-based EET mech-
members of the human microbiota (Clostridium spp., Enterococcus anism thus establishes further avenues for the study of electrochemical
spp., Streptococcus spp. and so on) and lactic acid bacteria that have activities throughout the microbial world.

4 O C T O B ER 2 0 1 8 | V O L 5 6 2 | N A T U RE | 1 4 3
© 2018 Springer Nature Limited. All rights reserved.
 RESEARCH Letter

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11. Kerscher, S., Dröse, S., Zickermann, V. & Brandt, U. The three families of
Acknowledgements We thank G. Chen, J.-D. Sauer, E. Stevens, M. Marco and
respiratory NADH dehydrogenases. Results Probl. Cell Differ. 45, 185–222
N. Freitag for providing bacterial strains; H. Carlson, A. Williamson and J. Coates
(2008).
for helpful feedback; and N. Garelis for experimental assistance. Research
12. Unden, G. & Bongaerts, J. Alternative respiratory pathways of Escherichia coli:
reported in this publication was supported by funding from the National
energetics and transcriptional regulation in response to electron acceptors.
Institute of Allergy and Infectious Diseases of the National Institutes
Biochim. Biophys. Acta 1320, 217–234 (1997).
of Health (F32AI136389 to S.H.L., 1P01 AI063302 to D.A.P., and 1R01 AI27655
13. Bertsova, Y. V. et al. Alternative pyrimidine biosynthesis protein ApbE is a flavin
to D.A.P.), the Office of Naval Research (N0001417WX01603 to C.M.A.-F.), and
transferase catalyzing covalent attachment of FMN to a threonine residue in
the China Scholarship Council (no. 201606090098 to L.S.).
bacterial flavoproteins. J. Biol. Chem. 288, 14276–14286 (2013).
A mass spectrometer used in this study was purchased with NIH support (grant
14. Deka, R. K., Brautigam, C. A., Liu, W. Z., Tomchick, D. R. & Norgard, M. V.
1S10OD020062-01). Work at the Molecular Foundry was supported by the
Evidence for posttranslational protein flavinylation in the syphilis
Office of Science, Office of Basic Energy Sciences, of the US Department of
spirochete Treponema pallidum: structural and biochemical insights from the
Energy under Contract No. DE-AC02-05CH11231.
catalytic core of a periplasmic flavin-trafficking protein. MBio 6, e00519-15
(2015).
15. Zückert, W. R. Secretion of bacterial lipoproteins: through the cytoplasmic Reviewer information Nature thanks N. Freitag, J. Gralnick, K. Nealson and
membrane, the periplasm and beyond. Biochim. Biophys. Acta 1843, G. Reguera for their contribution to the peer review of this work.
1509–1516 (2014).
16. Glasser, N. R., Saunders, S. H. & Newman, D. K. The colorful world of Author contributions S.H.L., A.T.I., C.M.A.-F. and D.A.P. designed the study.
extracellular electron shuttles. Annu. Rev. Microbiol. 71, 731–751 (2017). S.H.L, L.S. and J.A.C. performed electrochemical experiments. S.H.L. and A.T.I.
17. Brutinel, E. D. & Gralnick, J. A. Shuttling happens: soluble flavin mediators of performed mass spectrometric experiments. S.H.L., A.L. and R.R.-L. performed
extracellular electron transfer in Shewanella. Appl. Microbiol. Biotechnol. 93, microbiological and biochemical experiments. S.H.L. and D.A.P. wrote the
41–48 (2012). manuscript.
18. Marsili, E. et al. Shewanella secretes flavins that mediate extracellular electron
transfer. Proc. Natl Acad. Sci. USA 105, 3968–3973 (2008). Competing interests D.A.P. has a consulting relationship with and a financial
19. von Canstein, H., Ogawa, J., Shimizu, S. & Lloyd, J. R. Secretion of flavins by interest in Aduro Biotech; both he and the company stand to benefit from the
Shewanella species and their role in extracellular electron transfer. Appl. Environ. commercialization of this research.
Microbiol. 74, 615–623 (2008).
20. Kotloski, N. J. & Gralnick, J. A. Flavin electron shuttles dominate extracellular Additional information
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21. Powers, H. J. Riboflavin (vitamin B-2) and health. Am. J. Clin. Nutr. 77, 018-0498-z.
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Quantification of riboflavin, flavin mononucleotide, and flavin adenine Reprints and permissions information is available at http://www.nature.com/
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1 4 4 | N A T U RE | V O L 5 6 2 | 4 O C T O B ER 2 0 1 8
© 2018 Springer Nature Limited. All rights reserved.
 Letter RESEARCH

