1

Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing 1

effects on growth and the iron-sulfur enzyme MiaB 2

3

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

12

Running Title: Opposing effects of YgfZ and MnmEG 13

14

* 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

20

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

77

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

135

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

REFERENCES 263

1. Aas, P.A.,M. Otterlei, P.O. Falnes, C.B. Vagbo, F. Skorpen, M. Akbari, O. Sundheim, 264

M. Bjoras, G. Slupphaug, E. Seeberg, and H.E. Krokan. 2003. Human and bacterial 265

oxidative demethylases repair alkylation damage in both RNA and DNA. Nature. 421:859–266

863. 267

2. Anton, B.P., L. Saleh, J.S. Benner, E.A. Raleigh, S. Kasif, and R.J. Roberts. 2008. 268

RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of 269

ribosomal protein S12 in Escherichia coli. Proc. Natl. Acad. Sci. USA 105:1826-1831. 270

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

13

3. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. 271

Tomita, B. L. Wanner, and H. Mori. 2006. Construction of Escherichia coli K-12 in-272

frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2:2006.0008. 273

4. Bailly, M., M. Blaise, H. Roy, M. Deniziak, B. Lorber, C. Birck, H.D. Becker, and D. 274

Kern. 2008. tRNA-dependent asparagine formation in prokaryotes: Characterization, 275

isolation and structural and functional analysis of a ribonucleoprotein particle generating 276

Asn-tRNAAsn. Methods 44:146-163. 277

5. Beinert, H. 2000. Iron-sulfur proteins: ancient structures, still full of surprises. J. Biol. 278

Inorg. Chem. 5:2-15. 279

6. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram 280

quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-281

254. 282

7. Chen, J.W., C.M. Sun, W.L. Sheng, Y.C. Wang, and W.J. Syu. 2006. Expression 283

analysis of up-regulated genes responding to plumbagin in Escherichia coli. J. Bacteriol. 284

188:456-463. 285

8. Clarke, S. 2003. Aging as war between chemical and biochemical processes: protein 286

methylation and the recognition of age-damaged proteins for repair. Ageing Res. Rev. 287

2:263-285. 288

9. Datsenko, K.A., and B.L. Wanner. 2000. One-step inactivation of chromosomal genes in 289

Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645. 290

10. Fontecave, M., and S. Ollagnier de Choudens. 2008. Iron-sulfur cluster biosynthesis in 291

bacteria: Mechanisms of cluster assembly and transfer. Arch. Biochem. Biophys. 474:226-292

237. 293

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

14

11. Gelling, C.E. 2008. Tetrahydrofolate and iron-sulfur metabolism in Saccharomyces 294

cerevisiae. PhD thesis. University of New South Wales, Sydney Australia. 295

12. Gelling, C., I.W. Dawes, N. Richhardt, R. Lill, and U. Mühlenhoff. 2008. Mitochondrial 296

Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM 297

enzymes. Mol. Cell. Biol. 28:1851-1861. 298

13. Gonzalez, C.F., M. Proudfoot, G. Brown, Y. Korniyenko, M. Mori, A.V Savchenko, 299

and A.F. Yakunin. 2006. Molecular basis of formaldehyde detoxification: 300

Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and 301

YeiG. J. Biol. Chem. 281:14514-14522. 302

14. Guzman, L.-M., D. Belin, M.J. Carson, and J. Beckwith. 1995. Tight regulation, 303

modulation, and high-level expression by vectors containing the arabinose PBAD promoter. 304

J. Bacteriol. 177:4121-4130. 305

15. Halsey, K.H., A.E. Carter, and S.J. Giovannoni, 2011. Synergistic metabolism of a 306

broad range of C1 compounds in the marine methylotrophic bacterium HTCC2181. 307

Environ. Microbiol. doi: 10.1111/j.1462-2920.2011.02605.x 308

16. Hernández, H.L., F. Pierrel, E. Elleingand, R. García-Serres, B.H. Huynh, M.K. 309

Johnson, M. Fontecave, and M. Atta. 2007. MiaB, a bifunctional radical-S-adenosyl-310

methionine enzyme involved in the thiolation and methylation of tRNA, contains two 311

essential [4Fe-4S] clusters. Biochemistry 46:5140-5147. 312

17. Johnson, D.C., D.R. Dean, A.D. Smith, and M.K. Johnson. 2005. Structure, function, 313

and formation of biological iron-sulfur clusters. Annu. Rev. Biochem.74:247-281. 314

