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Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing 1
effects on growth and the iron-sulfur enzyme MiaB 2
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Jeffrey C. Waller1,4*, Kenneth W. Ellens1, Ghulam Hasnain1, Sophie Alvarez2, James R. Rocca3, 4
and Andrew D. Hanson1 5
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1Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611; 7
2Donald Danforth Plant Science Center, St. Louis, MI 63132; 8
3AMRIS Facility, McKnight Brain Institute, University of Florida, Gainesville, FL 32610; 9
4Present address, Department of Chemistry and Biochemistry, Mount Allison University, 10
Sackville, New Brunswick, E4L 1G8, Canada 11
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Running Title: Opposing effects of YgfZ and MnmEG 13
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* Room 119, Barclay Building, Dept. of Chemistry and Biochemistry, Mount Allison University, 15
Sackville, New Brunswick, E4L 1G8, Canada 16
Phone: (506) 364-2310 17
Fax: (506) 364-2313 18
Email: jwaller@mta.ca 19
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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.06226-11 JB Accepts, published online ahead of print on 11 November 2011
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ABSTRACT 21
The folate-dependent protein YgfZ of Escherichia coli participates in synthesis and repair of 22
iron-sulfur (Fe/S) clusters; it belongs to a family of enzymes that use folate to capture formalde-23
hyde units. Ablation of ygfZ is known to reduce growth, to increase sensitivity to oxidative 24
stress, and to lower the activities of MiaB and other Fe/S enzymes. It has been reported that the 25
growth phenotype can be suppressed by disrupting the tRNA modification gene mnmE. We first 26
confirmed the latter observation using deletions in a simpler, more defined genetic background. 27
We then showed that deleting mnmE substantially restores MiaB activity in ygfZ deletant cells, 28
and that overexpressing MnmE with its partner MnmG exacerbates the growth and MiaB activity 29
phenotypes of the ygfZ deletant. MnmE, with MnmG, normally mediates a folate-dependent 30
transfer of a formaldehyde unit to tRNA, and the MnmEG-mediated effects on the phenotypes of 31
the ΔygfZ mutant apparently require folate, as evidenced by the effect of eliminating all folates 32
by deleting folE. Expression of YgfZ was unaffected by deleting mnmE or overexpressing 33
MnmEG, or by folate status. Since formaldehyde transfer is a potential link between MnmEG 34
and YgfZ, we inactivated formaldehyde detoxification by deleting frmA. This deletion had little 35
effect on growth or MiaB activity in the ΔygfZ strain in the presence of formaldehyde, making it 36
unlikely that formaldehyde alone connects the actions of MnmEG and YgfZ. A more plausible 37
explanation is that MnmEG erroneously transfers a folate-bound formaldehyde unit to MiaB and 38
that YgfZ reverses this. 39
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INTRODUCTION 41
Iron-sulfur (Fe/S) clusters are versatile cofactors that are typically liganded to cysteine residues 42
in proteins (5, 19). Fe/S-clusters are highly versatile; the proteins that contain them play diverse 43
roles in electron transfer, enzyme catalysis, and transcriptional regulation (17). Although Fe/S 44
clusters are simple structures, a complex machinery involving over twenty proteins is needed for 45
their assembly, insertion into apoproteins, and maintenance (17). This machinery is incompletely 46
understood (10, 19). 47
A newly discovered component of this machinery is the COG0354 protein family. COG-48
0354 proteins are known to occur in all domains of life and to participate in the maturation of a 49
subset of Fe/S proteins and in combating oxidative stress (12, 20, 28, 38). Null mutation of 50
