Download - Downloaded from on May 11, 2020 by guestSep 04, 2012 · 150 Normal replication procedure with platinum (2 nm thickness) as first and carbon (20 151 nm thickness) as second layer

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Impact of vitamin B12 on the formation of the tetrachloroethene 1

reductive dehalogenase in Desulfitobacterium hafniense strain Y51 2

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Running Title: Repression of pce gene transposition by vitamin B12 5

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Anika Reinhold1‡, Martin Westermann2‡, Jana Seifert3, Martin von Bergen3, 4, 7

Torsten Schubert1, and Gabriele Diekert1# 8

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1 Department of Applied and Ecological Microbiology, Institute of Microbiology, 10

Friedrich-Schiller-University, Philosophenweg 12, D-07743 Jena, Germany 11

2 Electron Microscopy Center of the University Hospital Jena, Friedrich-Schiller-12

University, Ziegelmuehlenweg 1, D-07743 Jena, Germany 13

3 Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, 14

Permoserstrasse 15, D-04318 Leipzig, Germany 15

4 Department of Metabolomics, Helmholtz-Centre for Environmental Research-UFZ, 16

Permoserstrasse 15, D-04318 Leipzig, Germany 17

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‡Anika Reinhold and Martin Westermann contributed equally to this work. 19

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#Corresponding author: Prof. Gabriele Diekert 21

Friedrich Schiller University, Institute of Microbiology, Philosophenweg 12, D-07743 22

Jena, Germany 23

Tel.: +49 (0) 3641-949300; Fax: +49 (0) 3641-949302 24

e-mail: [email protected] 25

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02173-12 AEM Accepts, published online ahead of print on 7 September 2012

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ABSTRACT 26

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Corrinoids are essential cofactors of reductive dehalogenases in anaerobic 28

bacteria. Microorganisms mediating reductive dechlorination as part of their energy 29

metabolism are either capable of de novo corrinoid biosynthesis (e. g. 30

Desulfitobacterium spp.) or dependent on exogenous vitamin B12 (e. g. 31

Dehalococcoides spp.) In this study the impact of exogenous vitamin B12 32

(cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the 33

subcellular localization of reductive PCE dehalogenase was investigated in the gram-34

positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize 35

corrinoids de novo. PCE-depleted cells grown for several sub-cultivation steps on 36

fumarate as alternative electron acceptor lost the tetrachloroethene reductive 37

dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the 38

absence of vitamin B12, a gradual decrease of the PceA activity and protein amount 39

was observed; after 5 sub-cultivation steps with 10% inoculum, more than 90% of the 40

enzyme activity and of the PceA protein was lost. In the presence of vitamin B12, a 41

significant delay in the decrease of the PceA activity with a ~90% loss after 20 sub-42

cultivation steps was observed. This corresponded to the decrease in the pceA gene 43

level, indicating that exogenous vitamin B12 hampered the transposition of the pce 44

gene cluster. In the absence or presence of exogenous vitamin B12, the intracellular 45

corrinoid level decreased in fumarate-grown cells and the PceA precursor formed 46

catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that 47

exogenous vitamin B12 is not incorporated into the PceA precursor, even though it 48

affects the transposition of the pce gene cluster. 49

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INTRODUCTION 51

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The anaerobic reductive dehalogenation of organohalides is a metabolic 53

feature widespread among the genus Desulfitobacterium (35). Aliphatic and also 54

aromatic halogenated hydrocarbons (e. g. chlorinated or brominated ethenes, 55

polychlorinated phenols) are reductively dehalogenated by different 56

Desulfitobacterium strains. These strains possess different reductive dehalogenases 57

mediating the anaerobic dehalogenation. Almost all reductive dehalogenases 58

isolated so far harbor a corrinoid cofactor at the active site (11). 59

The gram-positive Desulfitobacterium hafniense strain Y51 was shown to have 60

a corrinoid-dependent reductive dehalogenase (PceA) that dechlorinates 61

tetrachloroethene (PCE) to cis-1,2-dichloroethene (33, 34). The gene encoding the 62

PceA enzyme is located in the pceABCT gene cluster that is flanked by insertion 63

sequences including transposase genes. The pceB gene product was proposed to 64

serve as a membrane anchor for PceA (24), although this role has never been 65

confirmed so far. The pceC gene shows homology to orfs encoding transmembrane 66

transcriptional regulators of the NirI/NosR-type involved in nitrite or nitrous oxide 67

reduction (28, 39). The pceT gene carries the genetic information for a peptidyl-prolyl 68

cis/trans isomerase. Recently, a role of the PceT protein in the maturation of PceA 69

was proposed (22) and its interaction with the Tat (twin arginine translocation) signal 70

peptide of the intracellular precursor of PceA was shown (20). 71

When D. hafniense strain Y51 is cultivated in the absence of PCE, the pce 72

gene cluster in whole or in part is irreversibly lost by transposition events (8). Circular 73

intermediates formed after the excision of the transposable elements were identified 74

in D. hafniense strain Y51 (8) and earlier in the closely related D. hafniense strain 75