18. Kallen, R.G. and W.P. Jencks. 1966. The mechanism of the condensation of formalde-315

hyde with tetrahydrofolic acid. J. Biol. Chem. 241:5851-5863. 316

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

15

19. Lill, R., and U. Mühlenhoff. 2008. Maturation of iron-sulfur proteins in eukaryotes: 317

mechanisms, connected processes, and diseases. Annu. Rev. Biochem. 77:669-700. 318

20. Lin, C.N., W.J. Syu, W.S. Sun, J.W. Chen, T.H. Chen, M.J. Don, and S.H. Wang. 319

2010. A role of ygfZ in the Escherichia coli response to plumbagin challenge. J. Biomed. 320

Sci. 17:84. 321

21. Ma, T. H., and M.M. Harris. 1988. Review of the genotoxicity of formaldehyde. 322

Mutat. Res. 196: 37–59. 323

22. Mason, R.P., J.K. Sanders, A. Crawford, and B.K. Hunter. 1986. Formaldehyde 324

metabolism by Escherichia coli. Detection by in vivo 13C NMR spectroscopy of S-325

(hydroxymethyl) glutathione as a transient intracellular intermediate. Biochemistry. 326

25:4504-4507. 327

23. Metz, B., G.F. Kersten, P. Hoogerhout, H.F. Brugghe, H.A. Timmermans, A. de Jong, 328

H. Meiring, J. ten Hove, W.E. Hennink, D.J. Crommelin, and W. Jiskoot. 2004. 329

Identification of formaldehyde-induced modifications in proteins: reactions with model 330

peptides. J. Biol. Chem. 279:6235-6243. 331

24. Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory 332

Press, Cold Spring Harbor, NY. 333

25. Moukadiri, I., S. Prado, J. Piera, A. Velázquez-Campoy, G.R. Björk, and M.E. 334

Armengod. 2009. Evolutionarily conserved proteins MnmE and GidA catalyze the format-335

ion of two methyluridine derivatives at tRNA wobble positions. Nucleic Acids Res. 336

37:7177-7193. 337

26. Nakahigashi, K., N. Kubo, S. Narita, T. Shimaoka, S. Goto, T. Oshima, M. Mori, M. 338

Maeda, C. Wada, and H. Inokuchi. 2002. HemK, a class of protein methyl transferase 339

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

16

with similarity to DNA methyl transferases, methylates polypeptide chain release factors, 340

and hemK knockout induces defects in translational termination. Proc. Natl. Acad. Sci. 341

USA 99:1473-1478. 342

27. Neidhardt, F.C, P.L. Bloch, and D.F. Smith. 1974. Culture medium for enterobacteria. J. 343

Bacteriol. 119:736-747. 344

28. Nichols, R.J., S. Sen, Y.J. Choo, P. Beltrao, M. Zietek, R. Chaba, S. Lee, K.M. 345

Kazmierczak, K.J. Lee,A. Wong, M. Shales, S. Lovett, M.E. Winkler, N.J. Krogan, A. 346

Typas, and C.A. Gross. 2011. Phenotypic landscape of a bacterial cell. Cell. 144:143-56. 347