COG0354 causes growth defects in Escherichia coli, particularly under oxidative stress (20, 30, 51
37 anemia in zebrafish embryos (29), a petite phenotype in yeast (12), and lethality in plants 52
(39). The E. coli COG0354 protein, YgfZ, has been shown to help maintain the activity of the 53
Fe/S enzyme MiaB, a tRNA modification enzyme that catalyzes the methylthiolation of N6-54
isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) (16, 30, 38). YgfZ 55
was also demonstrated to maintain the activities of the Fe/S enzymes succinate dehydrogenase, 56
dimethylsulfoxide reductase, 6-phosphogluconate dehydratase, and fumarase (38). The biochem-57
ical function of COG0354 proteins remains unknown but evidence from genetically manipul-58
ating in vivo folate contents indicates that E. coli YgfZ and its yeast counterpart Iba57p require a 59
tetrahydrofolate (THF) species, almost certainly THF itself (11, 38) rather than a one carbon-60
subsituted THF molecule. Consistent with this evidence, YgfZ binds a stable model folate in 61
vitro and its 3D structure contains a predicted folate binding site (35, 38). Moreover, COG0354 62
proteins are paralogous to the THF-dependent enzymes glycine cleavage T protein, sarcosine 63
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oxidase, and dimethylglycine oxidase (34, 35, 38). These three enzymes all use THF to accept a 64
formaldehyde unit, specifically yielding 5,10-methylene-THF. It is thus reasonable to hypoth-65
esize that YgfZ accepts a formaldehyde unit using a THF molecule, as do its paralogues. 66
Ote et al. (30) reported that a growth phenotype of an E. coli ygfZ disruptant was partially 67
suppressed by disrupting mnmE (trmE). MnmE and its partner MnmG (GidA) form the hetero-68
tetramer MnmEG, which uses 5,10-methylene-THF as formaldehyde donor for a tRNA modific-69
ation reaction (25). MnmEG thus mediates folate-dependent formaldehyde donation whereas 70
paralogs of YgfZ (and perhaps YgfZ itself) mediate folate-dependent formaldehyde removal. 71
Given this possible reciprocity of action, the opposing effects of MnmEG and YgfZ, if confirm-72
ed, seemed likely to provide insights into the biochemical function of YgfZ. Accordingly, in this 73
study we first used deletants in a simple, well-defined genetic background to validate and extend 74
the observations of Ote et al. (30), then applied genetic, comparative genomic, and biochemical 75
approaches to search for links between MnmEG and YgfZ. 76
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MATERIALS AND METHODS 78
Bioinformatics. Prokaryote genomes were analyzed using the SEED database and its tools (31). 79
Full results are available at http://theseed.uchicago.edu/FIG/ in the YgfZ subsystem. 80
81
Bacterial strains, plasmids, and media. Strains, plasmids, and primers are listed in Tables S1 82
and S2 in the supplemental material. Deletions from the Keio collection (3) were transferred to 83
E. coli K12 MG1655 by P1 transduction (24); for double deletants, kan cassettes were removed 84
by flippase-mediated recombination using the pCP20 plasmid (9). Deletions were verified by 85
sequencing. Cells were grown at 37°C in Antibiotic Medium 3 (Difco), LB medium, MOPS 86
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minimal medium plus 0.2% (wt/vol) glucose (27), or M9 minimal medium plus 0.2% (wt/vol) 87
glycerol as indicated. Media were solidified with 15 g of agar/liter; ampicillin was added to 50 88
μg/ml. Gene expression was induced with 0.02% (wt/vol) L-arabinose. Where indicated, 89
plumbagin or formaldehyde were added to give final concentrations of 30 µM or 0.2 mM, 90
respectively. Folate mutants were cultured as described (38). 91
92
Expression constructs. For the mnmEG construct, the mnmE ORF was amplified from E. coli 93
K12 MG1655 genomic DNA with primers mnmE-Fwd and -Rev, digested with BspHI and XbaI, 94
and ligated into similarly digested pBAD24 (14) to give pBAD24::mnmE. The mnmG ORF was 95
