Physiological and Transcriptome Response of the Polycyclic Aromatic

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Physiological and transcriptome response of the polycyclic aromatic hydrocarbon degrading Novosphingobium sp. LH128 after inoculation in soil Tekle Tafese Fida, Silvia K Moreno-Forero, Philip Breugelmans, Hermann Josef Heipieper, Wilfred F.M Röling, and Dirk Springael Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03822 • Publication Date (Web): 31 Dec 2016 Downloaded from http://pubs.acs.org on January 1, 2017

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Environmental Science & Technology

1

Physiological and transcriptome response of the polycyclic aromatic hydrocarbon

2

degrading Novosphingobium sp. LH128 after inoculation in soil

3 4

Tekle Tafese Fida1, Silvia K. Moreno-Forero2, Philip Breugelmans1, Hermann J. Heipieper3,

5

Wilfred F.M. Röling4, Dirk Springael1*

6 7

1

8

Belgium

9

2

Division of Soil and Water Management, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee,

Departement of Fundamental Microbiology, University of Lausanne, Bâtiment Biophore

10

Quartier Sorge, 1015 Lausanne, Switzerland

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3

12

UFZ, Permoserstr. 15, 04318 Leipzig, Germany

13

4

14

Amsterdam, the Netherlands

Department Environmental Biotechnology, Helmholtz Centre for Environmental Research–

Molecular Cell Physiology, FALW, VU University Amsterdam, De Boelelaan 1085, 1081 HV

15 16 17

*Corresponding author:

18

Dirk Springael

19

Division Soil and Water Management

20

KU Leuven

21

Kasteelpark Arenberg 20

22

3001 Heverlee

23

Belgium

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Tel. 32 16 32 16 04

25

Fax 32 16 32 19 97

26

E-mail: [email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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Soil bioaugmentation involves the inoculation of pollutant-degrading bacteria to accelerate

33

pollutant degradation. Often the inoculum shows a dramatic decrease in Colony Forming Units

34

(CFU) upon soil inoculation but this behavior is not well-understood. In this study, the

35

physiology and transcriptomic response of GFP tagged variant of Novosphingobium sp. LH128

36

was examined after inoculation into phenanthrene spiked soil. Four hours after inoculation, strain

37

LH128-GFP showed about 99% reduction in CFU while microscopic counts of GFP-expressing

38

cells were identical to the expected initial cell density, indicating that the reduction in CFU

39

number is explained by cells entering into a Viable But Non-Culturable (VBNC)-like state and

40

not by cell death. Transcriptome analysis showed a remarkably higher expression of

41

phenanthrene degradation genes 4 h after inoculation, compared to the inoculum suspension

42

concomitant with an increased expression of genes involved in stress response. This indicates

43

that the cells were active in phenanthrene degradation while experiencing stress. Between 4 h

44

and 10 days, CFU numbers increased to numbers comparable to the inoculated cell density. Our

45

results suggest that strain LH128-GFP enters a VBNC-like state upon inoculation into soil but is

46

metabolically active and that VBNC cells should be taken into account in evaluating

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bioaugmentation approaches.

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Size scattering of GFP cells

Heatmap of gene expression Inoculum

4h

Day 10

% of Max

day 10

4h

Inoculum

56

Forward size scatter

57

TOC/Abstract art

58 59 60 61 62 63 64 65 66 67 68 69



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70

INTRODUCTION

71

Microbial degradation is an important process in the removal of environmental contaminants

72

such as polycyclic aromatic hydrocarbons (PAHs) in soil1. Various bacteria display interesting

73

capacities for the catabolism of recalcitrant organic pollutants which make them of prime interest

74

for targeted bioaugmentation of polluted soils to accelerate biodegradation. When introduced

75

into soil, often the inoculated bacteria show a rapid decline in numbers as colony forming units

76

(CFU) mainly attributed to rapid cell death due to stress situations encountered in soil

77

Inoculated bacteria are suspected to be exposed to various physico-chemical and biological stress

78

factors in soil. Physico-chemical stress factors include moisture limitation, change in

79

temperature, pH, osmotic pressure, presence of toxic chemicals, and nutrient limitation while

80

biological stress factors include the presence of predator and competitor populations5,6,7. An

81

alternative explanation for such a decline in CFU numbers is the transition of the cells into a

82

Viable But Non-Culturable (VBNC)-like state. Such a transition to VBNC-like state has been

83

reported for more than 85 bacterial species as a response to different types of stresses including

84

starvation, extreme temperature, change of pH, sunlight, high salinity, desiccation, oxygen

85

limitations, sulfur dioxide, biocides, and heavy metals8,9,10,11,12,13. Moreover, various bacterial

86

species such as the plant pathogen Ralstonia solanacearum14,15 and the biocontrol strain

87

Pseudomonas fluorescens CHAO16 have been reported to become VBNC in soil.

2,3,4

.

88 89

Sphingomonads comprise a bacterial group whose members are often isolated as degraders of

90

recalcitrant organic pollutants including compounds like pesticides17, dioxins18 polychlorinated

91

biphenyls19, chlorinated phenols20 and polycyclic aromatic hydrocarbons21. These aerobic

92

organisms use the organic pollutants as sole source of carbon and energy by employing relevant

93

metabolic pathways. As they are relatively easy to culture in the laboratory, information is

94

available with regard to the physiology, stress response characteristics, and genetics of organic

95

pollutant catabolism in many contaminant-degrading Sphingomonads, making them prime

96

objects for bioremediation of contaminated soil21,22,23,24,25,26,27. Studies showed that inoculation of

97

soils with pollutant-degrading Sphingomonads was followed by a rapid decrease of number of

98

CFU in the soil, directly after inoculation2,28. As for other organic pollutant degrading strains that


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99 100


show immediate decrease in CFU after soil inoculation, this decrease in CFU number is not wellunderstood.