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TCE-1 (4, 19). An acquisition of the gene cluster by horizontal gene transfer has 76

been discussed (19). 77

For PCE-dependent growth of D. hafniense Y51 no exogenous corrinoid has 78

to be added (33). This can be explained by the presence of corrinoid biosynthetic 79

genes in the genome of the organism (25) indicating the de novo formation of the 80

corrinoid cofactor of PceA. In addition, corrinoid salvaging might occur via the 81

functional expression of a genome-encoded vitamin B12-specific ATP-binding 82

cassette (ABC) transporter (btuFCD). The structure of the PceA corrinoid cofactor in 83

D. hafniense strain Y51 has not been identified so far. The only corrinoid cofactor of 84

reductive dehalogenases identified as yet is that of the tetrachloroethene reductive 85

dehalogenase in the gram-negative Sulfurospirillum multivorans, which is a unique 86

Norpseudo-B12 (16). This cofactor is synthesized de novo by the organism 87

(unpublished results). Therefore, S. multivorans is able to grow with PCE in the 88

absence of exogenous corrinoids. In contrast, the organohalide respiring 89

Dehalococcoides mccartyi (Chloroflexi) is strictly dependent on the addition of vitamin 90

B12 to the growth medium (18). 91

The necessity of the corrinoid cofactor for reductive dehalogenase function 92

became eminently evident when the heterologous production of the enzyme in E. coli 93

was tested, an organism lacking de novo corrinoid biosynthesis (1). In such 94

experiments the non-active reductive dehalogenase apoprotein formed intracellular 95

protein aggregates (15, 24, 34). Recently it has been shown, that the solubility of the 96

heterologously produced PceA in E. coli can be increased by the co-production of its 97

dedicated chaperone PceT (20). However, no reductive dehalogenase enzyme 98

activity was reported, most probably due to the absence of the corrinoid cofactor. 99

The anaerobic reductively dehalogenating bacteria (11, 21, 35) were isolated 100

from different environments including soil and sediment and were envisaged as a tool 101


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in bioremediation of contaminated sites (5). Hence, the availability of corrinoids in 102

natural environments or at polluted sites and their effect on the reductive 103

dehalogenation may be relevant for the dechlorination potential in soil contaminated 104

with organohalides. Other anaerobic prokaryotes in these environments such as 105

acetogens or methanogens contain corrinoids, which, upon occasional lysis of the 106

organisms, are released and therefore available for dehalogenating bacteria. 107

The study presented here sheds light on the interplay between de novo 108

corrinoid biosynthesis and the formation of a catalytically active reductive 109

dehalogenase. The effect of exogenous corrinoids on the stability of the pceA gene, 110

its transcription and translation as well as the maturation of the PceA protein was 111

investigated. A model for the maturation of the protein in dependence on the 112

presence of corrinoids and of PCE was developed. 113

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MATERIALS AND METHODS 115

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Cultivation of the organism. Desulfitobacterium hafniense strain Y51 (33) 117

was cultivated under anoxic conditions in a defined medium described by Scholz-118

Muramatsu et al. (30). Pyruvate (40 mM) was added as electron donor and either 119

PCE (10 mM) or fumarate (40 mM) as electron acceptor. Per 50 ml medium 1 ml of 120

0.5 M PCE dissolved in hexadecane was applied. The cultivation was carried out at 121

28°C in serum glass bottles closed with butyl rubber stoppers or Teflon septa and 122

anaerobized with nitrogen. Unless stated otherwise, vitamin B12 (cyanocobalamin) 123

was not added to the growth medium. 124

125

PceA enzyme activity measurements. The activity of the tetrachloroethene 126

reductive dehalogenase (PceA) was measured according to Neumann et al. (23). 127

Cells from a 100 ml culture were sedimented and resuspended in 1 ml anoxic buffer 128

(50 mM Tris-HCl, pH 7.5). The cell suspension was mixed with an equal volume of 129

glass beads (0.25 - 0.5 mm diameter, Carl Roth GmbH, Karlsruhe, Germany). For 130

cell disruption a bead mill was used (5 min 25 Hz; Retsch Mixer Mill MM400, Haan, 131

Germany). Cell debris was removed by centrifugation (1 min, 5,250×g). The protein 132

concentration was determined according to Bradford (2) using the Roti-Nanoquant 133

reagent (Carl Roth GmbH, Karlsruhe, Germany). 134

135

Immunoblot analysis. For the detection of PceA protein, crude extracts of D. 136

hafniense Y51 (5 µg protein/lane) were subjected to SDS-PAGE (13.5% 137

polyacrylamide gels). The immunological analysis was done as described earlier (13, 138

14). PceA antibodies were generated by immunization of rabbits with heterologously 139

produced PceA from D. hafniense PCE-S. PceT antibodies were kindly provided by 140