amplified with primers mnmG-Fwd and-Rev, digested with NcoI and SalI, and ligated into 96
similarly digested pET28b(+) (Novagen), yielding pET28b(+)::mnmG. A XbaI/SalI fragment 97
(containing a 27 bp upstream spacer, the ribosome binding site, and mnmG) was excised from 98
pET28b(+)::mnmG and ligated downstream of mnmE in similarly digested pBAD24::mnmE to 99
give the bicistronic pBAD24::mnmEG construct. For pBAD24::mnmG, the NcoI and SalI-digest-100
ed mnmG amplicon was ligated into similarly digested pBAD24. All constructs were sequenced. 101
102
Immunoblot analysis. Cells were grown in Antibiotic Medium 3 to an OD600 nm of 1.0, harvest-103
ed by centrifugation (4,000 × g, 10 min, 4°C) and washed once in ice-cold phosphate-buffered 104
saline. Proteins were extracted from cell pellets by lysis with a Mini-Beadbeater in 0.1 M Tris-105
HCl (pH 7.5), 0.2 M KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM 106
benzamidine, 1 mM phenylmethylsulfonyl fluoride, 5 mM ε-aminocaproic acid. Extracts were 107
centrifuged (12,000 × g, 20 min, 4°C) to clear. Protein was estimated by dye-binding (6) with 108
bovine serum albumin as standard. Electrophoresis and immunoblotting were as described (36). 109
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Antiserum was raised in rabbits (Cocalico Biologicals Inc.) against hexahistidine-tagged, 110
denatured YgfZ prepared as described (38), and was diluted 1:1000. 111
112
MiaB activity analysis. Bulk nucleic acids were isolated from stationary phase cells cultured in 113
Antibiotic Medium 3 or MOPS medium plus glucose as above and enriched for tRNA (4) before 114
Nucleobond AXR 400 column purification (Machery-Nagel). Purified tRNA was then 115
hydrolyzed and analyzed by liquid chromatography-tandem mass spectrometry as described (32). 116
For statistical treatment, blocks were based on the replicates and were treated as random. Log-117
transformed data were analyzed by one-way ANOVA using Glimix procedures (SAS Institute 118
Inc., Cary, NC). Multiple comparisons were adjusted by Tukey-Kramer. 119
120
NMR analysis of E. coli cells incubated with [13C]formaldehyde. [13C]Formaldehyde (99% 121
atom percent) was from Cambridge Isotope Laboratories. Wild type and ΔfrmA strains were 122
grown in 100 ml of MOPS minimal medium to an OD600 of 1.0. Culture samples (30 ml) were 123
centrifuged (5,000 × g, 20 min, 4°C) and the pellets resuspended in 600 µl of 100 mM K-phos-124
phate buffer, pH 7.4, and kept on ice until analysis. Samples for NMR contained 525 µl of cell 125
suspension, 65 µl of D2O, and 10 µl of 0.6 M H13CHO (final concentration 10 mM, 22). 126
Directly detected 126 MHz 13C-NMR spectra were obtained at 37°C on a Bruker Avance-500 127
instrument equipped with a 5 mm broadband observe (BBO) probe. Each data set was acquired 128
in 600 scans over a spectral width of 240 ppm using a 45° pulse, a 1 sec acquisition time, and a 2 129
sec relaxation delay. Composite-pulse 1H-decoupling was employed only during the acquisition, 130
and not during the delay, using the “zgig” pulse program. The data as FID’s were zero-filled 131
once and processed with 2 Hz exponential line broadening before Fourier transformation. 132
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Stacked spectral plots were prepared using Bruker’s XWinPlot software. Spectra were acquired 133
at 30 min intervals. 134
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RESULTS AND DISCUSSION 136
Deleting mnmE partially reverses ΔygfZ growth and MiaB activity defects. The phenotype 137
exhibited by the ΔygfZ mutant in rich media is moderately slowed growth; either oxidative stress 138
(imposed with plumbagin) or growth in M9 minimal medium aggravates this defect (20, 30, 38). 139
We therefore tested whether the reported suppression by mnmE disruption of the moderate ygfZ 140
growth defect in rich medium (30) could be reproduced in oxidative stress conditions or in M9 141