101 102

In this study, we aimed to improve our understanding of the fate of the phenanthrene degrading

103

Novosphingobium sp. LH128 after inoculation into sterile soil containing the PAH compound

104

phenanthrene. Upon inoculation, strain LH128 shows a rapid decline in CFU numbers which

105

increased afterwards with concomitant removal of phenanthrene28.As a common practice in

106

bioaugmentation procedures, the strain was cultured on a carbon source, i.e., glucose, that allows

107

reaching relatively rapid high cell densities, and was inoculated at high cell densities. The reason

108

behind this decline in CFU numbers was explored by using a GFP-expressing variant of strain

109

LH128 (LH128-GFP) enabling cultivation-independent counting of LH128 in aqueous soil

110

extracts. An integrated approach involving CFU and single cell counting, flow cytometric

111

analysis, transcriptome analysis and analysis of phenanthrene degradation rates was used to

112

determine the physiology and activity of LH128-GFP cells after inoculation into soil.

113 114

MATERIALS AND METHODS

115 116

Bacterial strain and growth conditions. This study uses a green fluorescent protein (GFP)

117

tagged derivative of Novosphingobium sp. LH12828,29, designated as strain LH128-GFP, which

118

was grown at 25oC in phosphate buffered 0.2% glucose containing minimal medium (MM)30.

119

The bacterial strain was deposited in the BCCM/LMG culture collection (Ghent, Belgium) as LMG

120

28893.

121 122

Soil microcosms. The used soils were a silt loam soil TM from an agricultural field in Belgium,

123

and a sandy clay loam soil 151 from a grassland in the United Kingdom. The characteristics of

124

these soils have been previously described31. Briefly, they have pH of 6.8 and 6.4; total carbon

125

content of 1.0 and 4.4 mg kg-1; total nitrogen content of 0.098 and 0.57 mg kg-1; cation exchange

126

capacity of 9 and 23 cmolc kg-1; and clay content (%) of 15 and 21, respectively for TM and 151.

127

The soils were passed through a 2 mm pore-size sieve, well-mixed, dried in an oven at 105oC for

128

24 h, and sterilized 3 times by autoclaving at 121oC for 15 min with intermittent incubation at



129

20oC for 2 days. Sterility was confirmed by plating soil suspensions on R2A agar32 and

130

incubating the plates for at least 10 days. The moisture contents of the soils were adjusted by

131

adding sterile Milli-Q water till field capacity (corresponding to 32 and 42% moisture content for

132

soil TM and 151, respectively), taking into account the volume of inocula to be added later. Two

133

g of the sterile soils was added to a battery of sterile 15 ml glass Pyrex tubes and spiked with an

134

acetone solution of phenanthrene (10 g l-1) at a concentration of 500 mg kg-1. The soil in the

135

tubes was homogenized gently on a multivortex for 30 s at speed 7 (Multi-tube vortex VX-2500,

136

VWR) and acetone removed by evaporation in a laminar flow cabinet for 15 h. Strain LH128-

137

GFP was precultured in MM containing 2 g l-1 of glucose and harvested in the late exponential

138

growth phase (OD600 of 0.6). Five ml of cells were washed twice with 0.01 M MgSO4,

139

resuspended in 0.01 M MgSO4 at appropriate cell densities and added to the soil microcosms as a

140

volume of 160 µl cell suspension per tube at final cell densities ranging from 5.4 x 109 to 5 x 103

141

CFU per g soil for soil TM, and 4.0 x 107 for soil 151. The soils and cells were mixed by gentle

142

vortexing and incubated at 20°C. Triplicate tubes were sacrificed for CFU numbering,

143

microscopic analysis, and determination of residual phenanthrene concentrations at time points

144

that were selected based on a previous study28 i.e., after 4 h of incubation (the time when

145

detectable phenanthrene biodegradation in soil starts) and at day 10 of incubation (the time point

146

when most of the phenanthrene degraded). In another identical set-up of experiments, shorter

147

time point for sampling (2 min) was selected. Experiments with phenanthrene-free sterile soil

148

TM were done in an identical way as described above for the sterile phenanthrene amended soil

149

TM except that no phenanthrene was added. When appropriate, 160 µl of the LH128-GFP

150

suspensions used for inoculation of the soil microcosms were added to 2 ml of 0.01 M MgSO4 in

151

15 ml glass Pyrex tubes and incubated along with the soil microcosms at 20°C. Triplicate tubes

152

for analysis were then sacrificed at the same time points as done for the soil microcosms.

153 154

Determination of CFU numbers. To determine CFU numbers in the soil microcosms, 5 ml of

155

0.01 M MgSO4 was added to each tube and the tubes shaken end-over-end for 30 min. Soil

156

particles were settled for 10 min and 200 µl triplicate samples of the suspension were 10-fold

157

serially diluted in 0.01 M MgSO4. Five µl of each dilution were spotted in triplicate on 0.2%

158

glucose MM square agar plates. The limit of CFU detection was 1000 CFU per g of soil. CFU


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159

numbers in the LH128-GFP suspensions in 0.01 M MgSO4 were determined in an identical way

160

after further dilution in 0.01 M MgSO4. We also examined whether the LH128-GFP inoculum

161

density affected CFU numbers by inoculating sterilized TM soil with cell densities ranging from

162

5 x 107 to 5 x 103 CFU per g of soil and counting CFU numbers after 4 h.