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T. Futagami (Kyushu University, Fukuoka, Japan). The PceA antiserum was diluted 141

5,000-fold, the PceT antiserum 50,000- fold, and the antibodies were detected via a 142

secondary antibody coupled to alkaline phospatase. 143

144

Freeze-fracture replica immunogold labeling (FRIL). D. hafniense Y51 cells 145

were harvested in the late exponential growth phase by centrifugation (4,600×g, 5 146

min, 28°C) and washed with substrate-free medium. Resuspended cells were rested 147

in anaerobic glass vials for at most 30 minutes at 28°C. Freeze-fracture of the cells 148

was done according to the protocol described for Sulfurospirillum multivorans (13). 149

Normal replication procedure with platinum (2 nm thickness) as first and carbon (20 150

nm thickness) as second layer was used. Replicas were treated with 5 mg/ml 151

lysozyme for 30 minutes at 37°C. The replica immunolabeling was carried out as 152

described earlier (7, 13). The PceA antibody was diluted 1:50 in labeling blocking 153

buffer (LBB; 1% [w/v] bovine serum albumin, 0.5% gelatine, 0.005% Tween 20). 154

Images were taken as digital pictures with a Zeiss EM 902A electron microscope 155

(Zeiss, Oberkochen, Germany) operated at 80 kV using a FastScan TVIPS CCD-156

camera 1k x 1k (TVIPS, Munich, Germany). The digital camera was operated by the 157

E-Menu4 software (TVIPS, Munich, Germany). The number of immunogold 158

signals/µm2 was determined as average of the fractured areas of 10 cells. 159

160

DNA isolation. D. hafniense Y51 was sub-cultivated with either PCE or 161

fumarate; the sub-cultivation experiments were performed twice for each electron 162

acceptor. In the late exponential growth phase (OD578 ~ 0.6) samples for DNA 163

isolation were taken from the cultures and harvested by centrifugation (16,000×g, 5 164

min, 10°C). The cell pellets were stored at -20°C. DNA was extracted using the 165

InnuPREP Bacteria DNA Kit (Analytik Jena, Jena, Germany). An additional RNA 166


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digestion was carried out by Ribonuclease I treatment (Sigma-Aldrich, Hamburg, 167

Germany). The quality of isolated DNA was determined via agarose gel 168

electrophoresis and photometric analysis (UV/Vis Cary 100 spectrophotometer, 169

Agilent Technologies, Böblingen, Germany). Isolated DNA was stored at -20°C. 170

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RNA isolation and reverse transcription. Sample preparation was done 172

according to the DNA isolation protocol (see above) in the exponential growth phase 173

(OD578 ~ 0.4). Cell pellets were stored at -80°C. RNA was isolated using RNeasy Mini 174

Kit (Qiagen, Hilden, Germany). An additional DNA digestion was carried out by 175

treatment with DNase I (RNAse free; Roche, Mannheim, Germany). The quality of 176

isolated RNA was determined via agarose gel elelctrophoresis and photometric 177

analysis (UV/Vis Cary 100 spectrophotometer, Agilent Technologies, Böblingen, 178

Germany). Isolated RNA was stored at -80°C. Reverse transcription of 1.25 µg total 179

RNA was carried out for pceA using the RevertAidTM First Strand cDNA Synthesis Kit 180

(Fermentas, St. Leon-Rot, Germany). As reference the transcript of the rpoB gene 181

was used (for primer sequences see quantitative real-time PCR section). To exclude 182

DNA contamination RNA without reverse transcriptase treatment was applied to 183

quantitative real-time PCR. Isolated cDNA was stored at -20°C 184

185

Quantitative real-time PCR. Quantification of gene and transcript levels was 186

implemented in a CFX96 Real-Time PCR system (Bio-Rad, München, Germany) for 187

pceA (GenBank acc. no. AAW80323.1; forward primer 5’-GGA GTG TAA TCC CGC 188

TTT ATC-3’; reverse primer 5’-AAT TTC CAC TGT TGG CCT TGT-3’; 136 bp) and 189

for rpoB as reference gene (NCBI acc. no. YP_516696.1; forward primer 5’-GAT 190

TCG GGC TTT GGG TTA TGC-3’; reverse primer 5’-CGC AGA CGC TTG TAG ATT 191

TCC-3’; 138 bp). The rpoB gene and transcript level was chosen, because it remains 192


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stable during sub-cultivation. Reactions contained 5 µl 2x MaximaTM SYBR Green 193

qPCR Master Mix (Fermentas, St. Leon-Rot, Germany), 0.4 µl 10 pmol/µl of each 194

primer and 25 ng DNA or 0.8 µl cDNA (final volume 10 µl). Each reaction was carried 195

out and measured three times. A three-step cycling program with an initial 196

denaturation for 10 min at 95°C followed by 40 cycles 15 sec 95°C, 30 sec 60°C, 30 197

sec 72°C was chosen. Data acquisition was performed during the extension step. 198