Methods 0.5 mM FAD to one aliquot—incubated for 1 h at 37 °C. To test the effect of flavins,
L. monocytogenes strains and growth conditions. All L. monocytogenes strains riboflavin, FMN or FAD was titrated into cells resuspended in a LSM base that
used in this study were derived from wild-type 10403S (Supplementary Table 4). lacked flavins.
Transduction methods were used to introduce transposons into distinct genetic To prepare other species (detailed in Supplementary Table 4) for the ferric iron
backgrounds, as previously described37,38. L. monocytogenes cells were grown at reductase assay, cells were grown anaerobically in brain–heart infusion medium
37 °C and spectrophotometrically measured by optical density at a wavelength of for 36 h. Sub-cultures in brain–heart infusion medium supplemented with 25 mM
600 nm (OD600). Anaerobic conditions were achieved with the BD GasPak EZ ferric ammonium citrate were then grown to mid-log phase. Cells were washed
pouch system or an anaerobic chamber (Coy Laboratory Products) with an envi- twice, resuspended in fresh brain–heart infusion medium and cell densities were
ronment of 2% H2 balanced in N2. normalized to wild-type L. monocytogenes. Next, ferrozine was added to a final
Filter-sterilized brain–heart infusion medium (Difco) or variants of chemically concentration of 2 mM and 100 μl of cells were dispensed in a 96-well plate. The
defined Listeria synthetic medium (LSM)39 were used in all studies. Aerobic res- experiment was initiated by adding 100 μl of brain–heart infusion medium sup-
piration medium replaced the glucose in LSM with 50 mM glycerol. The require- plemented with 10 mM ferric ammonium citrate and OD562 measurements were
ment of an electron acceptor to support L. monocytogenes growth on xylitol was made as described for the L. monocytogenes ferric iron reductase assay.
identified by comparing aerobic versus anaerobic (absent an alternative electron L. monocytogenes growth on xylitol and ferric iron. To test electron acceptor
acceptor) growth on carbon sources, using PM1 and PM2A plates of the Phenotype usage capabilities, xylitol medium was inoculated with L. monocytogenes and
MicroArray (Biolog). ‘Xylitol medium’ replaced the glucose in LSM with 50 mM incubated at 25 °C in an anaerobic chamber. Conditions testing putative electron
xylitol. acceptors contained 50 mM ferric ammonium citrate or ferric (hydr)oxide, pre-
Gene name assignment. The identified EET locus is widely conserved in L. mono­ pared as previously described42. For the ferric ammonium citrate experiments,
cytogenes isolates and encompasses the genes lmrg_02179–lmrg_02186 in L. mono­ 50 mM sodium citrate was included in the control condition that lacked ferric
cytogenes 10403S (which correspond to lmo2634–lmo2641 in L. monocytogenes ammonium citrate and CFUs were enumerated following overnight incubation in
EGD-e). Identified genes in the EET locus were assigned dmk or fmn prefixes based a 96-well plate (Greiner Bio-One). Ferric (hydr)oxide experiments were conducted
on putative roles in demethylmenaquinone biosynthesis or PplA FMNylation, in a 6-well plate (Costar) and CFUs were enumerated 6 days after inoculation.
respectively. The eet prefix was assigned to the remaining genes, which at present NAD+ and NADH measurements. L. monocytogenes cells grown overnight in
lack high-confidence functional assignments. The only previously named gene, LSM were washed and resuspended in 500 μl of medium. Cells were then split
pplA, was so-called on the basis of the role of its cleaved signal peptide as a signal- and 50 mM ferric ammonium citrate was added to one aliquot. To test aerobic