163 164

Determination

165

concentrations, the soil suspension in 0.01 M MgSO4 from triplicate tubes was centrifuged for 10

166

min at 3,070 x g (Jouan centrifuge) and phenanthrene extracted from the corresponding triplicate

167

soil pellets using a hexane:acetone (4:1 ratio) solution. After adding the solution (2.5 ml), the

168

tubes were vortexed for 2 min, centrifuged at 3,070 x g for 10 min and the supernatant collected

169

in a 1.5 ml glass vial. The pelleted soil was extracted a second time and both extracts were

170

pooled. The phenanthrene concentration in the extracts was determined by means of HPLC

171

(LaChrom, Merck Hitachi) equipped with a Platinum EPS C18 100A 3U column and a UV

172

detector set at 254 nm. The mobile phase was Milli-Q H2O/CH3CN (25/75%) with a flow rate of

173

0.8 ml min-1 and the injection volume was 10 µl. Quantification was based on comparison to the

174

peak areas obtained with standard concentrations of phenanthrene in hexane between 1 mg l-1

175

and 100 mg l-1. Abiotic losses and loss in extraction efficiency of phenanthrene due to for

176

instance soil sorption, were checked in identically treated sterile none-inoculated soil

177

microcosms after 4 h, 10 and 20 days incubation and found insignificant (95 ± 10 %).

of

phenanthrene

concentrations.

To

determine

soil

phenanthrene

178 179

Microscopic counting of cells. To examine cell clumping, attachment to soil particles, cell death

180

or a transition into a VBNC-like state, microscopic counting of green fluorescent LH128-GFP

181

cells was done concomitantly with the CFU numbering performed on cell extracts recovered

182

from the microcosm experiment with sterile TM soil inoculated with 5.4 x 109 CFU per g of soil

183

after 4 h and after 10 days of incubation. Cells containing GFP and motile in soil solution and in

184

the LH128-GFP suspensions in 0.01 M MgSO4 were counted in the dilutions used for CFU

185

counting, in a Helber counting chamber by means of epifluorescence microscopy coupled with a

186

digital camera (Olympus BX51, Olympus Belgium N.V). For detection of GFP containing cells,

187

filter set U-M41001 composed of a 461-500 nm excitation filter and a 521-560 barrier filter was

188

used.



189 190

Flow cytometry. Flow cytometry analysis was used to examine whether LH128-GFP cells

191

overall exhibited physiological changes at 4 h or 10 days of incubation in sterilized soil TM. The

192

soil suspensions prepared in 0.01 M MgSO4 were centrifuged for 2 min at 750 x g to remove

193

large soil particles. The supernatant was carefully layered onto 1 ml of Nycodenz (Axis-Shield-

194

Lucron Bioproducts, BV) with a density of 1.3 g ml-1 in a sterile Falcon tube and centrifuged at

195

7,000 x g for 30 min. The Nycodenz-MgSO4 interface containing the bacteria was recovered,

196

diluted with 9 ml of 0.01 M MgSO4 and centrifuged at 3,070 x g for 10 min. The pellet was

197

resuspended in 2 ml of 0.01 M MgSO4 and used for flow cytometry analysis in a BD Influx (BD

198

Biosciences). The cell suspension was delivered at flow rate of 300 events per s and data were

199

acquired until approximately 50,000 events were counted. GFP-containing cells were excited

200

using a 488 nm band pass filter and collected through the FL1 detector channel using a 530 nm

201

band pass filter. Analysis of data was done with FlowJo version 7.6 software (Tree Star, Inc.

202

Ashland, OR). The background noise was determined using a non-inoculated soil suspension in

203

0.01 M MgSO4 prepared as for the inoculated soil.

204 205

RNA extraction from inoculated soil and the inoculum culture. Total RNA was extracted

206

from 3 replicate tubes containing 2 g soil of the phenanthrene spiked TM soil microcosm

207

experiment inoculated with 5.4 x 109 cells per g soil, after 4 h and 10 days of incubation. RNA

208

extraction was performed using the RNA PowerSoil Total RNA Isolation Kit (Mo Bio, Benelux,

209

BV) according to the manufacturer’s instructions, with minor modifications needed for obtaining

210

RNA suitable for microarray analysis. Briefly, 10 µl of β-mercaptoethanol per ml of bead

211

solution was added to the soil in the microcosm tube before adding the SR1 solution to avoid

212

RNA degradation. The samples were incubated for 30 min at room temperature instead of at 4oC

213

after addition of the SR4 solution. The RNA was eluted with 100 µl of SR7 solution. RNeasy

214

Protect Bacteria Mini Kit (Qiagen, Benelux, BV) was used to extract total RNA in triplicate

215

immediately from inoculum suspension in 0.01 M MgSO4 containing a number of cells that was

216

identical to this added to the soil or after 4 h from suspension in 0.01 M MgSO4 incubated along

217

with the soil microcosm and stored in RNA protect Bacteria Reagent (Qiagen, Benelux BV). The

218

RNeasy Protect Bacteria Mini Kit was used since application of the kit used for extracting RNA


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219

from the soil microcosms resulted in poor extraction efficiency. RNA was purified using an

220

illustra MicroSpin™ S-400 HR gel filtration column (GE Healthcare, Little Chalfont, UK) and

221

precipitated using sodium acetate and ethanol. The dried pellet was suspended in 100 µl of

222

RNase free water and co-extracted genomic DNA removed by digestion with TURBO DNA-free

223

DNase kit (Ambion). Absence of DNA was verified by performing PCR using the primers

224

(DOF1:

225

targeting phnA1f

226

RNeasy Mini Kit column (Qiagen, Benelux, BV) according to the manufacturer’s instructions.

227

Integrity and purity of the RNA extracts were examined using the Bioanalyzer 2100 (Agilent

228

Technologies).