After the program was finished, a melting curve analysis to specify the PCR product 199

was carried out. Data analysis was carried out via the 2-∆∆Ct method (17). As 200

calibrator, DNA or cDNA of D. hafniense Y51 cultivated on pyruvate and PCE was 201

used. In each experiment the Ct value of no-template controls (NTC) was about 30. 202

The difference between the Ct values of the samples with the lowest template 203

concentrations and of the NTCs did never fall below a minimum of 7 cycles. 204

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Isolation and analysis of corrinoids. Cells of D. hafniense Y51 were 206

harvested by centrifugation (10 min, 6,700×g, 10°C); 5 g wet cells were resuspended 207

in 30 ml 50 mM Tris-HCl (pH 7.5). The cells were disrupted in a French Pressure Cell 208

at 2,000 psi (French Pressure Cell Press, SIM-AMINCO, Spectronic Instruments, 209

New York). The corrinoid extraction protocol was based on the method described by 210

Stupperich et al. (32). The final concentration of KCN in the crude extract was raised 211

to 100 mM. The Amberlite® XAD4 (Sigma-Aldrich, München, Germany) was washed 212

with methanol and equilibrated with 0.1% acetic acid prior to use. Each ml cyanide 213

extract was mixed with 0.25 g XAD4 material (16 h, 22°C, 400 rpm). The XAD4 214

material was subsequently washed with distilled water. The corrinoids were eluted 215

with methanol and the eluate was evaporated to dryness in a Speed Vac 216

Concentrator (Speed Vac Concentrator 100H, Savant, USA). Concentrates were 217

dissolved in distilled water and applied to a column filled with 3 g neutral aluminum 218


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oxide. Elution was conducted with distilled water. The first 20 ml of the eluate were 219

concentrated to 100 µl in a vacuum concentrator. For analysis of isolated corrinoids a 220

UV/Vis absorption spectrum was recorded (UV/Vis Cary 100 spectrophotometer, 221

Agilent Technologies, Böblingen, Germany). 222

223

Isolation and purification of PceA aggregates. Cells of D. hafniense Y51 of 224

sub-culture 15 were harvested by centrifugation in the late exponential growth phase 225

(OD578 ~ 0.6). Cells resuspended in 50 mM Tris-HCl (pH 7.5) were disrupted in a 226

French Pressure Cell at 2,000 psi (French Pressure Cell Press, SIM-AMINCO, 227

Spectronic Instruments, New York). Cell debris and large membrane fragments were 228

sedimented in two centrifugation steps (5,250×g and 12,000×g, 20 min, 4°C). The 229

supernatant of the second centrifugation step was subjected to ultracentrifugation 230

(100,000×g, 45 min, 4°C) and thus separated into soluble extract and particulate 231

fraction. The latter fraction was washed twice with 50 mM Tris-HCl (pH 7.5) and 232

analyzed via negative staining electron microscopy (see below). 233

The extracted membrane fraction was resuspended in solubilization buffer (20 234

mM Tris-HCl pH 8.0, 0.2 M NaCl, 1% sodium desoxycholate). After 30 min incubation 235

at room temperature the suspension was centrifuged and the pellet was washed four 236

times with 0.25% sodium desoxycholate in 20 mM Tris-HCl pH 8.0. After repeated 237

electron microscopic control the pellet was resuspended in distilled water and 238

subjected to density gradient centrifugation. A discontinuous sucrose gradient (70, 239

72, 74, 76, 78, 80, 82, 84, 86, 100% (w/v); volume 2 ml, respectively) was created. 240

Immediately after centrifugation (3h; 100,000×g; 4°C) each layer (2 ml) of the 241

gradient was carefully removed and protein precipitation according to the method of 242

Wessel and Flügge (37) was conducted. The precipitated protein was resuspended in 243

30 µl 50 mM Tris-HCl (pH 7.5). Equal volumes of the fractions were analyzed via 244


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SDS-PAGE (5 µl for immunoblot analysis, 2 µl for silver staining). Silver stain was 245

carried out according to the method of Schägger (29). From the lane of the aggregate 246

containing fraction major bands were excised. As a control, the same areas were 247

excised from the lane of the aggregate-free fraction of PCE-grown cells. 248

249

Analysis of the PceA aggregates. The silver was removed by adding potassium 250

ferricyanide and sodium thiosulfate (9). Subsequently, the sample was proteolytically 251

digested as described earlier (12). Peptides were reconstituted in 0.1% formic acid, 252

injected by an autosampler and were concentrated on a trapping column 253

(nanoAcquity UPLC column, C18, 180 μm x 2 cm, 5 μm, Waters, Eschborn, 254

Germany) with water containing 0.1% formic acid at flow rates of 15 μl/min. After 4 255

min, the peptides were eluted onto the separation column (nanoAcquity UPLC 256

column, C18, 75 μm x 100 mm, 1.7 μm, Waters, Eschborn, Germany). 257

Chromatography was performed by using 0.1% formic acid in solvents A (100% 258

water) and B (100% acetonitrile), with peptides eluted over 30 min with an 8-40% 259

solvent B gradient using a nano-HPLC system (nanoAcquity, Waters) coupled to an 260

LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). Continuous scanning of 261

eluted peptide ions was carried out between 400-2000 m/z, automatically switching to 262

MS/MS collision-induced dissociation (CID) mode on ions exceeding an intensity of 263

3000. For MS/MS CID measurements, a dynamic precursor exclusion of 3 min was 264

enabled. 265

Raw data were applied to a database search using the Thermo Proteome 266

Discoverer software (v1.0 build 43) to carry out a tandem ion search algorithm from 267

the Mascot house server (v2.2.1) by database comparison against the genome 268

project entry of D. hafniense Y51 in the National Center for Biotechnology Information 269

(NCBInr database, September 2010) with a 10 ppm tolerance for the precursor and 270


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0.8 Da for MS2 fragments. Furthermore, trypsin with a maximum of two missed 271

cleavage sites was selected and variable modifications, such as methionine oxidation 272

and carbamidomethylation of cysteine, were allowed. Peptides were considered to be 273

identified by Mascot when a probability < 0.05 (probability based ion scores threshold 274

> 40) was achieved. Protein identification was positive when at least two peptides 275

were identified. 276

277

Negative staining electron microscopy. The aggregate-containing fraction was 278

adsorbed to carbon-coated 400 mesh copper grids for 10 min, washed in distilled 279

water and negatively stained with 2% uranyl acetate for 1 min. For microscope and 280

imaging parameters see above. 281


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RESULTS AND DISCUSSION 282

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PceA formation in fumarate-grown cells is influenced by vitamin B12 284

D. hafniense Y51 was routinely cultivated on a pyruvate/PCE-containing 285

medium (30) to select for cells functionally expressing the pce genes. For strain 286

maintenance the organism was grown on medium void of vitamin B12 287

(cyanocobalamin). D. hafniense Y51 is able to synthesize corrinoids de novo. 288

Pyruvate was used as electron donor and PCE as electron acceptor in the energy 289

metabolism of the organism. The addition of vitamin B12 (50 µg/L cyanocobalamin) to 290

the cultures had no influence on the PCE reductive dehalogenase (PceA) activity in 291

crude extracts or on the formation of the PceA protein as tested via immunoblot (Fig. 292

1A and B, lane 1, "with PCE"). This result pointed to an adequate supply of the PceA 293

enzyme with corrinoid cofactor produced by the de novo corrinoid biosynthesis in D. 294

hafniense Y51. PCE-grown cultures (with and without vitamin B12) were used to 295

inoculate media containing fumarate rather than PCE as electron acceptor. For the 296

subsequent long-term cultivation, an inoculum of 10% was used for each transfer. 297

The experiment was conducted in media either with or without vitamin B12. During the 298

sub-cultivation, PceA activity as well as the PceA protein level was determined in the 299

late exponential growth phase. It should be noted that two forms of PceA were 300

detected in the crude extracts (prePceA, with the Tat signal peptide, and PceA, 301

without the Tat signal peptide) (Fig. 1A and B). 302

After one cultivation step in the absence of PCE and vitamin B12, half of the 303

PceA activity was lost and after six transfers almost no activity was detectable (Fig. 304

1A). The same result was observed for the PceA protein. The loss of the PceA 305

activity was significantly delayed when vitamin B12 was present in the medium (Fig. 306

1B). After 10 transfers the enzyme activity was decreased by almost 50%. Until sub-307


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cultivation step 20, the PceA protein level was comparable to that of PCE-grown 308

cells; however, less than 10% of the enzyme activity remained. This experiment was 309

repeated several times and was reproducible for the sub-cultivation in the absence of 310

vitamin B12. In the presence of vitamin B12, the enzyme activity was reduced by 90% 311

after a minimum of 15-20 sub-cultivation steps; however, different results were 312

obtained for the number of transfers required for a complete loss of the PceA protein 313

(Fig. S1). The minimal transfer number was about 30 as shown in Fig. 1B. In other 314

experiments, the PceA level remained constant as determined by immunoblot. 315

Nonetheless, all experiments lead to the conclusion that inactive PceA protein is 316

formed in the course of the sub-cultivation in the absence of PCE and in the 317

presence of vitamin B12. Our results are in accordance with a recent study with D. 318

hafniense strain TCE-1, which showed a very slow decrease within 30 sub-319

cultivations in the presence of vitamin B12 (250 µg/L) (4). An earlier study with D. 320

hafniense Y51, however, showed a fast decrease of the pceA gene level within only 321

a few sub-cultivation steps (8). In this study, a vitamin B12-free medium was used. 322