medium, using full deletants in a well-defined (K12 MG1655) background. This proved to be the 142
case; deletion of mnmE in the ΔygfZ background largely reversed the severe ΔygfZ growth defect 143
observed in plumbagin-containing or M9 medium (Fig. 1A). Deletion of mnmE alone had no 144
effect (Fig. 1A). 145
To extend the analysis to the biochemical level, we measured the in vivo activity of MiaB, 146
which is known to depend upon YgfZ (30, 38). The ratio of MiaB product to substrate, i.e., the 147
ms2i6A/i6A ratio in tRNA, is a semiquantitative measure of MiaB activity (38). When cultured in 148
Antibiotic Medium 3, wild type E. coli typically has a ratio of 20-100, whereas that of ΔygfZ 149
mutants is <2 (30, 38). As expected, wild type cells showed a high ms2i6A/i6A ratio, as did and 150
ΔmnmE cells, and ΔygfZ cells showed a low one (Fig. 1B). Relative to the ΔygfZ single mutant, 151
the ΔmnmE ΔygfZ double mutant showed increased ms2i6A, decreased i6A, and restoration of the 152
ms2i6A/i6A ratio to 28% of the wild type level (Fig. 1B). Collectively, these data fit with the 153
possibility that MnmEG damages MiaB, contributing to growth defects, and is in some way 154
opposed by YgfZ. 155
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156
Overexpressing MnmEG depressess growth and MiaB activity. Since mnmE deletion, and 157
thus loss of the MnmEG complex, partially reversed the growth and MiaB activity phenotypes of 158
the ΔygfZ strain, we tested whether overexpression of MnmEG is detrimental. Overexpression of 159
MnmEG in the ΔygfZ background exacerbated the moderate growth defect in LB medium, and 160
also slightly reduced growth of the wild type (Fig. 2A). Similar effects were observed on MiaB 161
activity; MnmEG overexpression reduced the already low ms2i6A/i6A ratio in the ΔygfZ strain by 162
a further 72%, and caused a smaller (36%) but significant reduction in the much higher ratio in 163
the wild type strain (Fig. 2B). Expressing MnmE or MnmG alone did not affect growth of ΔygfZ 164
or wild type strains (not shown). These results again fit with MnmEG-mediated damage to 165
MiaB; they also indicate that this damage requires the enzymatically competent MnmEG 166
complex, not merely its individual subunits. 167
168
Evidence that the detrimental effect of MnmEG requires folate. As already noted, MnmEG 169
uses 5,10-methylene-THF in a tRNA modification reaction. We therefore investigated whether 170
MnmEG-mediated damage to MiaB is also folate-dependent. MiaB activity (ms2i6A/i6A ratio) 171
was measured in a ΔfolE mutant that lacks folates (38), and in the double ΔfolE ΔmnmE mutant. 172
Because MiaB activity depends upon folate being available to YgfZ, it is reduced in ΔfolE 173
strains (38), but the remaining activity is sufficient to test the influence of MnmEG. Were the 174
MnmEG-mediated reaction that damages MiaB folate-independent, deleting mnmE in the ΔfolE 175
background should raise MiaB activity because the damage would be removed. Eliminating 176
damage should enhance MiaB activity even though YgfZ is inactive for want of folate. Con-177
versely, if the MnmEG damage reaction was strictly folate-dependent, there should be no differ-178
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ence in MiaB activity between the ΔfolE and ΔfolE ΔmnmE strains because damage would 179
already be absent due to loss of folate. The latter result was observed: The ms2i6A/i6A ratios in 180
the ΔfolE and ΔfolE ΔmnmE mutants were the same (Fig. 3). 181
182
MnmEG expression and folate status do not affect YgfZ expression. The arguments in the 183
three preceding sections assume that MnmEG level and folate status do not impact YgfZ expr-184
ession. Both assumptions were validated by immunoblot analysis: There was no effect on YgfZ 185
expression of deleting mnmE or mnmG (Fig. S1A), of overexpressing MnmEG (Fig. S1B), of 186