AARGGYTTCATYTTCGGYTGC 33

and

DOR1:

TGSGTCCAKCCSACRTGAT)

. The RNA preparation was finally desalted and concentrated using an

229 230

Microarray analysis and Reverse Transcriptase quantitative PCR (RT-qPCR). Genome-

231

wide microarray analysis was performed on the above mentioned RNA extracts obtained from

232

triplicate of the microcosm experiment with sterile soil TM and the LH128-GFP inoculum. The

233

obtained transcriptomes were compared to this of the original inoculum culture extracted at the

234

time of inoculation. Design of microarray probes, cDNA labeling, array hybridization, data

235

processing, normalization and analysis were performed as described33 with minor changes in the

236

nucleic acid labeling procedure, i.e., the labeling reaction was performed using Superscript III

237

reverse transcriptase (Invitrogen) and the heating reaction was performed at 55oC. The Minimum

238

Information About a Microarray Experiment (MIAME) procedure34 was followed for microarray

239

analysis and deposited in the NCBI's Gene Expression Omnibus35 (GEO series accession number

240

GSE41705). RT-qPCR to assess the number of transcripts of phnA1f was performed as

241

described33.

242 243

Phenanthrene degradation rate and growth of LH128-GFP on phenanthrene in soil. In a

244

separate experiment but with a similar set-up (i.e. three independent tubes sacrificed per time

245

points 3, 48 and 120 h), degradation rates at time point x (Jx, in mg phenanthrene per h per kg of

246

soil) were determined on basis of the phenanthrene concentrations in soil TM at sampling time

247

point x and sampling time points before (x-1) and after (x+1), as follows:



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[ phn]x − 1 − [ phn]x tx − tx − 1

248

Jbefore =

249

Jafter =

250

Jx =

251

in which [phn] is the concentration of phenanthrene in the soil (in mg phenanthrene per kg soil,

252

averaged over replicates), and t is time (in h). To obtain an activity per culturable cell at time x

253

(qin situ, x in mg phenanthrene per cell per h) the overall degradation rate Jx was divided by the

254

CFU (per kg of soil) at time x:

255

qin situ, x =

256

The in situ activities were compared to the maximum activity of Novosphingobium sp. LH128-

257

GFP, which was determined by growing the strain in MM containing 0.2 g l-1 phenanthrene

258

crystals as sole source of carbon in triplicate and determination of CFU and phenanthrene

259

concentrations as described above. Data corresponding to the exponential growth phase were

260

used to determine maximum growth rate, revealing a maximum growth rate (µmax) of 0.060 ±

261

0.002 h-1. Data from the stationary phase were used to determine the growth yield on

262

phenanthrene (Yphe), which was calculated as 7.95 ± 0.25 x 109 cells per mg phenanthrene

263

consumed. Maximum activity was subsequently calculated as µmax/Yphe

264

phenanthrene per cell per h.

[ phn ]x − [ phn]x + 1 t x + 1 − tx

Jbefore + Jafter 2

Jx CFUx

=

7.5 x 10-12 mg

265 266

Statistical analysis. A two-tailed Student’s t-test was used using SPSS 11.5 version software

267

and a p-value cutoff of 0.05 was used to assess that differences between the inoculum culture and

268

the LH128-GFP populations in soil were significant. To test for significantly differentially

269

expressed genes, Welch’s t- test with unequal variances was first used to calculate p-values

270

followed by the Benjamini and Hochberg procedure to correct the p-vales for multiple

271

hypothesis testing and convert the p-values into false discovery rates (FDRs)36. Genes with


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272

significant differential expression between conditions were defined as FDR of less than 0.05 and

273

a fold difference in hybridization signal intensity of greater than or equal to two.

274 275

RESULTS

276 277

Survival and phenanthrene degradation of LH128-GFP in soil. CFU numbers were

278

drastically lower (by two log units, p < 0.001) 4 h after inoculation compared to the expected

279

number based on the inoculum density (Figure 1A). At day 10 of the incubation, CFU numbers

280

had increased to 2 ±1.3 x 109 CFU per g soil which is about 37% of the predicted initial cell

281

density at soil inoculation. Phenanthrene was concomitantly degraded with about 70% removal

282

at day 10 as shown in Figure 1B. In contrast, no significant reduction in CFU number was

283

observed in the inoculum culture after either 4 h or 10 days of storage in 0.01 M MgSO4 under

284

the same conditions as the soil microcosms (Figure 1A), suggesting that the decline in CFU

285

numbers observed after 4 h was due to exposure to the soil environment. In other identically set

286

up experiments, a similar rapid decline in CFU numbers (p < 0.001) was recorded when the cells

287

were extracted 2 min after inoculation into sterile phenanthrene amended soil TM (Figure 1C),

288

and 4 h after inoculation in sterile phenanthrene-free soil TM (Figure 1D). Even with the lower

289

inoculum density (5 x 104), a decline in CFU number occurred although the recovery % as CFU

290

appears to increase with lower inoculum densities (at least till 6 x 105) (Figure S1 in

291

supplementary material).

292 293

GFP-fluorescent cells could be easily recognized from soil debris in the soil extract. Live-dead

294

staining to discriminate between life and dead cells was unfortunately unsuccessful due to

295

background interferences. Cell aggregates were not observed. GFP-fluorescent cells recovered

296

from soil after 4 h incubation were clearly moving with frequent change in directions, a

297

movement which was distinct from soil particles and similar to the motility of washed cells

298

stored for 4 h in 0.01 M MgSO4, suggesting that the cells were alive. After 4 h of incubation, the

299

number of moving GFP-fluorescent cells in the soil was not significantly different from the

300

number of cells that had been inoculated and hence around two log higher than the CFU counts

301

(Figure 2). After 10 days of incubation in soil, the number of motile GFP-fluorescent cells in the



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302

soil was not significantly different from this after 4 h of incubation and similar to the CFU

303

number of LH128-GFP cell suspension in 0.01 M MgSO4 used for inoculation. In addition,

304

unlike the two-log reduction in CFU number, the content of total RNA extracted from the soil

305

was not statistically different between the extract recovered after 4 h of incubation from the soil

306

microcosm (26.8 ± 8.2 µg per soil microcosm) and the extract recovered from 2 ml of the

307

inoculum suspension in 0.01 M MgSO4 containing the same amount of cells as added to a soil

308

microcosm (19.1 ± 2.5 µg per inoculum).