The discrepancy of both preceding studies can now be explained by our finding that 323

vitamin B12 has an impact on the rate of PceA decrease. 324

325

The pceA gene level is affected by vitamin B12 326

The loss of PceA upon sub-cultivation of D. hafniense Y51 has earlier been 327

described and attributed to the excision of the pce gene cluster (8). To monitor the 328

loss of the pceA gene during the long-term sub-cultivation experiment depicted in Fig. 329

1, quantitative real-time PCR (qPCR) was used. After 2 sub-cultivation steps in the 330

absence of PCE and vitamin B12, a decrease of almost 50% of the pceA gene level 331

was observed (Fig. 2A). No pceA gene was detected after 8 transfers (corresponding 332

to ~27 generations). Concomitantly with the loss of the pceA gene the pceA transcript 333


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level was reduced as determined by Reverse Transcription qPCR (Fig. 2B). In the 334

presence of vitamin B12 a slow decrease of the gene number was observed (Fig. 2A). 335

In sub-culture 15, the pceA level was still about 50%. The complete loss of the pceA 336

gene in cultures supplemented with vitamin B12 occurred after 60 sub-cultivations 337

steps. Again, the pceA transcript level decreased almost in parallel to the pceA gene 338

level (Fig. 2B). These results point to a stabilizing effect of vitamin B12 on the pce 339

gene cluster in D. hafniense Y51, which also explains the effect of vitamin B12 on 340

PceA in the course of the long-term cultivation described above. This result was 341

unexpected and cannot be explained so far. 342

343

Localization of PceA in the presence or absence of PCE and vitamin B12 344

The finding that the PceA activity decreased faster than the level of the PceA 345

protein in cells grown in the presence of vitamin B12 raised the question of the fate of 346

the exogenous corrinoid on the one hand and of the PceA protein on the other hand. 347

Therefore, cells sub-cultivated for 3 and 6 steps in the absence or for 30 and 60 348

steps in the presence of vitamin B12 were subjected to corrinoid extraction. In both 349

cultivations, the corrinoid level decreased significantly (Fig. 3). With exogenous 350

vitamin B12, less than ~20% of the initial level was recovered in cells of sub-351

cultivation step 30 (Fig. 3A). In vitamin B12-depleted cells, only a minor decrease of 352

the corrinoid level was observed after 10 sub-cultivation steps (Fig. 3B). It is 353

assumed that a cofactor-free PceA apoprotein is formed when the cells are long-term 354

sub-cultivated with fumarate instead of PCE and in the presence of vitamin B12. 355

To investigate the subcellular localization of PceA under the different growth 356

conditions (pyruvate/PCE and pyruvate/fumarate either with or without vitamin B12) 357

by electron microscopy, we used PceA directed antibodies for freeze-fracture replica 358

immunogold labeling (FRIL). This method was shown earlier to be useful for the 359


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detection of membrane integral proteins or proteins attached either to the 360

protoplasmic or the exoplasmic face of the cytoplasmic membrane (6, 7, 13, 26, 31, 361

38). Exemplarily the results obtained for subculture 15 in the presence of vitamin B12 362

are shown in Fig. 4. 363

To compare the results obtained under different growth conditions, the PceA 364

signals per area were counted (Fig. 4). The PceA signal distribution shown in Fig. 4A-365

C for cells grown on pyruvate/PCE revealed the presence of PceA proteins in the 366

cytoplasm, at the protoplasmic fracture face, and predominantly at the exoplasmic 367

fracture face of the cell membrane. The PceA signals in all three detection areas 368

were almost evenly distributed and not accumulated in a certain cell section. In 369

contrast, in cells grown in the absence of PCE the PceA protein was detected in large 370

aggregates localized in the cytoplasm (Fig. 4D). In the majority of the analyzed cells 371

just one cytoplasmic PceA patch was discovered that seemed to be associated with 372

the membrane in each case. Under these conditions PceA signals were found neither 373

on the protoplasmic face nor on the exoplasmic face of the cytoplasmic membrane 374

(Fig. 4E and F). The absence of exoplasmic PceA in the freeze-fracture of whole 375

cells and the presence of both forms of PceA in the crude extract (see Fig. 1B) 376

seems contradictory at first glance. It may be explained by a partial processing of 377

prePceA by the cells' signal peptidase, which may occur upon lysis of the cells. The 378

results depicted in Fig. 4 point to a predominantly exoplasmic and membrane-bound 379

state of PceA in PCE-grown cells and an intracellular and aggregated state of PceA 380

in fumarate-grown cells. The aggregation of PceA in the cytoplasm was observed in 381

PCE-depleted cultures independent on the absence or presence of exogenous 382

vitamin B12 but never in cells cultivated with PCE. The aggregation was already 383

visible after one transfer to medium void of PCE (Fig. S2). When the closely related 384

strain D. hafniense PCE-S (35) was subjected to the same experimental procedure 385