deleting folE, or of other genetic perturbations to the folate pool (Fig. S1C). YgfZ is induced by 187
oxidative stress (7, 20) so the lack of effect of MnmEG overexpression (Fig. S1B) implies that 188
the damage caused by MnmE probably does not entail oxidative stress. 189
190
Examining formaldehyde-YgfZ-MiaB connections. Although 5,10-methylene-THF is normal-191
ly made enzymatically, it can also form spontaneously from THF and formaldehyde from cellul-192
ar reactions or the environment (1, 18, 21, 33). Formaldehyde also forms adducts with protein-193
bound thiol and amino groups spontaneously and readily (23), and so might in principle damage 194
MiaB independently of MnmEG. We therefore sought links between formaldehyde, YgfZ, and 195
MiaB using comparative genomic and experimental approaches. 196
Formaldehyde is metabolized mainly via its spontaneous adduct with glutathione (S-hydr-197
oxymethylglutathione), which is oxidized to S-formylglutathione by FrmA; S-formylglutathione 198
is then hydrolyzed by FrmB and YeiG, giving formate (Fig. 4A) (13). A survey of 858 bacterial 199
genomes in the SEED database (31) revealed no tendency for frmA or other formaldehyde 200
metabolism genes to cluster on the chromosome with ygfZ. Nor were frmA and ygfZ co-201
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distributed: ygfZ occurred without frmA in 197 genomes and frmA occurred without ygfZ in two; 202
303 genomes had both genes and 353 had neither. Comparative genomics thus detects no link 203
between formaldehyde metabolism genes and YgfZ. However, the methyltrophic bacterium 204
Methylophilales bacterium HTCC2181 has a COG0354 protein family member in an operon with 205
proteins involved in methanol dehydrogenase activity, a reaction known to generate 206
formaldehyde (15). 207
In our experimental approach, we first tested single and double ygfZ and frmA deletants 208
for sensitivity to supplied formaldehyde, selecting a concentration (0.2 mM) shown in pilot tests 209
to slightly inhibit growth of wild type cells in minimal medium. (Minimal medium was used to 210
avoid formaldehyde adduct formation with the organic constituents of rich media.) No deletant 211
strain was substantially more sensitive to formaldehyde than the wild type (Fig. 4A). We then 212
analyzed MiaB activities in the presence and absence of formaldehyde (Fig. 4B). As expected, 213
the ms2i6A/i6A was significantly depressed in the ΔygfZ strain although the magnitude differed 214
from that in Fig. 1B due to use of minimal medium. Deleting frmA had little effect alone or 215
combined with the ygfZ deletion, whether or not formaldehyde was supplied. These data indicate 216
that formaldehyde itself, as opposed to its folate-bound form 5,10-methylene-THF, does not 217
cause the damage to MiaB that YgfZ opposes. 218
The above inference rests on the assumptions that supplied formaldehyde reaches the 219
cytosol, rather than being intercepted by adduct formation in the cell wall or periplasm, and that 220
deleting frmA raises cytosolic formaldehyde levels. To test both assumptions, we gave [13C]form-221
aldehyde to wild type or ΔfrmA cells and followed its fate by NMR. In wild type cells, [13C]form-222
aldehyde, observed as its hydrate H213C(OH)2 and its glutathione adduct, was progressively meta-223
bolized to [13C]formate (Fig. S2). In the ΔfrmA strain, however, little [13C]formate was formed 224
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and the hydrate remained predominant (Fig. S2). These data thus validate the two assumptions, 225
although it should be noted that, in order to obtain strong NMR signals (22), a higher formalde-226
hyde level was used than in the experiments on growth and MiaB activity (10 mM vs. 0.2 mM). 227
228
Conclusions. Biochemical roles for YgfZ have been proposed in the past but were unsupported 229