309 310

Whole-genome expression analysis. At 4 h of incubation, about 7.2% of all targeted protein-

311

coding genes (7,033 genes) were differentially expressed (with 4.5% showing increased and

312

2.7% showing decreased expression) while at day 10, about 8.7% of the genes were differentially

313

expressed (4.5% showed increased and 4.2% showed decreased expression) of which only 1.3%

314

were common between 4 h and day 10. A list of genes differentially expressed in soil at 4 h and

315

day 10 is presented in Tables S2 and S3, respectively, of the supplementary material while a heat

316

map of all differentially expressed genes is presented in Figure 3. Interestingly, at 4 h incubation,

317

the majority of genes encoding enzymes involved in PAH-degradation showed increased

318

expression, including genes which specify the initial attack of phenanthrene (phnA1f and phnA2f,

319

phnA3, phnA4) and others suspected to encode further downstream steps (phnA1d, phnA2d,

320

phnA2b, bphC, bphD, bphE, xylE, xylF, and xylY). In LH128, phnA1f and phnA2f form one

321

operon while phnA3, phnA4, phnC, bphD, xylY, phnA1d, phnA2d xylF, and xylE are located in

322

another operon. RT-qPCR analysis confirmed the increased expression of phnA1f (95-fold

323

increase) in soil at 4 h incubation (data not shown). These results support the hypothesis that

324

VBNC-like cells of LH128-GFP were indeed metabolically active in phenanthrene degradation 4

325

h after inoculation. Genes that are putatively involved in the transport of ions across the

326

membrane, such as genes encoding homologues of cation/multidrug efflux pump proteins,

327

Na+/H+ antiporter, copper resistance protein, and bacterioferritin, also revealed increased

328

expression. Other genes with increased expression were a gene encoding the extracytoplasmic

329

function (ECF) subfamily RNA polymerase sigma-70 factor, and genes encoding homologues of

330

antioxidative stress response proteins such as a catalase and a 1-Cys peroxiredoxin. Genes

331

involved in ribosomal biogenesis revealed decreased expression 4 h after inoculation.


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332 333

At day 10, the expression pattern in soil was largely changed. Differential expression (in

334

comparison with the inoculum culture) of most of the genes associated with phenanthrene

335

degradation and showing increased expression at 4 h, was not observed including phnA1f and

336

phnA2f. Exceptions were phnA2b, bphD, xylE and xylY. In addition, genes encoding homologues

337

of proteins involved in osmotic stress response showed increased expression, including proteins

338

involved in biosynthesis of the compatible solute trehalose (such as trehalose-6-phosphatase), an

339

outer membrane protein (OpsA) and a carbohydrate-selective porin. Moreover, the differential

340

expression of a gene encoding the ECF subfamily RNA polymerase sigma-24 factor as well as

341

genes encoding different classes of efflux pumps and permeases, was observed. Furthermore,

342

expression of the genes specifying ribosomal biogenesis had decreased while the gene encoding

343

the putative 1-Cys peroxiredoxin showed increased expression.

344 345

Physiological responses of LH128-GFP in soil. Flow cytometry analysis was used to examine

346

whether LH128-GFP cells overall exhibited physiological changes at 4 h or 10 days of

347

incubation in sterilized soil TM. The analysis was performed in a new experiment in which

348

LH128-GFP was inoculated at OD600 of 0.6 into soil. Flow cytometry showed that LH128-GFP

349

decreased in cell size at both 4 h and 10 days of incubation in the soil as compared to the cells

350

used for inoculation (Figure 4).

351 352

Phenanthrene degradation per LH128-GFP cell in soil. It was next questioned whether the

353

VBNC-like cells were contributing to phenanthrene degradation. Therefore, we compared the

354

calculated in situ degradation rates per culturable cell to a theoretical maximum rate per cell

355

derived from the maximum growth rate and growth yield as determined in a pure culture study

356

(see Material and Methods). For that purpose, LH128-GFP CFU numbers and phenanthrene

357

degradation were followed in more detail in two different soils. Soils TM and 151 were

358

inoculated at cell densities of 9.33 x 107 and 4.0 x 107 CFU per g soil, respectively, and soil

359

samples were analyzed after 3, 48, and 120 h for CFU and phenanthrene concentrations (Table

360

S1 in supplementary material). In both soils, LH128-GFP behaved similarly as reported above in

361

soil TM (Figure 1A), i.e., showing a sharp decline in CFU (by two log units) at 3 h of incubation.



Page 14 of 30

362

The decreases in phenanthrene concentration during the first 3 h of incubation in soil were too

363

minor to separate it from the experimental error in determining phenanthrene concentrations, and

364

thus calculating in situ rates. After 48 h, 7-18% of phenanthrene was degraded in both soils and

365

the CFU number had not increased in soil 151. In soil TM, the number of CFUs had increased by

366

a factor 297 after 48 h when compared with the data at 3 h, and so had reached a cell density

367

similar to the initial inoculum density, but then dropped again (Table S1 in supplementary

368

material). The phenanthrene degradation rates per culturable cell in the two soils, based on CFU

369

numbers, were 4 to 183 times higher than the theoretical maximum (Table 1), indicating that

370

VBNC-like cells had contributed to the majority of phenanthrene degradation between time point

371

3 h and 48 h. In soils TM and 151 an increase in CFU numbers was observed after 48 h (Table

372

S1 in supplementary material).