ling pheromone40 (a function that seems to be unrelated to the mature protein). conditions, 14-ml tubes were placed in a shaking (200 r.p.m.) incubator. To achieve
Bioelectrochemical characterization and measurements. Chronoamperometry microaerophilic conditions, the headspace in the tube was purged with argon gas
and cyclic voltammetry were carried out using a Bio-Logic Science Instruments and the tightly capped tube was placed in a stationary incubator. After 1.5 h at
potentiostat model VSP-300. All measurements were performed using double 37 °C, bacteria were collected by centrifugation, resuspended in PBS and lysed by
chamber electrochemical cells (Extended Data Fig. 1a) and consisted of an vortexing with 0.1-mm-diameter zirconia–silica beads. NAD+ and NADH meas-
Ag/AgCl reference electrode (CH Instruments), a Pt wire counter electrode (Alfa urements were performed using the NAD/NADH-Glo Assay (Promega).
Aesar), and a 6.35-mm-thick graphite felt working electrode with a 16-mm radius Assay of FmnB FMN transferase activity. Constructs of fmnB and pplA that trun-
(Alfa Aesar). cated the signal peptide were subcloned into the pMCSG58 vector. Protein overex-
Electrochemical cells were prepared with 120 ml of modified LSM (containing pression and purification followed previously described protocols43. Purified PplA
0.8 μM FMN as the sole flavin) and an open circuit potential was performed in and FmnB were incubated overnight at a 10:1 molar ratio in assay buffer (0.5 M
the absence of bacteria. Once the current stabilized, the electrochemical cell was NaCl and 10 mM Tris, pH 8.3) with putative flavin substrates. Because homologous
inoculated to a final OD600 of ~0.1. The medium in the electrochemical chamber FMN transferases require a magnesium cofactor13, the effect of the chelator EDTA
was mixed with a magnetic stir bar for the course of the experiment. For current on activity was tested. Samples were analysed by SDS–PAGE and protein bands
acquisition, the applied potential was set at +0.4 V versus Ag/AgCl. To maintain with covalent flavin modifications were visualized by UV illumination.
anaerobic conditions, electrochemical cells were continuously purged with N2 gas. To identify the basis of post-translational modifications, intact protein mass
Cyclic voltammetry measurements in the potential region of −0.8 to +0.4 V versus measurements of PplA were made using a Synapt G2-Si mass spectrometer that was
Ag/AgCl and a scan rate of 10 mV s−1 were conducted immediately before inoc- equipped with an electrospray ionization source and a C4 protein ionKey (inner
ulation and 3 h later. Electric currents are reported as a function of the geometric diameter: 150 μm, length: 50 mm, particle size: 1.7 μm), and connected in-line
surface area of the electrode. To test the effect of flavins on electrochemical activity, with an Acquity M-class ultra-performance liquid chromatography system (UPLC;
FMN was injected into the L. monocytogenes-inoculated electrochemical chamber Waters). Acetonitrile, formic acid (Fisher Optima grade, 99.9%) and water purified
to a final concentration of 1 μM. to a resistivity of 18.2 MΩ·cm (at 25 °C) using a Milli-Q Gradient ultrapure water
For S. oneidensis experiments, the glucose in LSM was replaced with sodium purification system (Millipore) were used to prepare mobile phase solvents. Solvent
lactate and S. oneidensis was inoculated to an OD600 of 0.1. Growth-supporting A was 99.9% water/0.1% formic acid and solvent B was 99.9% acetonitrile/0.1%
L. monocytogenes experiments on xylitol medium were conducted in a similar formic acid (v/v). The elution program consisted of a linear gradient from 1% to
fashion, but the electrochemical cell was inoculated to an OD600 of ~0.002 and 10% B (v/v) over 1 min, a linear gradient from 10% to 90% B over 4 min, isocratic