by experimental data (30, 35). Here, we present genetic and biochemical evidence that the phen-230
otypes of ΔygfZ strains are, in part, due to a folate-dependent reaction catalyzed by MnmEG that 231
damages MiaB, and that the folate-dependent action of YgfZ counters this damage. Our data also 232
indicate that MnmEG is not the sole source of MiaB damage; were it so, deleting mnmE in the 233
ΔfolE strain would have fully restored MiaB activity (Fig. 1B), and MiaB activity in the ΔfolE 234
strain would have been higher than it was (Fig. 3). Moreover, YgfZ occurs in many genomes that 235
lack MnmEG (e.g. Actinobacteria), implying that MiaB is subject to additional types of damage. 236
The miaB deletant has relatively subtle growth phenotypes (28) whereas overexpressing 237
mnmEG in the ΔygfZ background causes quite severe growth and MiaB activity phenotypes. It 238
would consequently seem unlikely that MiaB is the only enzyme affected by the detrimental 239
action of MnmEG, just as it is not the only one benefited by YgfZ. It is therefore reasonable to 240
speculate that, in relation to MnmEG-mediated damage, MiaB is in effect a proxy for other 241
enzymes, as it is in relation to Fe/S enzyme activity loss when ygfZ is deleted (38). 242
Many cases are known where macro- or micromolecules are damaged by enzymatic mis-243
takes or spontaneous chemical reactions, and then enzymatically repaired (8, 37). The opposing 244
effects of MnmEG and YgfZ could reflect a system of this kind. Given that the MnmEG com-245
plex normally transfers a formaldehyde unit from folate to an tRNA (25), that close relatives of 246
RNA modification enzymes modify proteins instead (2, 26), and that proteins form formaldehyde 247
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adducts (23), it is conceivable that MnmEG occasionally and by mistake transfers a formalde-248
hyde unit to a sensitive residue in MiaB, and perhaps to such residues in other proteins. YgfZ 249
might strip this unit off and transfer it to THF. Evidence for such a system might in principle be 250
obtained from a proteomics search for modifications to MiaB that appear in the ΔygfZ mutant. 251
However, as formaldehyde adduct formation is reversible (18, 23) such a search would not be 252
straightforward. 253
254
ACKNOWLEDGEMENTS 255
This work was supported in part by National Science Foundation grant number MCB-0839926 256
and by an endowment from the C.V. Griffin Sr. Foundation. The authors gratefully acknowledge 257
the National Science Foundation through the National High Magnetic Field Laboratory, which 258
supported our NMR studies, in part, at the Advanced Magnetic Resonance Imaging and 259
Spectroscopy (AMRIS) facility in the McKnight Brain Institute of the University of Florida. We 260
thank S.M. Beverley, Arthur S. Edison, and V. de Crécy-Lagard for advice. Statistical analysis 261
was provided by Dongyan Wang of the IFAS Statistics Department at the University of Florida. 262
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ALB+plumbagin M9+glycerol
mnmEygfZ
ΔmnmEΔygfZ
WT
B
6 A r
atio
3
5
60
100
120
(x10
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unts
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s2i6
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ms2
i6A
/i6
ΔmnmE ΔygfZ
ΔmnmEWT ΔygfZ0
1
2
0
Pea
k ar
ea
i6A
40
20
FIG. 1. The effect of deleting mnmE on the growth and MiaB phenotype of the ΔygfZ strain. (A)FIG. 1. The effect of deleting mnmE on the growth and MiaB phenotype of the ΔygfZ strain. (A)Growth of three independent clones of wild type (WT), ΔmnmE, ΔygfZ, and ΔmnmE ΔygfZ strainson M9 medium plus 0.2% glycerol and 1 μM FeSO4 after 4 d at 22oC, or on LB medium with 30μM plumbagin after 12 h at 37oC. (B) LC-MS quantification of i6A and ms2i6A, and the ms2i6A/i6Aratio, in tRNA of wild type, ΔmnmE, ΔygfZ, and ΔmnmE ΔygfZ strains grown in Antibiotic Medium3. Data are means and standard errors for three independent cultures.