373 374

DISCUSSION

375 376

Our data showed that upon inoculation into soil, strain LH128-GFP CFU numbers rapidly

377

declined. Similar observations have been reported for other pollutant-degrading bacteria such as

378

the PAH-degrading Sphingomonas sp. B12 and the chlorophenol-degrading Arthrobacter

379

chlorophenolicus A637. The reduction in CFU at 4 h incubation in soil is best explained by the

380

conversion of the majority of LH128-GFP cells into a VNBC-like state rather than cell death, or

381

artifacts related to cell clumping and attachment to soil particles. This can in the first place be

382

concluded from the observation that 4 h after inoculation the number of GFP-containing cells in

383

soil was two orders of magnitude higher than CFUs, and similar to that of the inoculum. Second,

384

the total RNA yield obtained from soil after 4 h of incubation was not significantly less

385

compared with the RNA yield recovered from the inoculum cells, indicating that RNA was not

386

as such decayed. Transcription is well studied in Escherichia coli and has shown that the levels

387

of total RNA and mRNA depend on the levels of transcript synthesis and decay since mRNA is

388

rapidly degraded by ribonucleases when transcription stops38,39,40. If we assume that the half-life

389

of mRNA in LH128 is similar to this in E. coli, i.e., 6.8 min38, such half-life is far below the 4 h

390

of incubation in soil suggesting that the extracted soil RNA was contained within transcribing

391

and thus living LH128-GFP cells in the soil.


Page 15 of 30


392 393

The change in the VBNC-like state appears to be an immediate response and was due to

394

exposure to the soil environment as cells incubated in the 0.01 M MgSO4 solution that was also

395

used to inoculate and extract cells from soils, remained fully culturable. The transition to a

396

VBNC-like state is not related to exposure to phenanthrene since we observed an identical

397

behavior of LH128-GFP in sterile, phenanthrene-free TM soil (Figure 1D). Neither could it be

398

related to the changes in the physico-chemical characteristics of the soil resulting from

399

autoclaving, since a similar decline of CFU number was observed in non-sterile soil28. Moreover,

400

similar reduction in CFU numbers was recorded in gamma-irradiated soils including the TM soil

401

(Fida and Springael, unpublished results). We do not know the primary effector of the VBNC-

402

like response of strain LH128-GFP in soil. Previously, we observed that strain LH128 enters a

403

VBNC-like state under conditions of starvation; during storage in MgSO4 but only after a

404

prolonged storage of 6 months26. The percentage of the inoculated LH128-GFP cells that

405

remained culturable immediately after soil inoculation depended on the inoculum number,

406

although it never exceeded 10%. Improved survival of inoculated cells at lower inoculum

407

densities in soil was observed previously for the phenanthrene-degrading Arthrobacter

408

polychromogenes strain RP1741. This might be attributed to a decreased competition for scarce

409

resources required for growth in the soil in case of low inoculum densities. This suggested that

410

nutrient limitation could also contribute to VBNC-like state.

411 412

In the group of the α-proteobacteria to which Sphingomonads belong, entrance into a VBNC

413

state has been demonstrated for Agrobacterium tumefaciens and Rhizobium leguminosarum as a

414

response to copper stress42 and Sinorhizobium meliloti 1021 as a response to desiccation stress43.

415

The term VBNC in this study is used in a broad sense since the LH128-GFP VBNC cells might

416

represent impaired living cells that have for instance damaged membranes or might represent

417

actual healthy cells12,13,44,45,46. However, support for the latter comes from the transcriptomic,

418

flow cytometry, and degradation rate data, which suggest that the largest fraction of LH128-GFP

419

cells had changed their morphology and are active. Furthermore, the physiological response of

420

LH128-GFP cells was manifested by a considerable shrank after inoculation into soil as

421

previously reported for Pseudomonas putida KT244247, P. putida P8 and Enterobacter sp.



48

Page 16 of 30

, Listeria monocytogenes49, and Burkholderia pseudomallei3. The decrease in cell

422

VKGH12

423

size was also observed in LH128 during starvation stress26. Decreasing the cell size increases the

424

cell’s surface area to volume ratio that can increase uptake of scarce substrates50,51. The factors in

425

the soil that cause reduction in cell size are not well known. It could likely be due to reduced

426

cytoplasmic crowding by reducing the expression of ribosomal proteins or due to loss of water

427

from the cell as a result of the presence of solutes such as sodium, magnesium and others in the

428

soil52.

429 430

The transcriptomic data showed that, overall, LH128-GFP adapted its gene expression profile

431

upon inoculation. The transcriptome response of LH128-GFP at 4 h soil incubation concerned

432

the VBNC cells mainly and cannot solely be associated with the 1% fraction of cells that were

433

culturable, since (i) the observation that the inoculated cells were motile cells indicates that they

434

are metabolically active, and it is likely that they then produce transcripts and (ii) the overall

435

increased expression of phenanthrene catabolic mRNA was extremely high, i.e., till 100 times

436

more than in the cells in the 0.01 M MgSO4 solution used to perform the inoculation. Neither

437

could it be due to a non-soil associated change in transcriptome during 4 h of incubation. We

438

determined the transcriptome profile of washed LH128-GFP cells stored in 0.01 M MgSO4 for 4

439

h under the same conditions as the soil microcosms and the profile did not significantly change

440

during this time period compared to this of the initial inoculum used for inoculating the soil

441

microcosms.