the medium from the electrochemical chamber was sampled at regular intervals flow at 90% B for 5 min, a linear gradient from 90% to 1% B over 2 min, and iso-
for the enumeration of CFUs. cratic flow at 1% B for 18 min, at a flow rate of 2 μl/min. The ionKey column and
Screen of mutants with diminished ferric iron reductase activity. A previously the autosampler compartment were maintained at 40 °C and 6 °C, respectively.
described method was adapted to screen for L. monocytogenes mutants with dimin- Mass spectra were acquired in the positive ion mode and continuum format, oper-
ished ferric iron reductase activity6. Approximately 250 CFUs per plate of a pooled ating the time-of-flight analyser in resolution mode, with a scan time of 0.5 s, over
himar1 transposon library, generated as previously described38, were grown on the range m/z = 400 to 5,000. Mass spectral deconvolution was performed using
brain–heart infusion agar supplemented with 0.1 mg/ml ferric ammonium citrate. ProMass software (version 2.5 SR-1, Novatia).
After 24 h at 37 °C, plates were removed from the incubator and a 10-ml overlay L. monocytogenes protein trypsinization. One millilitre of L. monocytogenes cells
(0.8% agarose and 2 mM ferrozine) was applied. Colorimetric change resulting grown in LSM to mid-log phase was washed, resuspended in 100 μl of 100 mM
from ferrozine binding to Fe2+ was visually tracked for ~10 min. Colonies with NH4HCO3 (pH 7.5), and incubated at 100 °C for 10 min. Cells were lysed by bead
diminished colorimetric change were selected and the location of the transposon beating for 15 min at 4 °C. RapiGest SF (Waters) was added to lysed cells at a final
insertion identified by Sanger sequencing, as previously described41. concentration of 0.1% and the sample was incubated at 100 °C for 5 min. After
Ferrozine assay of ferric iron reductase activity. L. monocytogenes cells grown to adding 5 μl of 100 mM dithiothreitol, samples were incubated at 58 °C for 30 min.
mid-log phase were washed twice, normalized to an OD600 of 0.5, and resuspended Next, 15 μl of 100 mM iodoacetamide was added and samples were incubated for an
in fresh medium supplemented with 4 mM ferrozine. Experiments were initiated additional 30 min. Samples were then digested overnight with 10 μl Trypsin Gold
by adding 100 μl of cells to an equivalent volume of 50 mM ferric ammonium (Promega). The following morning, 10 μl of 5% trifluoroacetic acid was added and
citrate or ferric (hydr)oxide and were conducted in triplicate at 37 °C in 96-well samples were incubated at 37 °C for 90 min. Samples were centrifuged for 30 min
format using a plate reader. OD562 measurements were made every 30 s for up to an to remove hydrolysed RapiGest, and supernatant was collected.
hour. Maximal rates (typically over 2 min) calculated from a Fe2+ standard curve L. monocytogenes intracellular growth assays. Bone-marrow-derived
are reported. Assays were generally performed in LSM, with glucose serving as the macrophages prepared from 6- to 8-week-old female mice were plated overnight on
electron donor. However, because some of the respiratory mutants grew poorly coverslips and infected with L. monocytogenes strains at a multiplicity of infection
in these conditions, these strains were assayed in brain–heart infusion medium of 0.1. Macrophage monolayers were washed with PBS and fresh medium was
(with glucose remaining as the electron donor). For FAD complementation studies, added thirty minutes after infection. At 1 h post-infection, 50 μg/ml gentamicin
before washing steps, strains grown to mid-log were split and—after adding was added to kill extracellular bacteria. To enumerate L. monocytogenes CFUs,