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and pyruvate/fumarate-grown cells were investigated using FRIL, similar PceA-386

containing aggregates were detected (Fig. S3). An aggregation of PceA was also 387

observed upon long-term sub-cultivation of D. hafniense Y51 in the absence of 388

vitamin B12. 389

It is feasible that the aggregates formed in the absence of PCE serve as a 390

"reservoir" for prePceA, which may be maturated as soon as PCE becomes 391

available. In this case, PceT might be required as a chaperone for prePceA 392

maturation. Therefore, we investigated the localization of PceT by FRIL in the 393

absence or presence of PCE (Fig. S4) using a specific PceT antibody (22). The 394

experiments showed that in PCE-grown cells the PceT protein was evenly distributed 395

either in the cytoplasm (Fig. S4A) or on the protoplasmic face of the cytoplasmic 396

membrane (Fig. S4B). Almost no PceT signals were detected on the exoplasmic face 397

(Fig. S4C). In fumarate-grown cells of D. hafniense Y51, the PceT protein showed 398

aggregate formation (Fig. S4D) as detected for prePceA inside the cell (Fig. 4D and 399

E). While the prePceA protein was detected exclusively in the aggregates, still single 400

PceT signals were found in the cytoplasm or at the protoplasmic face of the 401

cytoplasmic membrane. No PceT signals were detected on the exoplasmic fracture 402

face of the cytoplasmic membrane (Fig. S4F). 403

It was proposed earlier that PceT keeps the prePceA protein in an open 404

conformation for the incorporation of the metal-containing cofactors (20). Such an 405

incomplete folding might support the protein aggregation in D. hafniense Y51 cells in 406

the absence of the corrinoid-cofactor. The earlier finding that PceT binding increased 407

the solubility of heterologously formed cofactor-free prePceA in E. coli (20) is not 408

necessarily contradictory to our observation. Other proteins or factors present in D. 409

hafniense Y51 might favour the aggregate formation. 410

411


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Analysis of the PceA aggregates 412

Upon subcellular fractionation of D. hafniense Y51 cells grown for 15 sub-413

cultivations on pyruvate/fumarate in the presence of vitamin B12, most of the PceA 414

protein sedimented with the membrane fraction, probably due to sedimentation of the 415

PceA containing aggregates. Immunoblot analysis of the membrane fraction revealed 416

that exclusively the precursor form of PceA was present. The isolated membrane 417

fraction containing the protein aggregates showed no significant PceA enzyme 418

activity and level of corrinoids (data not shown). Resuspended membrane fractions 419

derived from pyruvate/fumarate-grown cells were subjected to electron microscopy to 420

examine the size and shape of the prePceA-containing aggregates. The enriched 421

particles (Fig. 5A) exhibited an almost globular shape with a diameter of about 100 422

nm. In some of the particles a groove was visible with a central cavity the origin of 423

which is unknown. No aggregates were detected, when the aggregate enrichment 424

procedure was conducted with cells grown on pyruvate/PCE. When the LD variant (8) 425

that lacks the pce genes was applied to FRIL, no protein patches were found inside 426

the cells (Fig. S5). These results indicate that the protein aggregation in the D. 427

hafniense Y51 wild type cells is a PceA-dependent process. 428

To elucidate the major components of the intracellular protein aggregates, the 429

particles were extracted from the membrane fraction by stepwise washing with 430

detergent and purified via sucrose density gradient centrifugation. The fraction 431

containing the aggregates (86% [w/v] sucrose) was subjected to electron microscopy. 432

The size and shape of the particles seemed to be unaffected by the purification 433

procedure. Subsequently, the aggregate fraction was applied to one-dimensional 434

SDS-PAGE (Fig. 5B, lane 1). The most salient protein bands, visible after silver-435

staining, were excised and analyzed using LC-MS/MS measurements. In addition to 436

the precursor of PceA and the PceT protein, the presence of which was also proven 437


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via immunoblot (Fig. 5B, lanes 2 and 3), other proteins were found. Besides 438

elongation factor Tu (EF-Tu/DSY0469; Fig. 5B, lane 1), a nicotinate-nucleotide 439

dimethylbenzimidazole phosphoribosyltransferase (CobT/DSY2114; Fig. 5B, lane 1) 440

was unambiguously identified as component of the prePceA aggregates. This 441

enzyme is involved in the late steps of corrinoid biosynthesis (for a review see 36). 442

When PCE-grown cells were subjected to the same fractionation procedure, no 443

bands were detected on the SDS polyacrylamide gels. 444

The aggregate composition implicates an impeded biosynthesis and 445

maturation of the prePceA protein. The aggregation also indicates irregular folding of 446

the cofactor-less apoprotein and may precede protein degradation. The presence of 447

elongation factor Tu, a component of the translation machinery (27), and the absence 448

of corrinoids points to the formation of the protein aggregates either in parallel or 449

immediately after the translation of the reductive dehalogenase transcript and prior to 450

cofactor incorporation. The incorporation of the corrinoid cofactor into the enzyme is 451

expected to require the interaction of different proteins from the corrinoid cofactor 452

biosynthesis machinery with the prePceA apoprotein. One of these proteins might be 453

the nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase (CobT), 454

which catalyzes the activation of the lower ligand base in the late steps of corrinoid 455

biosynthesis (3). It is not yet known, however, if CobT is involved in corrinoid cofactor 456

incorporation into corrinoid-containing proteins. 457

From the data presented here and in previous publications (20, 22, 24) a 458

model for the maturation of PceA may be derived (Fig. 6). According to this model, 459

the pce genes are transcribed in PCE-grown cells, the PceA cofactor-free precursor 460

is formed and binds to the PceT chaperone (22). When corrinoid cofactor is provided 461

by de novo biosynthesis, it is incorporated into prePceA. After incorporation of this 462

cofactor and assembly and incorporation of the iron-sulfur clusters the precursor 463