442 443

The transcriptome suggested that the cells were catabolically active in phenanthrene degradation

444

as evidenced by the increased expression of phenanthrene catabolic genes. Previously, VBNC

445

cells have been described as metabolically dormant cells that only become active after

446

resuscitation when conditions become favorable45. However, other studies indicated that VBNC

447

cells are metabolically active and not dormant2,53,54. Naphthalene-degrading Sphingomonas

448

yanoikuyae B1 shows a similar increased expression of catabolic genes (bphC and xylE) after

449

inoculation in naphthalene spiked soil at a moment when the cells are declining in CFU

450

numbers2. This decline in CFU of B1 was suggested to be due to inefficient extraction of the

451

cells from soil due to strong adherence to soil particles but the authors show no evidence for that


Page 17 of 30


452

and hence the occurrence of active S. yanoikuyae B1 in a VBNC-like state might be an

453

alternative explanation. The occurrence of VBNC LH128-GFP cells active in phenanthrene

454

catabolism in soil upon inoculation was further supported by the observed degradation rates that

455

were in general much higher than theoretically possible, when only the culturable cells would

456

have been metabolically active. The theoretical maximum rate was determined by growing

457

LH128 in liquid medium containing phenanthrene crystals. We observed exponential growth,

458

indicating that phenanthrene was present in saturating concentrations and was not inducing

459

VBNC state. Therefore, we assume that these laboratory conditions provided optimal growth

460

conditions and hence allowed to establish the maximum theoretical rate per cell. The maximum

461

rate was calculated from the determined maximum growth rate and growth yield. A recent

462

literature compilation revealed that these laboratory-derived growth parameters enable

463

describing growth under in situ conditions55, and therewith to compare growth under laboratory

464

settings to growth in environmental settings.

465 466

Interestingly, several genes that were differentially expressed could be associated with responses

467

to stress situations indicating the LH128-GFP cells indeed experienced a stress situation 4 h after

468

inoculation in soil. Although studies were not available on the whole-genome transcriptome

469

response of pollutant-degrading bacteria in soil, the deferential expression of several genes in

470

LH128 was in agreement with pollutant- degrading Rhodococcus sp. TG13 and TN3 during cold-

471

stress in culture medium56. The increased expression of cation/multidrug efflux pumps and

472

Na+/H+ antiporters has been previously observed in LH128-GFP biofilm cells33 and other

473

bacteria such as Desulfovibrio vulgaris57,58 upon solute stress suggesting that the cells experience

474

osmotic or ionic stress in the soil. Moreover, the increased expression of genes encoding

475

oxidative stress response proteins, such as 1-Cys peroxiredoxin and catalase, suggested that

476

LH128-GFP experienced reactive oxygen species (ROS)59,60. ROS are generated by membrane

477

perturbation or altered enzyme activities that cause aberrant electron flows. In addition, the

478

degradation of aromatic compounds by mono- and dioxygenases can generate ROS in aerobic

479

organisms61,62.

480



Page 18 of 30

481

At day 10 of the incubation in soil, LH128-GFP CFU numbers had again increased to densities

482

comparable to the initial inoculum. It is not clear whether this increase in CFU number was due

483

to resuscitation of VBNC cells or to growth of the culturable cells or both implying an

484

equilibrium between cell death and growth. In Vibrio vulnificus, an increase in culturable fraction

485

was due to resuscitation of the VBNC population rather than of growth of the few culturable

486

cells63,64. At day 10 of incubation, the overall LH128-GFP transcriptome had significantly

487

changed compared to the 4 h incubation time point. Most of the catabolic genes had lost its

488

strong expression, likely due to the less bioavailable phenanthrene in the soil since phenanthrene

489

levels were reduced to 30% of the initial amount. S. yanoikuyae B1 showed a similar decrease in

490

expression of catabolic genes (bphC and xylE) in soil, with no detectable expression after 20

491

days of incubation when naphthalene was depleted 2. On the other hand, it is clear that, overall,

492

LH128-GFP still experienced a stress situation since at day 10, an increased expression of many

493

more genes (compared to time point 4 h) involved in general stress response and osmotic stress

494

was observed.

495 496

In conclusion, the physiological and genome-wide transcription analyses of LH128-GFP cells

497

inoculated into artificially PAH-contaminated soil strongly suggest a rapid entry of most LH128-

498

GFP cells into a VBNC-like state. The results indicate that those VBNC-like LH128-GFP cells

499

are metabolically active and appear to respond rapidly to the environmental stimuli in the soil by

500

expression of stress protective mechanisms. Our data emphasize on the importance of monitoring

501

bacterial activity in soil and show that VBNC cells should be taken into account in evaluating

502

bioaugmentation approaches.

503 504 505

ACKNOWLEDGMENTS

506 507

This research was supported by the EC FP7 Framework KBBE project BACSIN (grant KBBE-

508

211684) and by the Inter-University Attraction Pole (IUAP) “µ-manager” of the Belgian Science

509

Policy (BELSPO, P7/25). This article is dedicated to Wilfred Röling, who passed away on 25

510

September 2015.


Page 19 of 30


511

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(58) Mukhopadhyay, A.; He, Z.; Alm, E. J.; Arkin, A. P.; Baidoo, E. E.; Borglin, S. C.; Chen, W.; Hazen, T. C.; He, Q.; Holman, H.-Y.; Huang, K.; Huang, R.; Joyner, D. C.; Katz, N.; Keller, M.; Oeller, P.; Redding, A.; Sun, J.; Wall, J.; Wei, J.; Yang, Z.; Yen, H.-C.; Zhou, J.; Keasling, J. D., Salt Stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. J. Bacteriol. 2006, 188(11), 4068-4078.