© 2018 Springer Nature Limited. All rights reserved.

 RESEARCH Letter

macrophages were lysed by transferring coverslips to 10 ml of water, as previously Data acquisition was controlled using MassLynx software (version 4.1), and tryptic
described44. peptides were identified using Progenesis QI for Proteomics software (version
L. monocytogenes intravenous infections. Eight-week-old female C57BL/6 mice 4.0, Waters).
(The Jackson Laboratory) were infected with 1 × 105 CFUs in 200 μl of PBS by tail Bioinformatics analysis of identified genes in the EET locus. Ndh2 homologues
vein injection. Forty-eight hours post-infection, spleens and livers were collected, were identified by searching the sequence of the unique C-terminal domain of
homogenized and plated for the enumeration of CFUs. Ndh2 on the PSI-BLAST server52. To perform a phylogenetic analysis, represent-
L. monocytogenes oral infections. Previously described models of L. mono­ ative homologues were selected and aligned by ClustalW53. The maximum like-
cytogenes oral infection were adapted to address the role of EET in the intestinal lihood method was used to infer the evolutionary history of identified sequences
lumen45,46. Prior to infection, 5 mg/ml of streptomycin sulfate was added to the in Mega 7.0.26 and confidence limits of branch points were estimated by 1,000
drinking water of 8-week-old female C57BL/6 mice (The Jackson Laboratory). bootstrap replications54,55. The information about EET genetic loci summarized
After 24 h, mice were transferred to fresh cages and chow was removed to initiate in Supplementary Table 3 was acquired by analysing genomic context of identified
an overnight fast. Forty-eight hours after streptomycin addition to the water, mice genes in the PATRIC 3.5.1 database (https://www.patricbrc.org).
were isolated, fed a small piece of bread with 3 μl of butter and an inoculum with Statistics and reproducibility. No statistical methods were used to predetermine
108 CFUs of L. monocytogenes, and returned to cages containing standard drinking sample size. The experiments were not randomized and investigators were not
water and chow. To confine L. monocytogenes to the intestinal lumen, a Δhly paren- blinded to allocation during experiments and outcome assessment. Statistical anal-
tal strain (which has greatly reduced intracellular growth and spread) was used in yses were performed in Prism 5 for Mac OS X (GraphPad Software) and Progenesis
these experiments. Inoculums were prepared with a 1:1 ratio of Δhly and an eryth- QI for Proteomics version 4.0.
romycin-resistant Δhly strain (Δhly ermR, derived as previously described47) or Reporting summary. Further information on research design is available in
Δhly and Δhly ndh2::tn. Following infection, stools were collected, homogenized the Nature Research Reporting Summary linked to this paper.
and dilutions were plated. Because total parental strain CFUs did not statistically Data availability. The datasets generated during the current study are available
differ between conditions, results are simply reported as a competitive index (that from the corresponding author on reasonable request.
is, the ratio of streptomycin to erythromycin-resistant CFUs). These studies were
carried out in strict accordance with the recommendations in the Guide for the 37. Hodgson, D. A. Generalized transduction of serotype 1/2 and serotype 4b
Care and Use of Laboratory Animals of the National Institutes of Health. All pro- strains of Listeria monocytogenes. Mol. Microbiol. 35, 312–323 (2000).
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increase in (p)ppGpp. Cell Host Microbe 17, 788–798 (2015).
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© 2018 Springer Nature Limited. All rights reserved.

 Letter RESEARCH

Extended Data Fig. 1 | Electrochemical analyses of L. monocytogenes. labelled. b, Cyclic voltammograms of wild-type and ndh2::tn strains of
a, The double chamber cell used for electrochemical experiments. CE, L. monocytogenes. ‘Abiotic’ refers to an uninoculated control. Arrows
counter electrode; CEM, cation exchange membrane; RE, reference highlight the initiation of the catalytic wave. Results are representative of
electrode; WE, working electrode. Inlets and outlets for N2 gas are three independent experiments.

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter

Extended Data Fig. 2 | EET activity maintains cellular redox are expressed as mean ± s.e.m. A statistically significant difference
homeostasis. Ratio of NAD+ to NADH in wild-type and ndh2::tn between microaerophilic cells incubated with or without iron is indicated;
strains supplemented with ferric ammonium citrate under aerobic or *P = 0.0015, unpaired two-sided t-test.
microaerophilic conditions. Results from three independent experiments

© 2018 Springer Nature Limited. All rights reserved.

 Letter RESEARCH

Extended Data Fig. 3 | Evidence that a distinct menaquinone ± s.e.m. b, The L. monocytogenes hep operon. Notably, menG—which
derivative functions in aerobic respiration. a, Ferric iron reductase encodes demethylmenaquinone transferase (the enzyme that converts
activity of mutants described in Fig. 2 demonstrates that genes essential demethylmenaquinone to menaquione) (Fig. 2b)—neighbours the hepT
for growth on aerobic respiration medium are dispensable for EET. and hepS genes, which function in quinone biosynthesis and are essential
Results from three independent experiments are expressed as mean for aerobic respiration (Fig. 2c).

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RESEARCH Letter

Extended Data Fig. 4 | Recombinant FmnB FMNylates PplA at two FAD + FmnB (b). The observed molecular weight change (877 Da) is
discrete sites. a, b, Deconvoluted mass spectra from a single experiment consistent with two post-translational FMNylations (2 × 438.3 Da) on
of recombinant PplA (a) and recombinant PplA incubated with PplA.