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ApBAD in WTpBAD in ΔygfZ
B
0.20
ratio
ΔygfZ
2.0
2.5
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3.0
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ount
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pBAD::mnmEGin WT
pBAD::mnmEGin ΔygfZ
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pBAD mnmEG
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i6A
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r
0.5
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1.5
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0.15
WT
100x
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6.0
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io
i6A
0
40
60
120
Pea
k ar
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ts)
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100x
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80
100
pBAD mnmEG00
FIG. 2. Exacerbation of the growth and MiaB phenotypes of the ΔygfZ strain by overexpression ofg p yp yg y pmnmEG. (A) Growth of three independent clones of wild type (WT) and ΔygfZ strains transformedwith pBAD24 (pBAD) alone or pBAD::mnmEG. The plate contained LB medium plus 0.02% L-arabinose. (B) LC-MS quantification of i6A and ms2i6A, and the ms2i6A/i6A ratio, in tRNA of the wildtype and ΔygfZ strains transformed with pBAD24 alone or pBAD24::mnmEG. Strains were grown inAntibiotic Medium 3 plus 0.02% L-arabinose and 100 μg/ml ampicillin. Data are means andstandard errors for three independent cultures. For i6A and ms2i6A data, some error bars are toosmall to be visible The i6A data for wild type cells are shown in both normal (1×) and 100-foldsmall to be visible. The i A data for wild type cells are shown in both normal (1×) and 100 foldmagnified (100×) formats.
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ratio3
4
3
4
108
coun
ts)
s2i6 A
ΔfolE ΔfolE ΔmnmE
ms2
i6 A/i6
A r
i6 A
0
1
2
0
1
Pea
k ar
ea (x
1 ms
2
FIG. 3. Investigation of the folate dependence of the effect of MnmE on MiaB activity. LC-MSg p yquantification of i6A and ms2i6A, and the ms2i6A/i6A ratio, in tRNA of the ΔfolE and ΔfolE ΔmnmEstrains grown in Antibiotic Medium 3. Data are means and standard errors for three independentcultures.
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AGSH GS-CH2OH GS-CHO GSH + HCOOH
FrmAFrmBYeiGHCHO
GSH GS CH2OH GS CHO GSH HCOOH
WT ΔfrmA
- HCHO + HCHO
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200
A (
x106
)
100
200
Pea
k ar
ea (
coun
ts)
ms2
i6A
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300
ee e e
a
abcd
bd abcd
ee
abc ac
ee
bcd
d
40
120
80
160
ms2
i6A
/i6 A
rat
io
I6A
0WT WT
+HCHOΔfrmAΔygfZ
+HCHO
ΔfrmAΔygfZ
ΔfrmA ΔfrmA+HCHO
ΔygfZ ΔygfZ+HCHO
0
FIG. 4. Effect on formaldehyde on the growth and MiaB phenotypes of single or double ΔfrmA andy g p yp gΔygfZ mutant strains. Cells were grown in MOPS medium with 0.2% glucose, minus or plus 0.2 mMformaldehyde, for 48 h. At earlier times (e.g. 36 h) the double mutant showed marginally less growththan other strains. (A) Growth of three independent clones of wild type (WT), ΔfrmA, and ΔygfZstrains. (B) LC-MS quantification of i6A and ms2i6A, and the ms2i6A/i6A ratio, in tRNA of wild type,ΔfrmA, ΔygfZ, and ΔfrmA ΔygfZ strains. Data are means and standard errors for three independentcultures. Means not designated with the same letter were significantly different at P = 0.05 (seeMaterials and Methods) Note that these experiments used MOPS minimal medium to preventMaterials and Methods). Note that these experiments used MOPS minimal medium to preventformaldehyde titration by adduct formation with medium components; the ms2i6A/i6A ratios are thusnot directly comparable to those in other figures, where rich Antibiotic Medium A was used.
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