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protein is correctly folded and exported to the exoplasm by the Tat machinery. After 464

cleavage of the signal peptide the protein is bound to PceB, which has been 465

suggested to serve as a membrane anchor for PceA (10, 24, 35). In cells sub-466

cultivated for a few steps with fumarate instead of PCE corrinoid biosynthesis is 467

impeded; hence, this cofactor is not available for incorporation into prePceA. This 468

causes aggregation of the prePceA together with PceT and other proteins inside the 469

cells. Excision of the pce gene cluster occurs upon long-term cultivation with 470

fumarate. The loss of the gene cluster is delayed in the presence of exogenous 471

vitamin B12. To verify this model, further experiments are required, which are 472

currently underway in our laboratory. 473

474

475

ACKNOWLEDGEMENTS 476

477

This work was supported by grants from the Deutsche Forschungsgemeinschaft 478

(SCHM 2144/3-1 and FOR1530). The authors like to thank Taiki Futagami for 479

providing D. hafniense strain Y51, the LD variant of this bacterium and the PceT 480

antibody, Markus John and Denise Hinz for supplying the PceA antibody, and Peggy 481

Brand-Schön and Renate Kaiser for excellent technical assistance. 482


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610


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Figure 1: Protein level and specific activity of PceA during sub-cultivation of D. hafniense 1

Y51 on pyruvate and fumarate in the absence of PCE. The cultivation was carried out with 2

vitamin B12 (B; 50 µg/L) or without vitamin B12 (B) in the growth medium. The first lane 3

represents the pre-culture cultivated on pyruvate and PCE. Crude extracts (5 µg protein each 4

lane) were separated by SDS-PAGE and analyzed via immunoblot with PceA-specific 5

antibodies. pre: precursor form of PceA with the Tat signal peptide, mat: processed form of 6

PceA without the Tat signal peptide. 7

8

Figure 2: Relative pceA gene level (A) and transcript level (B) in cultures of D. hafniense 9

Y51 determined by quantitative PCR. Cells were sub-cultivated in the absence of PCE with 10

or without cyanocobalamin (vitamin B12, 50 µg/L) in the growth medium. 11

12

Figure 3: UV/Vis absorption spectra of isolated corrinoids from cells of D. hafniense Y51 13

sub-cultivated on pyruvate and fumarate in the absence of PCE. (A) Cells were cultivated 14

with vitamin B12 (50 µg/L) in the growth medium. (B) Cells were cultivated without vitamin B12 15

in the growth medium. sc - sub-culture. 16

17

Figure 4: Subcellular localization of PceA in D. hafniense Y51 determined by freeze-fracture 18

replica immunogold labeling (FRIL). (A-C) Cells cultivated on pyruvate and PCE, (D-F) cells 19

cultivated on pyruvate and fumarate in the absence of PCE (15 sub-cultivations). The 20

cultivation was carried out with vitamin B12 (50 µg/L) in the growth medium. The electron 21

micrographs show freeze-fractures through the cytoplasm (Cy), the protoplasmic fracture 22

face (PF), and exoplasmic fracture face (EF). The PceA protein was detected using PceA 23

specific antibodies and labeled with 10 nm gold-conjugated secondary antibodies (black 24

dots). The scale bar (A) represents 100 nm and is valid for all electron micrographs. 25


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Figure 5: Analysis of PceA aggregates enriched from cells of D. hafniense Y51 cultivated on 26

pyruvate and fumarate in the absence of PCE. The cultivation was carried out with vitamin 27

B12 (50 µg/L) in the growth medium. The enriched PceA aggregates were subjected to 28

electron microscopy (A, arrows) and SDS-PAGE (B). Lane 1: Silver stain; lanes 2 and 3: 29

Immunoblot analysis of the prePceA and the PceT protein using specific antibodies. prePceA 30

(DSY2839): precursor form of the PCE reductive dehalogenase, EF-Tu (DSY0469): 31

elongation factor Tu, PceT (DSY2836): peptidyl-prolyl cis/trans isomerase, CobT (DSY2114): 32

nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase. 33

34

Figure 6: Tentative model of PceA maturation in dependence on vitamin B12 and on the 35

electron acceptor. For a detailed explanation, see text. 36

37


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