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 Letter RESEARCH

Extended Data Fig. 5 | Proposed role of RibU and FmnA in FAD to promote FAD secretion. c, Ferric iron reductase activity of strains
secretion. a, Simplified adaptation of a previously proposed model of incubated with 0.5 mM FAD for 1 h. The ability of exogenous FAD to
L. monocytogenes riboflavin uptake through the RibU, EcfT, EcfA and specifically rescue ferric iron reductase activity in the fmnA::tn and
EcfA’ transporter10. According to this model, EcfT, EcfA and EcfA’ couple ribU::tn strains is consistent with FmnA and RibU functioning in FAD
ATP hydrolysis with conformational changes that result in substrate secretion. Results from three independent experiments are expressed as
bound to RibU being released into the cytosol. b, On the basis of protein mean ± s.e.m. Statistically significant differences between untreated and
homology (FmnA shares 50% sequence identity with EcfT) and the FAD-treated cells are indicated; *P = 0.038, **P < 0.0001, unpaired two-
expectation that extracellular FAD is required for FmnB to catalyse sided t-test.
FMNylation of PplA, we propose that the FmnA interacts with RibU

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RESEARCH Letter

Extended Data Fig. 6 | Flavin shuttles promote EET activity. with insoluble ferric (hydr)oxide (top) and soluble ferric ammonium
a, Chronoamperometry results from L. monocytogenes-inoculated citrate (bottom). With insoluble substrate the local iron concentration
electrochemical reactors with 1 μM FMN injections at the indicated time for most cells is low, whereas with soluble substrate the concentration
points. Results are representative of three independent experiments. b, The of iron in the direct vicinity of cells is high (insets). Results from three
effect of flavins on L. monocytogenes (Lm) ferric iron reductase activity independent experiments are expressed as mean ± s.e.m.

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 Letter RESEARCH

Extended Data Fig. 7 | EET supports anaerobic growth on ferric iron. growing cells. b, CFUs of L. monocytogenes strains anaerobically incubated
a, Growth following incubation of L. monocytogenes strains on xylitol in xylitol medium without (−) or with (+) ferric supplementation. Results
medium without (left) or with (right) ferric iron under aerobic (top) for soluble ferric ammonium citrate (top) and insoluble ferric (hydr)
or anaerobic (bottom) conditions. Results are representative of three oxide (bottom) are shown. Dashed lines denote the number of cells at the
independent experiments. Strain labels are coloured based on attributed start of the experiment. Results from three independent experiments are
deficiencies (Fig. 2d) in aerobic respiration (blue) or EET (red). Ndh1 and expressed as mean ± s.e.m. Statistically significant differences in the ferric
Ndh2 are probably functionally redundant under aerobic conditions, as a iron-supplemented condition are noted; ***P < 0.0001, unpaired two-
growth phenotype is only observed in the double mutant. Note the visual sided t-test.
evidence of ferrous iron production in the agar adjoining anaerobically

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RESEARCH Letter

Extended Data Fig. 8 | EET genes are dispensable for L. monocytogenes expressed as mean ± s.e.m. b, L. monocytogenes burdens in mouse organs
intracellular growth. a, Mouse bone-marrow-derived macrophages (n = 5) 48 h after intravenous infection. Representative results from two
were infected with L. monocytogenes, and CFUs were enumerated at independent experiments are expressed as median and s.e.
the indicated times. Results from three independent experiments are

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 Letter RESEARCH

Extended Data Fig. 9 | Identified EET loci are widespread in the those depicted in Extended Data Fig. 5a) that probably function with
Firmicutes phylum. a, Phylogenetic tree constructed from select Ndh2 RibU and FmnA subunits in flavin transport. The dmkA-like gene found
homologue sequences. A more comprehensive list of organisms that in Caldanaerobius fijiensis (and other genomes) lacks homology to dmkA,
possess an EET locus is provided in Supplementary Table 3. Labels on the but is annotated as catalysing the same reaction. The pplA variant in some
branches refer to the percentage of replicate trees that gave the depicted genomes contains a single FMNylated domain (rather than two) and
branch topology in a bootstrap test of 1,000 replicates. b, Distinct EET this property is indicated by a shorter arrow. A few bacteria (including
loci from select genomes are shown. Although the arrangement of genes Lactococcus spp.) lack a recognizable locus and distribute genes associated
varies, a locus with genes associated with EET is present in many genomes. with EET throughout the genome.
Some loci contain ECF transporter ATPase subunits (homologous to

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 nature research | reporting summary
Corresponding author(s): Daniel Portnoy

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