29. Nilsson, R., I.J. Schultz, E.L. Pierce, K.A. Soltis, A. Naranuntarat, D.M. Ward, J.M. 348

Baughman, P.N. Paradkar, P.D. Kingsley, V.C. Culotta, J. Kaplan, J. Palis, B.H. Paw, 349

and V.K. Mootha. 2009. Discovery of genes essential for heme biosynthesis through 350

large-scale gene expression analysis. Cell Metabolism 10:119-130. 351

30. Ote, T., M. Hashimoto, Y. Ikeuchi, M. Su'etsugu, T. Suzuki, T. Katayama, and J. 352

Kato. 2006. Involvement of the Escherichia coli folate-binding protein YgfZ in RNA 353

modification and regulation of chromosomal replication initiation. Mol. Microbiol. 59:265-354

275. 355

31. Overbeek, R., T. Begley, R. M. Butler, J. V. Choudhuri, H. Y. Chuang, M. Cohoon, 356

V. de Crécy-Lagard, N. Diaz, T. Disz, R. Edwards, M. Fonstein, E. D. Frank, S. 357

Gerdes, E. M. Glass, A. Goesmann, A. Hanson, D. Iwata-Reuyl, R. Jensen, N. 358

Jamshidi, L. Krause, M. Kubal, N. Larsen, B. Linke, A. C. McHardy, F. Meyer, H. 359

Neuweger, G. Olsen, R. Olson, A. Osterman, V. Portnoy, G. D. Pusch, D. A. 360

Rodionov, C. Ruckert, J. Steiner, R. Stevens, I. Thiele, O. Vassieva, Y. Ye, O. 361

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

17

Zagnitko, and V. Vonstein. 2005. The subsystems approach to genome annotation and 362

its use in the project to annotate 1,000 genomes. Nucleic Acids Res. 33:5691-5702. 363

32. Phillips, G., B. El Yacoubi, B. Lyons, S. Alvarez, D. Iwata-Reuyl, and V. de Crécy-364

Lagard. 2008. The biosynthesis of the 7-deazaguanosine modified tRNA nucleosides: A 365

new role for GTP cyclohydrolase I. J. Bacteriol. 190:7876-7884. 366

33. Precious, E., C.E. Gunn, and G.A. Lyles. 1988. Deamination of methylamine by 367

semicarbazide-sensitive amine oxidase in human umbilical artery and rat aorta. Biochem. 368

Pharmacol. 37:707–713. 369

34. Scrutton, N.S., and D. Leys. 2005. Crystal structure of DMGO provides a prototype for a 370

new tetrahydrofolate-binding fold. Biochem. Soc. Trans. 33:776-779. 371

35. Teplyakov, A., G. Obmolova, E. Sarikaya, S. Pullalarevu, W. Krajewski, A. Galkin, 372

A.J. Howard, O. Herzberg, and G.L. Gilliland. 2004. Crystal structure of the YgfZ 373

protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon 374

metabolism. J. Bacteriol. 186:7134-7140. 375

36. Turner, W.L., J.C. Waller, and W.A. Snedden. 2005. Identification, molecular cloning 376

and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale 377

cress). Biochem. J. 385:217-223. 378

37. Van Schaftingen, E., R. Rzem, and M. Veiga-da-Cunha M. 2009. L-2-Hydroxyglutaric 379

aciduria, a disorder of metabolite repair. J. Inherit. Metab. Dis. 32:135-142. 380

38. Waller, J.C., S. Alvarez, V. Naponelli, A. Lara-Nuñez, I.K. Blaby, V. Da Silva, M.J. 381

Ziemak, T.J. Vickers, S.M. Beverley, A.S. Edison, J.R. Rocca, J.F. Gregory, J.F. 3rd, 382

de Crécy-Lagard, V., and A.D. Hanson. 2010. A role for tetrahydrofolates in the 383

on Decem

ber 25, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

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metabolism of iron-sulfur clusters in all domains of life. Proc. Natl. Acad. Sci. USA 384

107:10412-10417. 385

39. Waller, J.C., K.W. Ellens, S. Alvarez, K. Loizeau, S. Ravanel, and A.D. Hanson. 2011. 386

Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-387

sulfur cluster metabolism. J. Exp. Bot. doi: 10.1093/jxb/err286 388

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ALB+plumbagin M9+glycerol

mnmEygfZ

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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

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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

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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

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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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