679 680 681

(59) Chang, W. S.; Li, X.; Halverson, L. J., Influence of water limitation on endogenous oxidative stress and cell death within unsaturated Pseudomonas putida biofilms. Environ.Microbiol. 2009, 11(6), 1482-1492.

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(60) Cytryn, E. J.; Sangurdekar, D. P.; Streeter, J. G.; Franck, W. L.; Chang, W. S.; Stacey, G.; Emerich, D. W.; Joshi, T.; Xu, D.; Sadowsky, M. J., Transcriptional and physiological responses



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of Bradyrhizobium japonicum to desiccation-induced stress. J. Bacteriol. 2007, 189(19), 67516762.

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(61) Tamburro, A.; Robuffo, I.; Heipieper, H. J.; Allocati, N.; Rotilio, D.; Di Ilio, C.; Favaloro, B., Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates. FEMS Microbiol. Lett. 2004, 241(2), 151-156.

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698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 24 ACS Paragon Plus Environment

Page 25 of 30

716


TABLES

717 718

Table 1. In situ degradation activity of Novosphingobium sp. LH128-GFP in soils TM and 151

719

after 48 h of incubation, as compared to a theoretical maximum determined by culturing (see

720

material and methods). Culture/soil

Activity (mg phenanthrene

Activity relative to

per cell per h)

theoretical maximum

7.55 10-12

1

TM

3.05 10-11

4

151

1.37 10-9

183

Culturing-based theoretical maximum

721 722

FIGURE LEGENDS

723

Figure 1. Dynamics of LH128-GFP CFU numbers and residual phenanthrene concentrations in

724

soil TM after inoculation. (A) CFU per gram of soil of Novosphingobium sp. LH128-GFP after

725

inoculation in sterile soil TM microcosms supplemented with about 500 mg kg-1 phenanthrene.

726

CFU numbers in soil (black bars) are compared with CFU numbers of the cell suspension in 0.01

727

M MgSO4 used for inoculation (white bars). (B) Residual phenanthrene concentrations in the

728

experiment whose CFU number data are reported in (A). (C) CFU per gram of soil of

729

Novosphingobium sp. LH128-GFP after incubation for 2 min in sterile soil TM microcosms

730

supplemented with 500 mg kg-1 phenanthrene. CFU numbers in soil (black bars) in comparison

731

with CFU numbers of the cell suspension in 0.01 M MgSO4 used for inoculation (white bars).

732

(D) CFU per gram of soil of Novosphingobium sp. LH128-GFP after inoculation in sterile soil

733

TM microcosms without phenanthrene amendment after 4 h of incubation. CFU numbers in soil



Page 26 of 30

734

(black bars) in comparison with CFU numbers of the cell suspension in 0.01 M MgSO4 used for

735

inoculation (white bars). In all cases, the inoculum suspension was incubated at 20°C (as done

736

for the soil microcosms) and samples for CFU counting taken at the same time as the soil

737

samples were analyzed (2 min, 4h and 10 days). The error bars indicate standard deviations

738

(n=3).

739

Figure 2. Total microscopic counts of GFP containing and motile Novosphingobium sp. LH128-

740

GFP cells recovered 4 h and 10 days after inoculation in sterile TM soil supplemented with 500

741

mg kg-1 phenanthrene and in 2 ml of the LH128-GFP suspension in 0.01 M MgSO4 (representing

742

the same amount of cells as added to the soil microcosms) after 4 and 10 days incubation under

743

the same conditions as the soil microcosms. White bars: inoculum suspension in 0.01 M MgSO4;

744

black bars: cell suspension recovered from soil in 0.01 M MgSO4. The error bars indicate

745

standard deviations (n=3).

746

Figure 3. Dendrogram of heatmap of gene expression in strain LH128-GFP in the suspension

747

used for inoculation (A), 4 h after inoculation in soil TM (B) and 10 days after inoculation in soil

748

TM (C), as measured by micro-array analysis. The color scale indicates relative gene expression

749

where red indicates high expression and blue represents low expression Result are shown for

750

each of the 3 biological replicates. The position of phenanthrene catabolic genes is indicated by

751

the red arrow.

752

Figure 4. Flow cytometry analysis of size scattering (forward scatter, FSC) of GFP containing

753

cells implying differences in cell size. Line symbols: (green line) inoculum culture before

754

addition in soil; (blue line), cells extracted from soil after 4 h; (violet line), cells extracted from

755

soil after day 10. The values in the Y-axis are in “% of Max” in which “Max” refers to the cells

756

which showed the most frequently encountered FSC value.



1.0E+10

A

1.0E+10

1.0E+09

CFU per gram of soil

CFU per gram of soil or ml of culture

Page 27 of 30

1.0E+08 1.0E+07 1.0E+06 1.0E+05

C

1.0E+09 1.0E+08 1.0E+07 1.0E+06 1.0E+05 1.0E+04

1.0E+04

1.0E+03

1.0E+03 0h

4h

0 min

Day 10

Time of incubation

600

Time of incubation

1.0E+10

B CFU per gram of soil

Residual phenanthrene concentration (mg kg-1)

400 300 200 100

1.0E+08 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03

0 0h

758

D

1.0E+09

500

4h

Day 10

Time of incubation

757

2 min

Figure 1.

759


0h Time of incubation

4h

Total number of GFP-expressing and motile LH128-GFP cells in soil or inoculum suspension


1.0E+10 1.0E+09 1.0E+08 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 0h

761

4h Time of incubation

760

Page 28 of 30

Figure 2.

762 763 764 765


day 10

Page 29 of 30


A

B

C

766 767 768

Figure 3.

769



Page 30 of 30

100

% MAX

80

60

40

20

20K FSC (PER)

770 771

40K

Figure 4.

772 773 774


60K

Physiological and Transcriptome Response of the Polycyclic Aromatic

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